bims-nimamd Biomed News
on Neuroimmunity and neuroinflammation in ageing and metabolic disease
Issue of 2022‒11‒13
93 papers selected by
Fawaz Alzaïd
Sorbonne Université
  1. CD1d-dependent rewiring of lipid metabolism in macrophages regulates innate immune responses.
  2. Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics.
  3. A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience.
  4. Enhanced T cell effector activity by targeting the Mediator kinase module.
  5. AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.
  6. Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.
  7. Mouse long-lived plasma cells develop from multiple origins.
  8. A nascent peptide code for translational control of mRNA stability in human cells.
  9. BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.
  10. SAMHD1 deacetylation by SIRT1 promotes DNA end resection by facilitating DNA binding at double-strand breaks.
  11. REC drives recombination to repair double-strand breaks in animal mtDNA.
  12. Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.
  13. Error, reproducibility and uncertainty in experiments for electrochemical energy technologies.
  14. Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR.
  15. Age-induced prostaglandin E2 impairs mitochondrial fitness and increases mortality to influenza infection.
  16. The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis.
  17. Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia.
  18. Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants.
  19. Non-viral precision T cell receptor replacement for personalized cell therapy.
  20. Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3.
  21. Influenza A virus reassortment in mammals gives rise to genetically distinct within-host subpopulations.
  22. Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist.
  23. Ferritin triggers neutrophil extracellular trap-mediated cytokine storm through Msr1 contributing to adult-onset Still's disease pathogenesis.
  24. Deep autoencoder for interpretable tissue-adaptive deconvolution and cell-type-specific gene analysis.
  25. Notch gives early T cell progenitors time to grow up.
  26. Impaired regulatory T cell-dendritic cell interactions contribute to autoimmunity in leukocyte adhesion deficiency type-1.
  27. Hi-TrAC reveals division of labor of transcription factors in organizing chromatin loops.
  28. Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.
  29. CK2-mediated phosphorylation of SUZ12 promotes PRC2 function by stabilizing enzyme active site.
  30. Dusp6 deficiency attenuates neutrophil-mediated cardiac damage in the acute inflammatory phase of myocardial infarction.
  31. Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.
  32. Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.
  33. PD-L1 negatively regulates antifungal immunity by inhibiting neutrophil release from bone marrow.
  34. Transcription factor antagonism regulates heterogeneity in embryonic stem cell states.
  35. Capture at the ER-mitochondrial contacts licenses IP3 receptors to stimulate local Ca2+ transfer and oxidative metabolism.
  36. Structural basis for the activation of the lipid scramblase TMEM16F.
  37. Structural basis of human SNAPc recognizing proximal sequence element of snRNA promoter.
  38. Analysis of clinically relevant variants from ancestrally diverse Asian genomes.
  39. Genetic regulation of serum IgA levels and susceptibility to common immune, infectious, kidney, and cardio-metabolic traits.
  40. ADRA1A-Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP.
  41. No sugar, just protein please - says the fly.
  42. Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.
  43. Single-cell immune profiling reveals thymus-seeding populations, T cell commitment, and multilineage development in the human thymus.
  44. Skip to the end: metastasis before tumorigenesis.
  45. Harnessing introns to monitor gene expression.
  46. Single cell sequencing reveals that CD39 inhibition mediates changes to the tumor microenvironment.
  47. The phenotypic landscape of essential human genes.
  48. Tuft-cell-intrinsic and -extrinsic mediators of norovirus tropism regulate viral immunity.
  49. The first mitotic division of human embryos is highly error prone.
  50. In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.
  51. Amino acid variability, tradeoffs and optimality in human diet.
  52. CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia.
  53. The memory B cell response to influenza vaccination is impaired in older persons.
  54. Platr4 is an early embryonic lncRNA that exerts its function downstream on cardiogenic mesodermal lineage commitment.
  55. Splitting up differentiation and cell cycle exit.
  56. RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4.
  57. Structures of a mobile intron retroelement poised to attack its structured DNA target.
  58. Robust replication initiation from coupled homeostatic mechanisms.
  59. Structural basis of organic cation transporter-3 inhibition.
  60. Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity.
  61. dcHiC detects differential compartments across multiple Hi-C datasets.
  62. Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells.
  63. Determining Complete Chromosomal Haplotypes by mLinker.
  64. Cell division drives DNA methylation loss in late-replicating domains in primary human cells.
  65. Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling.
  66. Association of apolipoprotein M and sphingosine-1-phosphate with brown adipose tissue after cold exposure in humans.
  67. Activating NO-sGC crosstalk in the mouse vascular niche promotes vascular integrity and mitigates acute lung injury.
  68. Omics goes spatial epigenomics.
  69. We are worthy.
  70. A gut-retching discovery.
  71. An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.
  72. Mechanoreceptor signal convergence and transformation in the dorsal horn flexibly shape a diversity of outputs to the brain.
  73. CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever.
  74. NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.
  75. Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans.
  76. Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.
  77. Systematic tissue annotations of genomics samples by modeling unstructured metadata.
  78. Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.
  79. CTCF blocks antisense transcription initiation at divergent promoters.
  80. CRTC1 deficiency, specifically in melanocortin-4 receptor-expressing cells, induces hyperphagia, obesity, and insulin resistance.
  81. Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.
  82. Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.
  83. An atypical RNA quadruplex marks RNAs as vectors for gene silencing.
  84. A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of pro-inflammatory interleukin 12 family cytokines.
  85. Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells.
  86. A loyal "G"uard.
  87. The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling.
  88. An allosteric modulator activates BK channels by perturbing coupling between Ca2+ binding and pore opening.
  89. METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury.
  90. CAR-T cells tackle autoimmunity.
  91. DNA replication initiation shapes the mutational landscape and expression of the human genome.
  92. Chromatin oxygen sensing by histone H3 prolyl hydroxylation.
  93. Ensuring Progress Toward Racial Equity in Pulse Oximetry-Reply.
  1. Nat Commun. 2022 Nov 07. 13(1): 6723
    CD1d-dependent rewiring of lipid metabolism in macrophages regulates innate immune responses.
    Phillip M Brailey, Lauren Evans, Juan Carlos López-Rodríguez, Anthony Sinadinos, Victoria Tyrrel, Gavin Kelly, Valerie O'Donnell, Peter Ghazal, Susan John, Patricia Barral.
      Alterations in cellular metabolism underpin macrophage activation, yet little is known regarding how key immunological molecules regulate metabolic programs in macrophages. Here we uncover a function for the antigen presenting molecule CD1d in the control of lipid metabolism. We show that CD1d-deficient macrophages exhibit a metabolic reprogramming, with a downregulation of lipid metabolic pathways and an increase in exogenous lipid import. This metabolic rewiring primes macrophages for enhanced responses to innate signals, as CD1d-KO cells show higher signalling and cytokine secretion upon Toll-like receptor stimulation. Mechanistically, CD1d modulates lipid import by controlling the internalization of the lipid transporter CD36, while blocking lipid uptake through CD36 restores metabolic and immune responses in macrophages. Thus, our data reveal CD1d as a key regulator of an inflammatory-metabolic circuit in macrophages, independent of its function in the control of T cell responses.
    DOI:  https://doi.org/10.1038/s41467-022-34532-x
  2. Cell. 2022 Nov 08. pii: S0092-8674(22)01360-5. [Epub ahead of print]
    Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics.
    John Janetzko, Ryoji Kise, Benjamin Barsi-Rhyne, Dirk H Siepe, Franziska M Heydenreich, Kouki Kawakami, Matthieu Masureel, Shoji Maeda, K Christopher Garcia, Mark von Zastrow, Asuka Inoue, Brian K Kobilka.
      Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with β-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in β-arrestin recruitment and GPCR-β-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for β-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of β-arrestin and stabilize GPCR-β-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-β-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for β-arrestin recruitment, this provides a mechanism for β-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
    Keywords:  GPCR; arrestin; conformational dynamics; endocytosis; fluorescence spectroscopy; phosphoinositides; signaling
    DOI:  https://doi.org/10.1016/j.cell.2022.10.018
  3. Nat Commun. 2022 Nov 10. 13(1): 6805
    A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience.
    Changnan Wang, Bingying Wang, Taruna Pandey, Yong Long, Jianxiu Zhang, Fiona Oh, Jessica Sima, Ruyin Guo, Yun Liu, Chao Zhang, Shaeri Mukherjee, Michael Bassik, Weichun Lin, Huichao Deng, Goncalo Vale, Jeffrey G McDonald, Kang Shen, Dengke K Ma.
      Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through conserved homeostatic mechanisms. Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that mediates lipid trafficking to confer cold resilience. We identify lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7. LPD-3 bridges the endoplasmic reticulum (ER) and plasma membranes (PM), forming a structurally predicted hydrophobic tunnel for lipid trafficking. lpd-3 mutants exhibit abnormal phospholipid distribution, diminished FAT-7 abundance, organismic vulnerability to cold, and are rescued by Lecithin comprising unsaturated phospholipids. Deficient lpd-3 homologues in Zebrafish and mammalian cells cause defects similar to those observed in C. elegans. As mutations in BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, LPD-3 family proteins may serve as evolutionarily conserved highway bridges critical for ER-associated non-vesicular lipid trafficking and resilience to cold stress in eukaryotic cells.
    DOI:  https://doi.org/10.1038/s41467-022-34450-y
  4. Science. 2022 Nov 11. 378(6620): eabn5647
    Enhanced T cell effector activity by targeting the Mediator kinase module.
    Katherine A Freitas, Julia A Belk, Elena Sotillo, Patrick J Quinn, Maria C Ramello, Meena Malipatlolla, Bence Daniel, Katalin Sandor, Dorota Klysz, Jeremy Bjelajac, Peng Xu, Kylie A Burdsall, Victor Tieu, Vandon T Duong, Micah G Donovan, Evan W Weber, Howard Y Chang, Robbie G Majzner, Joaquin M Espinosa, Ansuman T Satpathy, Crystal L Mackall.
      T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.
    DOI:  https://doi.org/10.1126/science.abn5647
  5. Sci Adv. 2022 Nov 11. 8(45): eabo7956
    AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.
    Lisa Tilokani, Fiona M Russell, Stevie Hamilton, Daniel M Virga, Mayuko Segawa, Vincent Paupe, Anja V Gruszczyk, Margherita Protasoni, Luis-Carlos Tabara, Mark Johnson, Hanish Anand, Michael P Murphy, D Grahame Hardie, Franck Polleux, Julien Prudent.
      Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.
    DOI:  https://doi.org/10.1126/sciadv.abo7956
  6. Nat Commun. 2022 Nov 07. 13(1): 6719
    Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.
    Kazuki Kato, Sae Okazaki, Soumya Kannan, Han Altae-Tran, F Esra Demircioglu, Yukari Isayama, Junichiro Ishikawa, Masahiro Fukuda, Rhiannon K Macrae, Tomohiro Nishizawa, Kira S Makarova, Eugene V Koonin, Feng Zhang, Hiroshi Nishimasu.
      Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.
    DOI:  https://doi.org/10.1038/s41467-022-34378-3
  7. Nat Immunol. 2022 Nov 10.
    Mouse long-lived plasma cells develop from multiple origins.
      
    DOI:  https://doi.org/10.1038/s41590-022-01346-4
  8. Nat Commun. 2022 Nov 11. 13(1): 6829
    A nascent peptide code for translational control of mRNA stability in human cells.
    Phillip C Burke, Heungwon Park, Arvind Rasi Subramaniam.
      Stability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers mRNA destabilization. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro slowdown ribosomes and reduce mRNA levels in vivo. The resulting nascent peptide code underlies the mRNA effects of hundreds of endogenous peptide sequences in the human proteome. Our work suggests an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.
    DOI:  https://doi.org/10.1038/s41467-022-34664-0
  9. Sci Transl Med. 2022 Nov 09. 14(670): eabn7336
    BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.
    In-Young Jung, Vivek Narayan, Sierra McDonald, Andrew J Rech, Robert Bartoszek, Gwanui Hong, Megan M Davis, Jun Xu, Alina C Boesteanu, Julie S Barber-Rotenberg, Gabriela Plesa, Simon F Lacey, Julie K Jadlowsky, Donald L Siegel, Dana M Hammill, Park F Cho-Park, Shelley L Berger, Naomi B Haas, Joseph A Fraietta.
      Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]-expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.
    DOI:  https://doi.org/10.1126/scitranslmed.abn7336
  10. Nat Commun. 2022 Nov 07. 13(1): 6707
    SAMHD1 deacetylation by SIRT1 promotes DNA end resection by facilitating DNA binding at double-strand breaks.
    Priya Kapoor-Vazirani, Sandip K Rath, Xu Liu, Zhen Shu, Nicole E Bowen, Yitong Chen, Ramona Haji-Seyed-Javadi, Waaqo Daddacha, Elizabeth V Minten, Diana Danelia, Daniela Farchi, Duc M Duong, Nicholas T Seyfried, Xingming Deng, Eric A Ortlund, Baek Kim, David S Yu.
      Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. These findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.
    DOI:  https://doi.org/10.1038/s41467-022-34578-x
  11. J Cell Biol. 2023 Jan 02. pii: e202201137. [Epub ahead of print]222(1):
    REC drives recombination to repair double-strand breaks in animal mtDNA.
    Anna Klucnika, Peiqiang Mu, Jan Jezek, Matthew McCormack, Ying Di, Charles R Bradshaw, Hansong Ma.
      Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.
    DOI:  https://doi.org/10.1083/jcb.202201137
  12. Cell. 2022 Nov 02. pii: S0092-8674(22)01318-6. [Epub ahead of print]
    Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.
    Haopeng Xiao, Luiz H M Bozi, Yizhi Sun, Christopher L Riley, Vivek M Philip, Mandy Chen, Jiaming Li, Tian Zhang, Evanna L Mills, Margo P Emont, Wenfei Sun, Anita Reddy, Ryan Garrity, Jiani Long, Tobias Becher, Laura Potano Vitas, Dina Laznik-Bogoslavski, Martha Ordonez, Xinyue Liu, Xiong Chen, Yun Wang, Weihai Liu, Nhien Tran, Yitong Liu, Yang Zhang, Aaron M Cypess, Andrew P White, Yuchen He, Rebecca Deng, Heiko Schöder, Joao A Paulo, Mark P Jedrychowski, Alexander S Banks, Yu-Hua Tseng, Paul Cohen, Linus T Tsai, Evan D Rosen, Samuel Klein, Maria Chondronikola, Fiona E McAllister, Nick Van Bruggen, Edward L Huttlin, Bruce M Spiegelman, Gary A Churchill, Steven P Gygi, Edward T Chouchani.
      Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.
    DOI:  https://doi.org/10.1016/j.cell.2022.10.003
  13. Nat Commun. 2022 Nov 11. 13(1): 6832
    Error, reproducibility and uncertainty in experiments for electrochemical energy technologies.
    Graham Smith, Edmund J F Dickinson.
      
    DOI:  https://doi.org/10.1038/s41467-022-34594-x
  14. Nat Commun. 2022 Nov 05. 13(1): 6681
    Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR.
    Emily Sempou, Valentyna Kostiuk, Jie Zhu, M Cecilia Guerra, Leonid Tyan, Woong Hwang, Elena Camacho-Aguilar, Michael J Caplan, David Zenisek, Aryeh Warmflash, Nick D L Owens, Mustafa K Khokha.
      Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.
    DOI:  https://doi.org/10.1038/s41467-022-34363-w
  15. Nat Commun. 2022 Nov 09. 13(1): 6759
    Age-induced prostaglandin E2 impairs mitochondrial fitness and increases mortality to influenza infection.
    Judy Chen, Jane C Deng, Rachel L Zemans, Karim Bahmed, Beata Kosmider, Min Zhang, Marc Peters-Golden, Daniel R Goldstein.
      Aging impairs the immune responses to influenza A virus (IAV), resulting in increased mortality to IAV infections in older adults. However, the factors within the aged lung that compromise host defense to IAV remain unknown. Using a murine model and human samples, we identified prostaglandin E2 (PGE2), as such a factor. Senescent type II alveolar epithelial cells (AECs) are overproducers of PGE2 within the aged lung. PGE2 impairs the proliferation of alveolar macrophages (AMs), critical cells for defense against respiratory pathogens, via reduction of oxidative phosphorylation and mitophagy. Importantly, blockade of the PGE2 receptor EP2 in aged mice improves AM mitochondrial function, increases AM numbers and enhances survival to IAV infection. In conclusion, our study reveals a key mechanism that compromises host defense to IAV, and possibly other respiratory infections, with aging and suggests potential new therapeutic or preventative avenues to protect against viral respiratory disease in older adults.
    DOI:  https://doi.org/10.1038/s41467-022-34593-y
  16. Nat Commun. 2022 Nov 05. 13(1): 6696
    The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis.
    Pengfei Guo, Nam Hoang, Joseph Sanchez, Elaine H Zhang, Keshari Rajawasam, Kristiana Trinidad, Hong Sun, Hui Zhang.
      The assembly of mammalian SWI/SNF chromatin remodeling complexes is developmentally programed, and loss/mutations of SWI/SNF subunits alter the levels of other components through proteolysis, causing cancers. Here, we show that mouse Lsd1/Kdm1a deletion causes dramatic dissolution of SWI/SNF complexes and that LSD1 demethylates the methylated lysine residues in SMARCC1 and SMARCC2 to preserve the structural integrity of SWI/SNF complexes. The methylated SMARCC1/SMARCC2 are targeted for proteolysis by L3MBTL3 and the CRL4DCAF5 ubiquitin ligase complex. We identify SMARCC1 as the critical target of LSD1 and L3MBTL3 to maintain the pluripotency and self-renewal of embryonic stem cells. L3MBTL3 also regulates SMARCC1/SMARCC2 proteolysis induced by the loss of SWI/SNF subunits. Consistently, mouse L3mbtl3 deletion causes striking accumulation of SWI/SNF components, associated with embryonic lethality. Our studies reveal that the assembly/disassembly of SWI/SNF complexes is dynamically controlled by a lysine-methylation dependent proteolytic mechanism to maintain the integrity of the SWI/SNF complexes.
    DOI:  https://doi.org/10.1038/s41467-022-34348-9
  17. JCI Insight. 2022 Nov 08. pii: e154948. [Epub ahead of print]7(21):
    Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia.
    Matilde I Conte, M Cecilia Poli, Angelo Taglialatela, Giuseppe Leuzzi, Ivan K Chinn, Sandra A Salinas, Emma Rey-Jurado, Nixa Olivares, Liz Veramendi-Espinoza, Alberto Ciccia, James R Lupski, Juan Carlos Aldave Becerra, Emily M Mace, Jordan S Orange.
      Human NK cell deficiency (NKD) is a primary immunodeficiency in which the main clinically relevant immunological defect involves missing or dysfunctional NK cells. Here, we describe a familial NKD case in which 2 siblings had a substantive NKD and neutropenia in the absence of other immune system abnormalities. Exome sequencing identified compound heterozygous variants in Go-Ichi-Ni-San (GINS) complex subunit 4 (GINS4, also known as SLD5), an essential component of the human replicative helicase, which we demonstrate to have a damaging impact upon the expression and assembly of the GINS complex. Cells derived from affected individuals and a GINS4-knockdown cell line demonstrate delayed cell cycle progression, without signs of improper DNA synthesis or increased replication stress. By modeling partial GINS4 depletion in differentiating NK cells in vitro, we demonstrate the causal relationship between the genotype and the NK cell phenotype, as well as a cell-intrinsic defect in NK cell development. Thus, biallelic partial loss-of-function mutations in GINS4 define a potentially novel disease-causing gene underlying NKD with neutropenia. Together with the previously described mutations in other helicase genes causing NKD, and with the mild defects observed in other human cells, these variants underscore the importance of this pathway in NK cell biology.
    Keywords:  Cell Biology; Cell cycle; Immunology; Monogenic diseases; NK cells
    DOI:  https://doi.org/10.1172/jci.insight.154948
  18. Nat Commun. 2022 Nov 09. 13(1): 6786
    Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants.
    Songhai Tian, Xiaozhe Xiong, Ji Zeng, Siyu Wang, Benjamin Jean-Marie Tremblay, Peng Chen, Baohua Chen, Min Liu, Pengsheng Chen, Kuanwei Sheng, Daniel Zeve, Wanshu Qi, David T Breault, César Rodríguez, Ralf Gerhard, Rongsheng Jin, Andrew C Doxey, Min Dong.
      Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.
    DOI:  https://doi.org/10.1038/s41467-022-33964-9
  19. Nature. 2022 Nov 10.
    Non-viral precision T cell receptor replacement for personalized cell therapy.
    Susan P Foy, Kyle Jacoby, Daniela A Bota, Theresa Hunter, Zheng Pan, Eric Stawiski, Yan Ma, William Lu, Songming Peng, Clifford L Wang, Benjamin Yuen, Olivier Dalmas, Katharine Heeringa, Barbara Sennino, Andy Conroy, Michael T Bethune, Ines Mende, William White, Monica Kukreja, Swetha Gunturu, Emily Humphrey, Adeel Hussaini, Duo An, Adam J Litterman, Boi Bryant Quach, Alphonsus H C Ng, Yue Lu, Chad Smith, Katie M Campbell, Daniel Anaya, Lindsey Skrdlant, Eva Yi-Hsuan Huang, Ventura Mendoza, Jyoti Mathur, Luke Dengler, Bhamini Purandare, Robert Moot, Michael C Yi, Roel Funke, Alison Sibley, Todd Stallings-Schmitt, David Y Oh, Bartosz Chmielowski, Mehrdad Abedi, Yuan Yuan, Jeffrey A Sosman, Sylvia M Lee, Adam J Schoenfeld, David Baltimore, James R Heath, Alex Franzusoff, Antoni Ribas, Arati V Rao, Stefanie J Mandl.
      The T cell receptor (TCR) provides the fine specificity of T cells to recognize mutations in cancer cells <sup>1-3</sup>. We developed a clinical-grade approach based on CRISPR/Cas9 non-viral precision genome editing to simultaneously knock-out the two endogenous TCR genes, TCRα (TRAC) and TCRβ (TRBC), and insert in the TRAC locus the two chains of a neoantigen-specific TCR (neoTCR), isolated from the patient's own circulating T cells using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with refractory solid cancers received up to three distinct neoTCR-transgenic cell products, each expressing a patient-specific neoTCR, in a cell dose-escalation, first-in-human phase 1 clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome, and one grade 3 encephalitis. All had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease, and the other 11 had disease progression as best response on therapy. NeoTCR-transgenic T cells were detected in tumour biopsies post-infusion at frequencies higher than the native TCRs pre-infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs recognizing mutational neoantigens, the simultaneous knock-out of the endogenous TCR and knock-in of the neoTCRs using single-step, non-viral precision genome editing, the manufacturing of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene edited neoTCR T cell products, and the ability of the transgenic T cells to traffic to the patients' tumours.
    DOI:  https://doi.org/10.1038/s41586-022-05531-1
  20. Nat Commun. 2022 Nov 08. 13(1): 6737
    Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3.
    Luciano G Dolce, Aubree A Zimmer, Laura Tengo, Félix Weis, Mary Anne T Rubio, Juan D Alfonzo, Eva Kowalinski.
      The essential deamination of adenosine A34 to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.
    DOI:  https://doi.org/10.1038/s41467-022-34441-z
  21. Nat Commun. 2022 Nov 11. 13(1): 6846
    Influenza A virus reassortment in mammals gives rise to genetically distinct within-host subpopulations.
    Ketaki Ganti, Anish Bagga, Silvia Carnaccini, Lucas M Ferreri, Ginger Geiger, C Joaquin Caceres, Brittany Seibert, Yonghai Li, Liping Wang, Taeyong Kwon, Yuhao Li, Igor Morozov, Wenjun Ma, Juergen A Richt, Daniel R Perez, Katia Koelle, Anice C Lowen.
      Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission.
    DOI:  https://doi.org/10.1038/s41467-022-34611-z
  22. Nat Commun. 2022 Nov 05. 13(1): 6700
    Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist.
    Francois Moreau, Nicholas S Kirk, Fa Zhang, Vasily Gelfanov, Edward O List, Martina Chrudinová, Hari Venugopal, Michael C Lawrence, Veronica Jimenez, Fatima Bosch, John J Kopchick, Richard D DiMarchi, Emrah Altindis, C Ronald Kahn.
      Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism.
    DOI:  https://doi.org/10.1038/s41467-022-34391-6
  23. Nat Commun. 2022 Nov 10. 13(1): 6804
    Ferritin triggers neutrophil extracellular trap-mediated cytokine storm through Msr1 contributing to adult-onset Still's disease pathogenesis.
    Jinchao Jia, Mengyan Wang, Jianfen Meng, Yuning Ma, Yang Wang, Naijun Miao, Jialin Teng, Dehao Zhu, Hui Shi, Yue Sun, Honglei Liu, Xiaobing Cheng, Yutong Su, Junna Ye, Huihui Chi, Tingting Liu, Zhuochao Zhou, Liyan Wan, Xia Chen, Fan Wang, Hao Zhang, Jingjing Ben, Jing Wang, Chengde Yang, Qiongyi Hu.
      Hyperferritinemic syndrome, an overwhelming inflammatory condition, is characterized by high ferritin levels, systemic inflammation and multi-organ dysfunction, but the pathogenic role of ferritin remains largely unknown. Here we show in an animal model that ferritin administration leads to systemic and hepatic inflammation characterized by excessive neutrophil leukocyte infiltration and neutrophil extracellular trap (NET) formation in the liver tissue. Ferritin-induced NET formation depends on the expression of peptidylarginine deiminase 4 and neutrophil elastase and on reactive oxygen species production. Mechanistically, ferritin exposure increases both overall and cell surface expression of Msr1 on neutrophil leukocytes, and also acts as ligand to Msr1 to trigger the NET formation pathway. Depletion of neutrophil leukocytes or ablation of Msr1 protect mice from tissue damage and the hyperinflammatory response, which further confirms the role of Msr1 as ferritin receptor. The relevance of the animal model is underscored by the observation that enhanced NET formation, increased Msr1 expression and signalling on neutrophil leukocytes are also characteristic to adult-onset Still's disease (AOSD), a typical hyperferritinemic syndrome. Collectively, our findings demonstrate an essential role of ferritin in NET-mediated cytokine storm, and suggest that targeting NETs or Msr1 may benefit AOSD patients.
    DOI:  https://doi.org/10.1038/s41467-022-34560-7
  24. Nat Commun. 2022 Nov 08. 13(1): 6735
    Deep autoencoder for interpretable tissue-adaptive deconvolution and cell-type-specific gene analysis.
    Yanshuo Chen, Yixuan Wang, Yuelong Chen, Yuqi Cheng, Yumeng Wei, Yunxiang Li, Jiuming Wang, Yingying Wei, Ting-Fung Chan, Yu Li.
      Single-cell RNA-sequencing has become a powerful tool to study biologically significant characteristics at explicitly high resolution. However, its application on emerging data is currently limited by its intrinsic techniques. Here, we introduce Tissue-AdaPtive autoEncoder (TAPE), a deep learning method connecting bulk RNA-seq and single-cell RNA-seq to achieve precise deconvolution in a short time. By constructing an interpretable decoder and training under a unique scheme, TAPE can predict cell-type fractions and cell-type-specific gene expression tissue-adaptively. Compared with popular methods on several datasets, TAPE has a better overall performance and comparable accuracy at cell type level. Additionally, it is more robust among different cell types, faster, and sensitive to provide biologically meaningful predictions. Moreover, through the analysis of clinical data, TAPE shows its ability to predict cell-type-specific gene expression profiles with biological significance. We believe that TAPE will enable and accelerate the precise analysis of high-throughput clinical data in a wide range.
    DOI:  https://doi.org/10.1038/s41467-022-34550-9
  25. Nat Immunol. 2022 Nov 10.
    Notch gives early T cell progenitors time to grow up.
      
    DOI:  https://doi.org/10.1038/s41590-022-01323-x
  26. JCI Insight. 2022 Nov 08. pii: e162580. [Epub ahead of print]
    Impaired regulatory T cell-dendritic cell interactions contribute to autoimmunity in leukocyte adhesion deficiency type-1.
    Tanja Klaus, Alicia S Wilson, Elisabeth Vicari, Eva Hadaschik, Matthias Klein, Sara Salome Helbich, Nadine Kamenjarin, Katrin Hodapp, Jenny Schunke, Maximilian Haist, Florian Butsch, Hans C Probst, Alexander H Enk, Karsten Mahnke, Ari Waisman, Monika Bednarczyk, Matthias Bros, Tobias Bopp, Stephan Grabbe.
      Leukocyte Adhesion Deficiency Type-1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common β-chain of the ß2 integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. At the same time, LAD-1 patients are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ regulatory T cells (Treg) are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell - dendritic cell (DC) interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extra-lymphatic organs but resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs, and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.
    Keywords:  Autoimmune diseases; Autoimmunity; T cells
    DOI:  https://doi.org/10.1172/jci.insight.162580
  27. Nat Commun. 2022 Nov 05. 13(1): 6679
    Hi-TrAC reveals division of labor of transcription factors in organizing chromatin loops.
    Shuai Liu, Yaqiang Cao, Kairong Cui, Qingsong Tang, Keji Zhao.
      The three-dimensional genomic structure plays a critical role in gene expression, cellular differentiation, and pathological conditions. It is pivotal to elucidate fine-scale chromatin architectures, especially interactions of regulatory elements, to understand the temporospatial regulation of gene expression. In this study, we report Hi-TrAC as a proximity ligation-free, robust, and sensitive technique to profile genome-wide chromatin interactions at high-resolution among regulatory elements. Hi-TrAC detects chromatin looping among accessible regions at single nucleosome resolution. With almost half-million identified loops, we reveal a comprehensive interaction network of regulatory elements across the genome. After integrating chromatin binding profiles of transcription factors, we discover that cohesin complex and CTCF are responsible for organizing long-range chromatin loops, related to domain formation; whereas ZNF143 and HCFC1 are involved in structuring short-range chromatin loops between regulatory elements, which directly regulate gene expression. Thus, we introduce a methodology to identify a delicate and comprehensive network of cis-regulatory elements, revealing the complexity and a division of labor of transcription factors in organizing chromatin loops for genome organization and gene expression.
    DOI:  https://doi.org/10.1038/s41467-022-34276-8
  28. Cell. 2022 Nov 02. pii: S0092-8674(22)01320-4. [Epub ahead of print]
    Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.
    Jinfan Wang, Byung-Sik Shin, Carlos Alvarado, Joo-Ran Kim, Jonathan Bohlen, Thomas E Dever, Joseph D Puglisi.
      How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.
    Keywords:  5′ UTR; RNA helicases; RNA secondary structures; dynamics; eukaryotic ribosome; near-cognate start codons; scanning; single-molecule fluorescence spectroscopy; translation initiation; translation regulation
    DOI:  https://doi.org/10.1016/j.cell.2022.10.005
  29. Nat Commun. 2022 Nov 09. 13(1): 6781
    CK2-mediated phosphorylation of SUZ12 promotes PRC2 function by stabilizing enzyme active site.
    Lihu Gong, Xiuli Liu, Lianying Jiao, Xin Yang, Andrew Lemoff, Xin Liu.
      Polycomb repressive complex 2 (PRC2) plays a key role in maintaining cell identity during differentiation. Methyltransferase activity of PRC2 on histone H3 lysine 27 is regulated by diverse cellular mechanisms, including posttranslational modification. Here, we report a unique phosphorylation-dependent mechanism stimulating PRC2 enzymatic activity. Residue S583 of SUZ12 is phosphorylated by casein kinase 2 (CK2) in cells. A crystal structure captures phosphorylation in action: the flexible phosphorylation-dependent stimulation loop harboring S583 becomes engaged with the catalytic SET domain through a phosphoserine-centered interaction network, stabilizing the enzyme active site and in particular S-adenosyl-methionine (SAM)-binding pocket. CK2-mediated S583 phosphorylation promotes catalysis by enhancing PRC2 binding to SAM and nucleosomal substrates and facilitates reporter gene repression. Loss of S583 phosphorylation impedes PRC2 recruitment and H3K27me3 deposition in pluripotent mESCs and compromises the ability of PRC2 to maintain differentiated cell identity.
    DOI:  https://doi.org/10.1038/s41467-022-34431-1
  30. Nat Commun. 2022 Nov 05. 13(1): 6672
    Dusp6 deficiency attenuates neutrophil-mediated cardiac damage in the acute inflammatory phase of myocardial infarction.
    Xiaohai Zhou, Chenyang Zhang, Xueying Wu, Xinli Hu, Yan Zhang, Xuelian Wang, Lixia Zheng, Peng Gao, Jianyong Du, Wen Zheng, Haibao Shang, Keping Hu, Zhengfan Jiang, Yu Nie, Shengshou Hu, Rui-Ping Xiao, Xiaojun Zhu, Jing-Wei Xiong.
      Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPβ signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.
    DOI:  https://doi.org/10.1038/s41467-022-33631-z
  31. Nature. 2022 Nov 09.
    Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.
    Dominantly Inherited Alzheimer Network
      Macrophages are important players in the maintenance of tissue homeostasis1. Perivascular and leptomeningeal macrophages reside near the central nervous system (CNS) parenchyma2, and their role in CNS physiology has not been sufficiently well studied. Given their continuous interaction with the cerebrospinal fluid (CSF) and strategic positioning, we refer to these cells collectively as parenchymal border macrophages (PBMs). Here we demonstrate that PBMs regulate CSF flow dynamics. We identify a subpopulation of PBMs that express high levels of CD163 and LYVE1 (scavenger receptor proteins), closely associated with the brain arterial tree, and show that LYVE1+ PBMs regulate arterial motion that drives CSF flow. Pharmacological or genetic depletion of PBMs led to accumulation of extracellular matrix proteins, obstructing CSF access to perivascular spaces and impairing CNS perfusion and clearance. Ageing-associated alterations in PBMs and impairment of CSF dynamics were restored after intracisternal injection of macrophage colony-stimulating factor. Single-nucleus RNA sequencing data obtained from patients with Alzheimer's disease (AD) and from non-AD individuals point to changes in phagocytosis, endocytosis and interferon-γ signalling on PBMs, pathways that are corroborated in a mouse model of AD. Collectively, our results identify PBMs as new cellular regulators of CSF flow dynamics, which could be targeted pharmacologically to alleviate brain clearance deficits associated with ageing and AD.
    DOI:  https://doi.org/10.1038/s41586-022-05397-3
  32. Sci Transl Med. 2022 Nov 09. 14(670): eabm1463
    Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.
    Daniel B Goodman, Camillia S Azimi, Kendall Kearns, Alexis Talbot, Kiavash Garakani, Julie Garcia, Nisarg Patel, Byungjin Hwang, David Lee, Emily Park, Vivasvan S Vykunta, Brian R Shy, Chun Jimmie Ye, Justin Eyquem, Alexander Marson, Jeffrey A Bluestone, Kole T Roybal.
      Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.
    DOI:  https://doi.org/10.1126/scitranslmed.abm1463
  33. Nat Commun. 2022 Nov 11. 13(1): 6857
    PD-L1 negatively regulates antifungal immunity by inhibiting neutrophil release from bone marrow.
    Yao Yu, Rong-Rong Wang, Nai-Jun Miao, Jia-Jie Tang, Yun-Wei Zhang, Xiang-Ran Lu, Pei-Yi Yan, Jing Wang, Xin-Ming Jia.
      Programmed death ligand 1 (PD-L1) has been shown to be inducibly expressed on neutrophils to suppress host immunity during polymicrobial sepsis, virus and parasite infections. However, the role of PD-L1 on neutrophil-mediated antifungal immunity remains wholly unknown. Here, we show that the expression of PD-L1 on murine and human neutrophils was upregulated upon the engagement of C-type lectin receptor Dectin-1 with its ligand β-glucans, exposed on fungal pathogen Candida albicans yeast. Moreover, β-glucan stimulation induced PD-L1 translocation into nucleus to regulate the production of chemokines CXCL1 and CXCL2, which control neutrophil mobilization. Importantly, C. albicans infection-induced expression of PD-L1 leads to neutrophil accumulation in bone marrow, through mediating their autocrine secretion of CXCL1/2. Furthermore, neutrophil-specific deficiency of PD-L1 impaired CXCL1/2 secretion, which promoted neutrophil migration from bone marrow into the peripheral circulation, thereby conferring host resistance to C. albicans infection. Finally, either PD-L1 blockade or pharmacological inhibition of PD-L1 expression significantly increased neutrophil release from bone marrow to enhance host antifungal immunity. Our data together indicate that activation of Dectin-1/PD-L1 cascade by β-glucans inhibits neutrophil release from bone marrow reserve, contributing to the negative regulation of antifungal innate immunity, which functions as a potent immunotherapeutic target against life-threatening fungi infections.
    DOI:  https://doi.org/10.1038/s41467-022-34722-7
  34. Mol Cell. 2022 Nov 04. pii: S1097-2765(22)01022-X. [Epub ahead of print]
    Transcription factor antagonism regulates heterogeneity in embryonic stem cell states.
    Sofia Hu, Emily Metcalf, Dig Bijay Mahat, Lynette Chan, Noor Sohal, Meenakshi Chakraborty, Maxwell Hamilton, Arundeep Singh, Abhyudai Singh, Jacqueline A Lees, Phillip A Sharp, Salil Garg.
      Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.
    Keywords:  Klf4; Zfp281; cell-to-cell variation; embryonic stem cell state; enhancer RNA; gene expression variation; transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2022.10.022
  35. Nat Commun. 2022 Nov 09. 13(1): 6779
    Capture at the ER-mitochondrial contacts licenses IP3 receptors to stimulate local Ca2+ transfer and oxidative metabolism.
    Máté Katona, Ádám Bartók, Zuzana Nichtova, György Csordás, Elena Berezhnaya, David Weaver, Arijita Ghosh, Péter Várnai, David I Yule, György Hajnóczky.
      Endoplasmic reticulum-mitochondria contacts (ERMCs) are restructured in response to changes in cell state. While this restructuring has been implicated as a cause or consequence of pathology in numerous systems, the underlying molecular dynamics are poorly understood. Here, we show means to visualize the capture of motile IP3 receptors (IP3Rs) at ERMCs and document the immediate consequences for calcium signaling and metabolism. IP3Rs are of particular interest because their presence provides a scaffold for ERMCs that mediate local calcium signaling, and their function outside of ERMCs depends on their motility. Unexpectedly, in a cell model with little ERMC Ca2+ coupling, IP3Rs captured at mitochondria promptly mediate Ca2+ transfer, stimulating mitochondrial oxidative metabolism. The Ca2+ transfer does not require linkage with a pore-forming protein in the outer mitochondrial membrane. Thus, motile IP3Rs can traffic in and out of ERMCs, and, when 'parked', mediate calcium signal propagation to the mitochondria, creating a dynamic arrangement that supports local communication.
    DOI:  https://doi.org/10.1038/s41467-022-34365-8
  36. Nat Commun. 2022 Nov 05. 13(1): 6692
    Structural basis for the activation of the lipid scramblase TMEM16F.
    Melanie Arndt, Carolina Alvadia, Monique S Straub, Vanessa Clerico Mosina, Cristina Paulino, Raimund Dutzler.
      TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca2+. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.
    DOI:  https://doi.org/10.1038/s41467-022-34497-x
  37. Nat Commun. 2022 Nov 11. 13(1): 6871
    Structural basis of human SNAPc recognizing proximal sequence element of snRNA promoter.
    Jianfeng Sun, Xue Li, Xuben Hou, Sujian Cao, Wenjin Cao, Ye Zhang, Jinyang Song, Manfu Wang, Hao Wang, Xiaodong Yan, Zengpeng Li, Robert G Roeder, Wei Wang.
      In eukaryotes, small nuclear RNAs (snRNAs) function in many fundamental cellular events such as precursor messenger RNA splicing, gene expression regulation, and ribosomal RNA processing. The snRNA activating protein complex (SNAPc) exclusively recognizes the proximal sequence element (PSE) at snRNA promoters and recruits RNA polymerase II or III to initiate transcription. In view that homozygous gene-knockout of SNAPc core subunits causes mouse embryonic lethality, functions of SNAPc are almost housekeeping. But so far, the structural insight into how SNAPc assembles and regulates snRNA transcription initiation remains unclear. Here we present the cryo-electron microscopy structure of the essential part of human SNAPc in complex with human U6-1 PSE at an overall resolution of 3.49 Å. This structure reveals the three-dimensional features of three conserved subunits (N-terminal domain of SNAP190, SNAP50, and SNAP43) and explains how they are assembled into a stable mini-SNAPc in PSE-binding state with a "wrap-around" mode. We identify three important motifs of SNAP50 that are involved in both major groove and minor groove recognition of PSE, in coordination with the Myb domain of SNAP190. Our findings further elaborate human PSE sequence conservation and compatibility for SNAPc recognition, providing a clear framework of snRNA transcription initiation, especially the U6 system.
    DOI:  https://doi.org/10.1038/s41467-022-34639-1
  38. Nat Commun. 2022 Nov 05. 13(1): 6694
    Analysis of clinically relevant variants from ancestrally diverse Asian genomes.
    SG10K_Health Consortium
      Asian populations are under-represented in human genomics research. Here, we characterize clinically significant genetic variation in 9051 genomes representing East Asian, South Asian, and severely under-represented Austronesian-speaking Southeast Asian ancestries. We observe disparate genetic risk burden attributable to ancestry-specific recurrent variants and identify individuals with variants specific to ancestries discordant to their self-reported ethnicity, mostly due to cryptic admixture. About 27% of severe recessive disorder genes with appreciable carrier frequencies in Asians are missed by carrier screening panels, and we estimate 0.5% Asian couples at-risk of having an affected child. Prevalence of medically-actionable variant carriers is 3.4% and a further 1.6% harbour variants with potential for pathogenic classification upon additional clinical/experimental evidence. We profile 23 pharmacogenes with high-confidence gene-drug associations and find 22.4% of Asians at-risk of Centers for Disease Control and Prevention Tier 1 genetic conditions concurrently harbour pharmacogenetic variants with actionable phenotypes, highlighting the benefits of pre-emptive pharmacogenomics. Our findings illuminate the diversity in genetic disease epidemiology and opportunities for precision medicine for a large, diverse Asian population.
    DOI:  https://doi.org/10.1038/s41467-022-34116-9
  39. Nat Commun. 2022 Nov 11. 13(1): 6859
    Genetic regulation of serum IgA levels and susceptibility to common immune, infectious, kidney, and cardio-metabolic traits.
    Lili Liu, Atlas Khan, Elena Sanchez-Rodriguez, Francesca Zanoni, Yifu Li, Nicholas Steers, Olivia Balderes, Junying Zhang, Priya Krithivasan, Robert A LeDesma, Clara Fischman, Scott J Hebbring, John B Harley, Halima Moncrieffe, Leah C Kottyan, Bahram Namjou-Khales, Theresa L Walunas, Rachel Knevel, Soumya Raychaudhuri, Elizabeth W Karlson, Joshua C Denny, Ian B Stanaway, David Crosslin, Thomas Rauen, Jürgen Floege, Frank Eitner, Zina Moldoveanu, Colin Reily, Barbora Knoppova, Stacy Hall, Justin T Sheff, Bruce A Julian, Robert J Wyatt, Hitoshi Suzuki, Jingyuan Xie, Nan Chen, Xujie Zhou, Hong Zhang, Lennart Hammarström, Alexander Viktorin, Patrik K E Magnusson, Ning Shang, George Hripcsak, Chunhua Weng, Tatjana Rundek, Mitchell S V Elkind, Elizabeth C Oelsner, R Graham Barr, Iuliana Ionita-Laza, Jan Novak, Ali G Gharavi, Krzysztof Kiryluk.
      Immunoglobulin A (IgA) mediates mucosal responses to food antigens and the intestinal microbiome and is involved in susceptibility to mucosal pathogens, celiac disease, inflammatory bowel disease, and IgA nephropathy. We performed a genome-wide association study of serum IgA levels in 41,263 individuals of diverse ancestries and identified 20 genome-wide significant loci, including 9 known and 11 novel loci. Co-localization analyses with expression QTLs prioritized candidate genes for 14 of 20 significant loci. Most loci encoded genes that produced immune defects and IgA abnormalities when genetically manipulated in mice. We also observed positive genetic correlations of serum IgA levels with IgA nephropathy, type 2 diabetes, and body mass index, and negative correlations with celiac disease, inflammatory bowel disease, and several infections. Mendelian randomization supported elevated serum IgA as a causal factor in IgA nephropathy. African ancestry was consistently associated with higher serum IgA levels and greater frequency of IgA-increasing alleles compared to other ancestries. Our findings provide novel insights into the genetic regulation of IgA levels and its potential role in human disease.
    DOI:  https://doi.org/10.1038/s41467-022-34456-6
  40. Nat Metab. 2022 Nov 07.
    ADRA1A-Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP.
    Janane F Rahbani, Charlotte Scholtes, Damien M Lagarde, Mohammed F Hussain, Anna Roesler, Christien B Dykstra, Jakub Bunk, Bozena Samborska, Shannon L O'Brien, Emma Tripp, Alain Pacis, Anthony R Angueira, Olivia S Johansen, Jessica Cinkornpumin, Ishtiaque Hossain, Matthew D Lynes, Yang Zhang, Andrew P White, William A Pastor, Maria Chondronikola, Labros Sidossis, Samuel Klein, Anastasia Kralli, Aaron M Cypess, Steen B Pedersen, Niels Jessen, Yu-Hua Tseng, Zachary Gerhart-Hines, Patrick Seale, Davide Calebiro, Vincent Giguère, Lawrence Kazak.
      Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of β-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and β3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A-Gαq-futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.
    DOI:  https://doi.org/10.1038/s42255-022-00667-w
  41. Nat Metab. 2022 Nov 07.
    No sugar, just protein please - says the fly.
    Afroditi Petsakou, Norbert Perrimon.
      
    DOI:  https://doi.org/10.1038/s42255-022-00665-y
  42. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01479-6. [Epub ahead of print]41(6): 111610
    Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.
    Shintaro Ide, Kana Ide, Koki Abe, Yoshihiko Kobayashi, Hiroki Kitai, Jennifer McKey, Sarah A Strausser, Lori L O'Brien, Aleksandra Tata, Purushothama Rao Tata, Tomokazu Souma.
      In both humans and mice, repair of acute kidney injury is worse in males than in females. Here, we provide evidence that this sexual dimorphism results from sex differences in ferroptosis, an iron-dependent, lipid-peroxidation-driven regulated cell death. Using genetic and single-cell transcriptomic approaches in mice, we report that female sex confers striking protection against ferroptosis, which was experimentally induced in proximal tubular (PT) cells by deleting glutathione peroxidase 4 (Gpx4). Single-cell transcriptomic analyses further identify the NFE2-related factor 2 (NRF2) antioxidant protective pathway as a female resilience mechanism against ferroptosis. Genetic inhibition and pharmacological activation studies show that NRF2 controls PT cell fate and plasticity by regulating ferroptosis. Importantly, pharmacological NRF2 activation protects male PT cells from ferroptosis and improves cellular plasticity as in females. Our data highlight NRF2 as a potential therapeutic target to prevent failed renal repair after acute kidney injury in both sexes by modulating cellular plasticity.
    Keywords:  CP: Molecular biology; GPX4; NRF2; acute kidney injury; cell fate; cellular plasticity; ferroptosis; glutathione Peroxidase 4; kidney injury and repair; sexual dimorphism; single-cell transcriptomics
    DOI:  https://doi.org/10.1016/j.celrep.2022.111610
  43. Sci Immunol. 2022 Nov 18. 7(77): eade0182
    Single-cell immune profiling reveals thymus-seeding populations, T cell commitment, and multilineage development in the human thymus.
    Martijn Cordes, Kirsten Canté-Barrett, Erik B van den Akker, Federico A Moretti, Szymon M Kiełbasa, Sandra A Vloemans, Laura Garcia-Perez, Cristina Teodosio, Jacques J M van Dongen, Karin Pike-Overzet, Marcel J T Reinders, Frank J T Staal.
      T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.
    DOI:  https://doi.org/10.1126/sciimmunol.ade0182
  44. Nat Genet. 2022 Nov 11.
    Skip to the end: metastasis before tumorigenesis.
    Ana Rita Nobre, Adrienne Boire.
      
    DOI:  https://doi.org/10.1038/s41588-022-01228-3
  45. Nat Cell Biol. 2022 Nov;24(11): 1571-1573
    Harnessing introns to monitor gene expression.
    Salima Benbarche, Omar Abdel-Wahab.
      
    DOI:  https://doi.org/10.1038/s41556-022-01003-w
  46. Nat Commun. 2022 Nov 08. 13(1): 6740
    Single cell sequencing reveals that CD39 inhibition mediates changes to the tumor microenvironment.
    Lilong Liu, Yaxin Hou, Changqi Deng, Zhen Tao, Zhaohui Chen, Junyi Hu, Ke Chen.
      Single-cell sequencing technologies have noteworthily improved our understanding of the genetic map and molecular characteristics of bladder cancer (BC). Here we identify CD39 as a potential therapeutic target for BC via single-cell transcriptome analysis. In a subcutaneous tumor model and orthotopic bladder cancer model, inhibition of CD39 (CD39i) by sodium polyoxotungstate is able to limit the growth of BC and improve the overall survival of tumor-bearing mice. Via single cell RNA sequencing, we find that CD39i increase the intratumor NK cells, conventional type 1 dendritic cells (cDC1) and CD8 + T cells and decrease the Treg abundance. The antitumor effect and reprogramming of the tumor microenvironment are blockaded in both the NK cells depletion model and the cDC1-deficient Batf3-/- model. In addition, a significant synergistic effect is observed between CD39i and cisplatin, but the CD39i + anti-PD-L1 (or anti-PD1) strategy does not show any synergistic effects in the BC model. Our results confirm that CD39 is a potential target for the immune therapy of BC.
    DOI:  https://doi.org/10.1038/s41467-022-34495-z
  47. Cell. 2022 Nov 02. pii: S0092-8674(22)01359-9. [Epub ahead of print]
    The phenotypic landscape of essential human genes.
    Luke Funk, Kuan-Chung Su, Jimmy Ly, David Feldman, Avtar Singh, Brittania Moodie, Paul C Blainey, Iain M Cheeseman.
      Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
    Keywords:  CRISPR-Cas9; cell division; essential genes; functional genomics; high-content screening; in situ sequencing; microscopy; mitosis; morphology; optical pooled screening
    DOI:  https://doi.org/10.1016/j.cell.2022.10.017
  48. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01458-9. [Epub ahead of print]41(6): 111593
    Tuft-cell-intrinsic and -extrinsic mediators of norovirus tropism regulate viral immunity.
    Madison S Strine, Mia Madel Alfajaro, Vincent R Graziano, Jaewon Song, Leon L Hsieh, Ryan Hill, Jun Guo, Kelli L VanDussen, Robert C Orchard, Megan T Baldridge, Sanghyun Lee, Craig B Wilen.
      Murine norovirus (MNoV) is a model for human norovirus and for interrogating mechanisms of viral tropism and persistence. We previously demonstrated that the persistent strain MNoVCR6 infects tuft cells, which are dispensable for the non-persistent strain MNoVCW3. We now show that diverse MNoV strains require tuft cells for chronic enteric infection. We also demonstrate that interferon-λ (IFN-λ) acts directly on tuft cells to cure chronic MNoVCR6 infection and that type I and III IFNs signal together via STAT1 in tuft cells to restrict MNoVCW3 tropism. We then develop an enteroid model and find that MNoVCR6 and MNoVCW3 similarly infect tuft cells with equal IFN susceptibility, suggesting that IFN derived from non-epithelial cells signals on tuft cells in trans to restrict MNoVCW3 tropism. Thus, tuft cell tropism enables MNoV persistence and is determined by tuft cell-intrinsic factors (viral receptor expression) and -extrinsic factors (immunomodulatory signaling by non-epithelial cells).
    Keywords:  CP: Immunology; CP: Microbiology; immune evasion; interferon; mucosal immunity; norovirus; tuft cells; viral persistence; viral tropism
    DOI:  https://doi.org/10.1016/j.celrep.2022.111593
  49. Nat Commun. 2022 Nov 08. 13(1): 6755
    The first mitotic division of human embryos is highly error prone.
    Cerys E Currie, Emma Ford, Lucy Benham Whyte, Deborah M Taylor, Bettina P Mihalas, Muriel Erent, Adele L Marston, Geraldine M Hartshorne, Andrew D McAinsh.
      Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.
    DOI:  https://doi.org/10.1038/s41467-022-34294-6
  50. JCI Insight. 2022 Nov 08. pii: e161698. [Epub ahead of print]7(21):
    In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.
    Kim Anthony-Gonda, Alex Ray, Hang Su, Yuge Wang, Ying Xiong, Danica Lee, Ariele Block, Vanessa Chilunda, Jessica Weiselberg, Lily Zemelko, Yen Y Wang, Sarah Kleinsorge-Block, Jane S Reese, Marcos de Lima, Christina Ochsenbauer, John C Kappes, Dimiter S Dimitrov, Rimas Orentas, Steven G Deeks, Rachel L Rutishauser, Joan W Berman, Harris Goldstein, Boro Dropulić.
      HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).
    Keywords:  AIDS/HIV; Immunotherapy; Therapeutics
    DOI:  https://doi.org/10.1172/jci.insight.161698
  51. Nat Commun. 2022 Nov 05. 13(1): 6683
    Amino acid variability, tradeoffs and optimality in human diet.
    Ziwei Dai, Weiyan Zheng, Jason W Locasale.
      Studies at the molecular level demonstrate that dietary amino acid intake produces substantial effects on health and disease by modulating metabolism. However, how these effects may manifest in human food consumption and dietary patterns is unknown. Here, we develop a series of algorithms to map, characterize and model the landscape of amino acid content in human food, dietary patterns, and individual consumption including relations to health status, covering over 2,000 foods, ten dietary patterns, and over 30,000 dietary profiles. We find that the type of amino acids contained in foods and human consumption is highly dynamic with variability far exceeding that of fat and carbohydrate. Some amino acids positively associate with conditions such as obesity while others contained in the same food negatively link to disease. Using linear programming and machine learning, we show that these health trade-offs can be accounted for to satisfy biochemical constraints in food and human eating patterns to construct a Pareto front in dietary practice, a means of achieving optimality in the face of trade-offs that are commonly considered in economic and evolutionary theories. Thus this study may enable the design of human protein quality intake guidelines based on a quantitative framework.
    DOI:  https://doi.org/10.1038/s41467-022-34486-0
  52. Nat Commun. 2022 Nov 08. 13(1): 6739
    CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia.
    Awatef Allouch, Laurent Voisin, Yanyan Zhang, Syed Qasim Raza, Yann Lecluse, Julien Calvo, Dorothée Selimoglu-Buet, Stéphane de Botton, Fawzia Louache, Françoise Pflumio, Eric Solary, Jean-Luc Perfettini.
      Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy.
    DOI:  https://doi.org/10.1038/s41467-022-34548-3
  53. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01482-6. [Epub ahead of print]41(6): 111613
    The memory B cell response to influenza vaccination is impaired in older persons.
    Alice R Burton, Stephane M Guillaume, William S Foster, Adam K Wheatley, Danika L Hill, Edward J Carr, Michelle A Linterman.
      Influenza infection imparts an age-related increase in mortality and morbidity. The most effective countermeasure is vaccination; however, vaccines offer modest protection in older adults. To investigate how aging impacts the memory B cell response, we track hemagglutinin-specific B cells by indexed flow sorting and single-cell RNA sequencing (scRNA-seq) in 20 healthy adults that were administered the trivalent influenza vaccine. We demonstrate age-related skewing in the memory B cell compartment 6 weeks after vaccination, with younger adults developing hemagglutinin-specific memory B cells with an FcRL5+ "atypical" phenotype, showing evidence of somatic hypermutation and positive selection, which happened to a lesser extent in older persons. We use publicly available scRNA-seq from paired human lymph node and blood samples to corroborate that FcRL5+ atypical memory B cells can derive from germinal center (GC) precursors. Together, this study shows that the aged human GC reaction and memory B cell response following vaccination is defective.
    Keywords:  B cell; CP: Immunology; aging; antibody selection; influenza; memory; vaccination
    DOI:  https://doi.org/10.1016/j.celrep.2022.111613
  54. Dev Cell. 2022 Nov 07. pii: S1534-5807(22)00721-3. [Epub ahead of print]57(21): 2450-2468.e7
    Platr4 is an early embryonic lncRNA that exerts its function downstream on cardiogenic mesodermal lineage commitment.
    Rasmani Hazra, Lily Brine, Libia Garcia, Brian Benz, Napon Chirathivat, Michael M Shen, John Erby Wilkinson, Scott K Lyons, David L Spector.
      The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs), many of which are developmentally regulated and differentially expressed across tissues, suggesting their potential roles in cellular differentiation. Despite this expression pattern, little is known about how lncRNAs influence lineage commitment at the molecular level. Here, we demonstrate that perturbation of an embryonic stem cell/early embryonic lncRNA, pluripotency-associated transcript 4 (Platr4), directly influences the specification of cardiac-mesoderm-lineage differentiation. We show that Platr4 acts as a molecular scaffold or chaperone interacting with the Hippo-signaling pathway molecules Yap and Tead4 to regulate the expression of a downstream target gene, Ctgf, which is crucial to the cardiac-lineage program. Importantly, Platr4 knockout mice exhibit myocardial atrophy and valve mucinous degeneration, which are both associated with reduced cardiac output and sudden heart failure. Together, our findings provide evidence that Platr4 is required in cardiac-lineage specification and adult heart function in mice.
    Keywords:  Ctgf; Hippo-signaling pathway; Platr4; Tead4; cardiac function; lncRNA; mesendoderm lineage
    DOI:  https://doi.org/10.1016/j.devcel.2022.10.002
  55. Nat Cell Biol. 2022 Nov 10.
    Splitting up differentiation and cell cycle exit.
    Albert Herms, Philip H Jones.
      
    DOI:  https://doi.org/10.1038/s41556-022-01022-7
  56. Nat Commun. 2022 Nov 05. 13(1): 6688
    RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4.
    Hiroshi Okuda, Ryo Miyamoto, Satoshi Takahashi, Takeshi Kawamura, Juri Ichikawa, Ibuki Harada, Tomohiko Tamura, Akihiko Yokoyama.
      Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins.
    DOI:  https://doi.org/10.1038/s41467-022-34558-1
  57. Science. 2022 Nov 11. 378(6620): 627-634
    Structures of a mobile intron retroelement poised to attack its structured DNA target.
    Kevin Chung, Ling Xu, Pengxin Chai, Junhui Peng, Swapnil C Devarkar, Anna Marie Pyle.
      Group II introns are ribozymes that catalyze their self-excision and function as retroelements that invade DNA. As retrotransposons, group II introns form ribonucleoprotein (RNP) complexes that roam the genome, integrating by reversal of forward splicing. Here we show that retrotransposition is achieved by a tertiary complex between a structurally elaborate ribozyme, its protein mobility factor, and a structured DNA substrate. We solved cryo-electron microscopy structures of an intact group IIC intron-maturase retroelement that was poised for integration into a DNA stem-loop motif. By visualizing the RNP before and after DNA targeting, we show that it is primed for attack and fits perfectly with its DNA target. This study reveals design principles of a prototypical retroelement and reinforces the hypothesis that group II introns are ancient elements of genetic diversification.
    DOI:  https://doi.org/10.1126/science.abq2844
  58. Nat Commun. 2022 Nov 07. 13(1): 6556
    Robust replication initiation from coupled homeostatic mechanisms.
    Mareike Berger, Pieter Rein Ten Wolde.
      The bacterium Escherichia coli initiates replication once per cell cycle at a precise volume per origin and adds an on average constant volume between successive initiation events, independent of the initiation size. Yet, a molecular model that can explain these observations has been lacking. Experiments indicate that E. coli controls replication initiation via titration and activation of the initiator protein DnaA. Here, we study by mathematical modelling how these two mechanisms interact to generate robust replication-initiation cycles. We first show that a mechanism solely based on titration generates stable replication cycles at low growth rates, but inevitably causes premature reinitiation events at higher growth rates. In this regime, the DnaA activation switch becomes essential for stable replication initiation. Conversely, while the activation switch alone yields robust rhythms at high growth rates, titration can strongly enhance the stability of the switch at low growth rates. Our analysis thus predicts that both mechanisms together drive robust replication cycles at all growth rates. In addition, it reveals how an origin-density sensor yields adder correlations.
    DOI:  https://doi.org/10.1038/s41467-022-33886-6
  59. Nat Commun. 2022 Nov 07. 13(1): 6714
    Structural basis of organic cation transporter-3 inhibition.
    Basavraj Khanppnavar, Julian Maier, Freja Herborg, Ralph Gradisch, Erika Lazzarin, Dino Luethi, Jae-Won Yang, Chao Qi, Marion Holy, Kathrin Jäntsch, Oliver Kudlacek, Klaus Schicker, Thomas Werge, Ulrik Gether, Thomas Stockner, Volodymyr M Korkhov, Harald H Sitte.
      Organic cation transporters (OCTs) facilitate the translocation of catecholamines, drugs and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 Å resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.
    DOI:  https://doi.org/10.1038/s41467-022-34284-8
  60. Nat Commun. 2022 Nov 09. 13(1): 6757
    Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity.
    Chun Jing Wang, Lina Petersone, Natalie M Edner, Frank Heuts, Vitalijs Ovcinnikovs, Elisavet Ntavli, Alexandros Kogimtzis, Astrid Fabri, Yassin Elfaki, Luke P Houghton, Ralf J Hosse, David A Schubert, Andreas P Frei, Ellen M Ross, Lucy S K Walker.
      Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first.
    DOI:  https://doi.org/10.1038/s41467-022-34477-1
  61. Nat Commun. 2022 Nov 11. 13(1): 6827
    dcHiC detects differential compartments across multiple Hi-C datasets.
    Abhijit Chakraborty, Jeffrey G Wang, Ferhat Ay.
      The compartmental organization of mammalian genomes and its changes play important roles in distinct biological processes. Here, we introduce dcHiC, which utilizes a multivariate distance measure to identify significant changes in compartmentalization among multiple contact maps. Evaluating dcHiC on four collections of bulk and single-cell contact maps from in vitro mouse neural differentiation (n = 3), mouse hematopoiesis (n = 10), human LCLs (n = 20) and post-natal mouse brain development (n = 3 stages), we show its effectiveness and sensitivity in detecting biologically relevant changes, including those orthogonally validated. dcHiC reported regions with dynamically regulated genes associated with cell identity, along with correlated changes in chromatin states, subcompartments, replication timing and lamin association. With its efficient implementation, dcHiC enables high-resolution compartment analysis as well as standalone browser visualization, differential interaction identification and time-series clustering. dcHiC is an essential addition to the Hi-C analysis toolbox for the ever-growing number of bulk and single-cell contact maps. Available at: https://github.com/ay-lab/dcHiC .
    DOI:  https://doi.org/10.1038/s41467-022-34626-6
  62. Nat Commun. 2022 Nov 10. 13(1): 6796
    Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells.
    Wei Chen, Pingting Liu, Dong Liu, Haoliang Huang, Xue Feng, Fang Fang, Liang Li, Jian Wu, Liang Liu, David E Solow-Cordero, Yang Hu.
      When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.
    DOI:  https://doi.org/10.1038/s41467-022-34682-y
  63. Methods Mol Biol. 2023 ;2590 149-159
    Determining Complete Chromosomal Haplotypes by mLinker.
    Sumit Sinha, Cheng-Zhong Zhang.
      Haplotype ("haploid genotype") phase is the combination of genotypes at sites of genetic variation along a chromosome [1]. We previously demonstrated that the complete chromosomal haplotype of diploid human genomes can be determined using molecular linkage from Hi-C sequencing and linked-reads sequencing [2]. In this chapter, we present a step-by-step guide to perform this analysis using mLinker, a software package for haplotype inference.
    Keywords:  Haplotype phasing; Hi-C sequencing; Long-read sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-2819-5_10
  64. Nat Commun. 2022 Nov 08. 13(1): 6659
    Cell division drives DNA methylation loss in late-replicating domains in primary human cells.
    Jamie L Endicott, Paula A Nolte, Hui Shen, Peter W Laird.
      DNA methylation undergoes dramatic age-related changes, first described more than four decades ago. Loss of DNA methylation within partially methylated domains (PMDs), late-replicating regions of the genome attached to the nuclear lamina, advances with age in normal tissues, and is further exacerbated in cancer. We present here experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Within PMDs, loss of DNA methylation at low-density CpGs in A:T-rich immediate context (PMD solo-WCGWs) tracks cumulative population doublings in primary cell culture. Cell cycle deceleration results in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halts methylation loss. Loss of methylation continues unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increases the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).
    DOI:  https://doi.org/10.1038/s41467-022-34268-8
  65. J Exp Med. 2023 Jan 02. pii: e20220342. [Epub ahead of print]220(1):
    Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling.
    Virginia Andreani, Senthilkumar Ramamoorthy, Reinhard Fässler, Rudolf Grosschedl.
      Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The adhesion of MZ B cells to the marginal sinus of the spleen is governed by integrins. Here, we address the question of whether β1-integrin has additional functions by analyzing Itgb1fl/flCD21Cre mice in which the β1-integrin gene is deleted in mature B cells. We find that integrin β1-deficient mice have a defect in the differentiation of MZ B cells and plasma cells. We show that integrin β1-deficient transitional B cells, representing the precursors of MZ B cells, have enhanced B cell receptor (BCR) signaling, altered PI3K and Ras/ERK pathways, and an enhanced interaction of integrin-linked kinase (ILK) with the adaptor protein Grb2. Moreover, the MZ B cell defect of integrin β1-deficient mice could, at least in part, be restored by a pharmacological inhibition of the PI3K pathway. Thus, β1-integrin has an unexpected function in the differentiation and function of MZ B cells.
    DOI:  https://doi.org/10.1084/jem.20220342
  66. Sci Rep. 2022 Nov 05. 12(1): 18753
    Association of apolipoprotein M and sphingosine-1-phosphate with brown adipose tissue after cold exposure in humans.
    Anna Borup, Ida Donkin, Mariëtte R Boon, Martin Frydland, Borja Martinez-Tellez, Annika Loft, Sune H Keller, Andreas Kjaer, Jesper Kjaergaard, Christian Hassager, Romain Barrès, Patrick C N Rensen, Christina Christoffersen.
      The HDL-associated apolipoprotein M (apoM) and its ligand sphingosine-1-phosphate (S1P) may control energy metabolism. ApoM deficiency in mice is associated with increased vascular permeability, brown adipose tissue (BAT) mass and activity, and protection against obesity. In the current study, we explored the connection between plasma apoM/S1P levels and parameters of BAT as measured via 18F-FDG PET/CT after cold exposure in humans. Fixed (n = 15) vs personalized (n = 20) short-term cooling protocols decreased and increased apoM (- 8.4%, P = 0.032 vs 15.7%, P < 0.0005) and S1P (- 41.0%, P < 0.0005 vs 19.1%, P < 0.005) plasma levels, respectively. Long-term cooling (n = 44) did not affect plasma apoM or S1P levels. Plasma apoM and S1P did not correlate significantly to BAT volume and activity in the individual studies. However, short-term studies combined, showed that increased changes in plasma apoM correlated with BAT metabolic activity (β: 0.44, 95% CI [0.06-0.81], P = 0.024) after adjusting for study design but not BAT volume (β: 0.39, 95% CI [- 0.01-0.78], P = 0.054). In conclusion, plasma apoM and S1P levels are altered in response to cold exposure and may be linked to changes in BAT metabolic activity but not BAT volume in humans. This contrasts partly with observations in animals and highlights the need for further studies to understand the biological role of apoM/S1P complex in human adipose tissue and lipid metabolism.
    DOI:  https://doi.org/10.1038/s41598-022-21938-2
  67. J Exp Med. 2023 Feb 06. pii: e20211422. [Epub ahead of print]220(2):
    Activating NO-sGC crosstalk in the mouse vascular niche promotes vascular integrity and mitigates acute lung injury.
    Hao He, Wu Yang, Nan Su, Chuankai Zhang, Jianing Dai, Feng Han, Mahak Singhal, Wenjuan Bai, Xiaolan Zhu, Jing Zhu, Zhen Liu, Wencheng Xia, Xiaoting Liu, Chonghe Zhang, Kai Jiang, Wenhui Huang, Dan Chen, Zhaoyin Wang, Xueyang He, Frank Kirchhoff, Zhenyu Li, Cong Liu, Jingning Huan, Xiaohong Wang, Wu Wei, Jing Wang, Hellmut G Augustin, Junhao Hu.
      Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC-pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC-pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO-sGC) is impaired in ALI. Indeed, stimulating the NO-sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC-pericyte interactions. Collectively, our data demonstrate that impaired NO-sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.
    DOI:  https://doi.org/10.1084/jem.20211422
  68. Cell. 2022 Nov 10. pii: S0092-8674(22)01329-0. [Epub ahead of print]185(23): 4253-4255
    Omics goes spatial epigenomics.
    Florian Schueder, Joerg Bewersdorf.
      Spatial omics techniques generate spatially resolved, comprehensive data about molecules that define the identity and function of cells in tissues. Epigenetic multiplexing approaches such as Multiplexed Error-robust FISH (MERFISH), introduced by Lu et al.1 in this issue of Cell, now allows researchers to study the epigenomic regulation of gene expression in a tissue-region specific manner.
    DOI:  https://doi.org/10.1016/j.cell.2022.10.014
  69. Science. 2022 Nov 11. 378(6620): 674
    We are worthy.
    Lan Nguyen Chaplin.
      
    DOI:  https://doi.org/10.1126/science.adf6612
  70. Cell. 2022 Nov 10. pii: S0092-8674(22)01357-5. [Epub ahead of print]185(23): 4249-4251
    A gut-retching discovery.
    Jane Y Chen, Richard D Palmiter.
      In this issue of Cell, Xie et al. identify a gut-to-brain pathway that triggers retching after toxic food ingestion or emetic agent administration. Their results shed light on how peripheral signals reach the brain to orchestrate appropriate behavioral responses and facilitate learning to prevent repeated ingestion of harmful substances.
    DOI:  https://doi.org/10.1016/j.cell.2022.10.015
  71. Nat Commun. 2022 Nov 10. 13(1): 6808
    An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.
    Kaushal Asrani, Juhyung Woo, Adrianna A Mendes, Ethan Schaffer, Thiago Vidotto, Clarence Rachel Villanueva, Kewen Feng, Lia Oliveira, Sanjana Murali, Hans B Liu, Daniela C Salles, Brandon Lam, Pedram Argani, Tamara L Lotan.
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.
    DOI:  https://doi.org/10.1038/s41467-022-34617-7
  72. Cell. 2022 Nov 01. pii: S0092-8674(22)01327-7. [Epub ahead of print]
    Mechanoreceptor signal convergence and transformation in the dorsal horn flexibly shape a diversity of outputs to the brain.
    Anda M Chirila, Genelle Rankin, Shih-Yi Tseng, Alan J Emanuel, Carmine L Chavez-Martinez, Dawei Zhang, Christopher D Harvey, David D Ginty.
      The encoding of touch in the spinal cord dorsal horn (DH) and its influence on tactile representations in the brain are poorly understood. Using a range of mechanical stimuli applied to the skin, large-scale in vivo electrophysiological recordings, and genetic manipulations, here we show that neurons in the mouse spinal cord DH receive convergent inputs from both low- and high-threshold mechanoreceptor subtypes and exhibit one of six functionally distinct mechanical response profiles. Genetic disruption of DH feedforward or feedback inhibitory motifs, comprised of interneurons with distinct mechanical response profiles, revealed an extensively interconnected DH network that enables dynamic, flexible tuning of postsynaptic dorsal column (PSDC) output neurons and dictates how neurons in the primary somatosensory cortex respond to touch. Thus, mechanoreceptor subtype convergence and non-linear transformations at the earliest stage of the somatosensory hierarchy shape how touch of the skin is represented in the brain.
    Keywords:  dorsal horn inhibition; dorsal horn outputs; mechanoreceptor convergence; spinal cord dorsal horn
    DOI:  https://doi.org/10.1016/j.cell.2022.10.012
  73. Nat Commun. 2022 Nov 09. 13(1): 6756
    CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever.
    Bo Shu, Justin S G Ooi, Aaron W K Tan, Thiam-Seng Ng, Wanwisa Dejnirattisai, Juthathip Mongkolsapaya, Guntur Fibriansah, Jian Shi, Victor A Kostyuchenko, Gavin R Screaton, Shee-Mei Lok.
      Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional. The intracellular dimeric NS1 (iNS1) forms part of the viral replication complex. Previous studies suggest the extracellular secreted NS1 (sNS1), which is a major factor contributing to DHF, exists as hexamers. The structure of the iNS1 is well-characterised but not that of sNS1. Here we show by cryoEM that the recombinant sNS1 exists in multiple oligomeric states: the tetrameric (stable and loose conformation) and hexameric structures. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated β-sheet or β-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.
    DOI:  https://doi.org/10.1038/s41467-022-34415-1
  74. JCI Insight. 2022 Nov 08. pii: e164603. [Epub ahead of print]
    NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.
    Daniel R Calabrese, Tasha Tsao, Mélia Magnen, Colin Valet, Ying Gao, Beñat Mallavia, Jennifer J Tian, Emily A Aminian, Kristin M Wang, Avishai Shemesh, Elman B Punzalan, Aartik Sarma, Carolyn S Calfee, Stephanie A Christenson, Charles R Langelier, Steven R Hays, Jeff A Golden, Lorriana E Leard, Mary E Kleinhenz, Nicholas A Kolaitis, Rupal J Shah, Aida Venado, Lewis L Lanier, John R Greenland, David M Sayah, Abbas Ardehali, Jasleen Kukreja, S Sam Weigt, John A Belperio, Jonathan P Singer, Mark R Looney.
      Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first post-transplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. Natural killer (NK) cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. Together, these data support an aberrant NK cell-neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.
    Keywords:  Immunology; NK cells; Neutrophils; Organ transplantation; Pulmonology
    DOI:  https://doi.org/10.1172/jci.insight.164603
  75. Nat Commun. 2022 Nov 11. 13(1): 6825
    Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans.
    Hagar Setty, Yehuda Salzberg, Shadi Karimi, Elisheva Berent-Barzel, Michael Krieg, Meital Oren-Suissa.
      How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.
    DOI:  https://doi.org/10.1038/s41467-022-34661-3
  76. Cell. 2022 Nov 04. pii: S0092-8674(22)01367-8. [Epub ahead of print]
    Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.
    Xiaonan Fu, Li Sun, Runze Dong, Jane Y Chen, Runglawan Silakit, Logan F Condon, Yiing Lin, Shin Lin, Richard D Palmiter, Liangcai Gu.
      Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.
    Keywords:  DNA array; DNA stamping; Pixel-seq; chronic pain; microcontact printing; olfactory bulb; parabrachial nucleus; polony gel; polony sequencing; spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.cell.2022.10.021
  77. Nat Commun. 2022 Nov 08. 13(1): 6736
    Systematic tissue annotations of genomics samples by modeling unstructured metadata.
    Nathaniel T Hawkins, Marc Maldaver, Anna Yannakopoulos, Lindsay A Guare, Arjun Krishnan.
      There are currently >1.3 million human -omics samples that are publicly available. This valuable resource remains acutely underused because discovering particular samples from this ever-growing data collection remains a significant challenge. The major impediment is that sample attributes are routinely described using varied terminologies written in unstructured natural language. We propose a natural-language-processing-based machine learning approach (NLP-ML) to infer tissue and cell-type annotations for genomics samples based only on their free-text metadata. NLP-ML works by creating numerical representations of sample descriptions and using these representations as features in a supervised learning classifier that predicts tissue/cell-type terms. Our approach significantly outperforms an advanced graph-based reasoning annotation method (MetaSRA) and a baseline exact string matching method (TAGGER). Model similarities between related tissues demonstrate that NLP-ML models capture biologically-meaningful signals in text. Additionally, these models correctly classify tissue-associated biological processes and diseases based on their text descriptions alone. NLP-ML models are nearly as accurate as models based on gene-expression profiles in predicting sample tissue annotations but have the distinct capability to classify samples irrespective of the genomics experiment type based on their text metadata. Python NLP-ML prediction code and trained tissue models are available at https://github.com/krishnanlab/txt2onto .
    DOI:  https://doi.org/10.1038/s41467-022-34435-x
  78. Nat Commun. 2022 Nov 11. 13(1): 6869
    Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.
    Chujiao Lin, Qiyuan Yang, Dongsheng Guo, Jun Xie, Yeon-Suk Yang, Sachin Chaugule, Ngoc DeSouza, Won-Taek Oh, Rui Li, Zhihao Chen, Aijaz A John, Qiang Qiu, Lihua Julie Zhu, Matthew B Greenblatt, Sankar Ghosh, Shaoguang Li, Guangping Gao, Cole Haynes, Charles P Emerson, Jae-Hyuck Shim.
      Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity.
    DOI:  https://doi.org/10.1038/s41467-022-34694-8
  79. Nat Struct Mol Biol. 2022 Nov 11.
    CTCF blocks antisense transcription initiation at divergent promoters.
    Jing Luan, Marit W Vermunt, Camille M Syrett, Allison Coté, Jacob M Tome, Haoyue Zhang, Anran Huang, Jennifer M Luppino, Cheryl A Keller, Belinda M Giardine, Shiping Zhang, Margaret C Dunagin, Zhe Zhang, Eric F Joyce, John T Lis, Arjun Raj, Ross C Hardison, Gerd A Blobel.
      Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.
    DOI:  https://doi.org/10.1038/s41594-022-00855-y
  80. FASEB J. 2022 Dec;36(12): e22645
    CRTC1 deficiency, specifically in melanocortin-4 receptor-expressing cells, induces hyperphagia, obesity, and insulin resistance.
    Shigenobu Matsumura, Motoki Miyakita, Haruka Miyamori, Satomi Kyo, Fuka Ishikawa, Tsutomu Sasaki, Tomoki Jinno, Jin Tanaka, Kotomi Fujita, Takumi Yokokawa, Tsuyoshi Goto, Keiko Momma, Shigeo Takenaka, Kazuo Inoue.
      Melanocortin-4 receptor (MC4R) is a critical regulator of appetite and energy expenditure in rodents and humans. MC4R deficiency causes hyperphagia, reduced energy expenditure, and impaired glucose metabolism. Ligand binding to MC4R activates adenylyl cyclase, resulting in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a secondary messenger that regulates several cellular processes. Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cAMP and is reportedly involved in obesity. However, the precise mechanism through which CRTC1 regulates energy metabolism remains unknown. Additionally, there are no reports linking CRTC1 and MC4R, although both CRTC1 and MC4R are known to be involved in obesity. Here, we demonstrate that mice lacking CRTC1, specifically in MC4R cells, are sensitive to high-fat diet (HFD)-induced obesity and exhibit hyperphagia and increased body weight gain. Moreover, the loss of CRTC1 in MC4R cells impairs glucose metabolism. MC4R-expressing cell-specific CRTC1 knockout mice did not show changes in body weight gain, food intake, or glucose metabolism when fed a normal-chow diet. Thus, CRTC1 expression in MC4R cells is required for metabolic adaptation to HFD with respect to appetite regulation. Our results revealed an important protective role of CRTC1 in MC4R cells against dietary adaptation.
    Keywords:  high-fat diet; melanocortins; obesity; transcription factors
    DOI:  https://doi.org/10.1096/fj.202200617R
  81. Nat Commun. 2022 Nov 07. 13(1): 6704
    Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.
    Ayesha Sen, Sebastian Kallabis, Felix Gaedke, Christian Jüngst, Julia Boix, Julian Nüchel, Kanjanamas Maliphol, Julia Hofmann, Astrid C Schauss, Marcus Krüger, Rudolf J Wiesner, David Pla-Martín.
      Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.
    DOI:  https://doi.org/10.1038/s41467-022-34205-9
  82. Nat Commun. 2022 Nov 09. 13(1): 6783
    Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.
    Jacob Gordon, Fleur L Chapus, Elizabeth G Viverette, Jason G Williams, Leesa J Deterding, Juno M Krahn, Mario J Borgnia, Joseph Rodriguez, Alan J Warren, Robin E Stanley.
      PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members include WDR18, TEX10, and SENP3. We reconstitute the mammalian Rix1 complex and identified a stable sub-complex comprised of the conserved PELP1 Rix1 domain and WDR18. We determine a 2.7 Å cryo-EM structure of the subcomplex revealing an interconnected tetrameric assembly and the architecture of PELP1's signaling motifs, including eleven LxxLL motifs previously implicated in SR signaling and coactivation of Estrogen Receptor alpha (ERα) mediated transcription. However, the structure shows that none of these motifs is in a conformation that would support SR binding. Together this work establishes that PELP1 scaffolds the Rix1 complex, and association with WDR18 may direct PELP1's activity away from SR coactivation.
    DOI:  https://doi.org/10.1038/s41467-022-34610-0
  83. Nat Struct Mol Biol. 2022 Nov 09.
    An atypical RNA quadruplex marks RNAs as vectors for gene silencing.
    Saeed Roschdi, Jenny Yan, Yuichiro Nomura, Cristian A Escobar, Riley J Petersen, Craig A Bingman, Marco Tonelli, Rahul Vivek, Eric J Montemayor, Marv Wickens, Scott G Kennedy, Samuel E Butcher.
      The addition of poly(UG) ('pUG') repeats to 3' termini of mRNAs drives gene silencing and transgenerational epigenetic inheritance in the metazoan Caenorhabditis elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA polymerase (RdRP) that synthesizes small interfering RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4) structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. The compact pUG fold binds six potassium ions and brings the RNA ends into close proximity. The biological importance of the pUG fold is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific 7-deaza substitutions that disrupt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA structural motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in noncoding regions of human RNAs, suggesting that the fold probably has biological roles beyond gene silencing.
    DOI:  https://doi.org/10.1038/s41594-022-00854-z
  84. J Biol Chem. 2022 Nov 03. pii: S0021-9258(22)01120-6. [Epub ahead of print] 102677
    A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of pro-inflammatory interleukin 12 family cytokines.
    Yonatan G Mideksa, Isabel Aschenbrenner, Anja Fux, Dinah Kaylani, Caroline A M Weiß, Tuan-Anh Nguyen, Nina C Bach, Kathrin Lang, Stephan A Sieber, Matthias J Feige.
      Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photo-crosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone:client complexes for mass spectrometry. Our analysis reveals that a large set of ER chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:β assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific ER chaperones.
    Keywords:  chaperone; genetic code expansion; interleukin; protein disulfide isomerase; protein folding
    DOI:  https://doi.org/10.1016/j.jbc.2022.102677
  85. Nature. 2022 Nov 09.
    Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells.
    Adrià Cañellas-Socias, Carme Cortina, Xavier Hernando-Momblona, Sergio Palomo-Ponce, Eoghan J Mulholland, Gemma Turon, Lidia Mateo, Sefora Conti, Olga Roman, Marta Sevillano, Felipe Slebe, Diana Stork, Adrià Caballé-Mestres, Antonio Berenguer-Llergo, Adrián Álvarez-Varela, Nicola Fenderico, Laura Novellasdemunt, Laura Jiménez-Gracia, Tamara Sipka, Lidia Bardia, Patricia Lorden, Julien Colombelli, Holger Heyn, Xavier Trepat, Sabine Tejpar, Elena Sancho, Daniele V F Tauriello, Simon Leedham, Camille Stephan-Otto Attolini, Eduard Batlle.
      Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
    DOI:  https://doi.org/10.1038/s41586-022-05402-9
  86. Cell Host Microbe. 2022 Nov 09. pii: S1931-3128(22)00472-3. [Epub ahead of print]30(11): 1505-1507
    A loyal "G"uard.
    Ryan Kenneally, Yangnan Gu.
      G proteins are conserved eukaryotic signal transducers that play crucial roles in plant development and responses to environmental stimuli. In this issue of Cell Host & Microbe, Ma et al. (2022) discover a plant-specific family of kinases that act as bona fide nuclear effectors for G-protein signaling during plant immune activation.
    DOI:  https://doi.org/10.1016/j.chom.2022.09.016
  87. J Cell Biol. 2023 Jan 02. pii: e202108101. [Epub ahead of print]222(1):
    The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling.
    Gonzalo Manuel Sanchez, Tugce Ceren Incedal, Juan Prada, Paul O'Callaghan, Oleg Dyachok, Santiago Echeverry, Özge Dumral, Phuoc My Nguyen, Beichen Xie, Sebastian Barg, Johan Kreuger, Thomas Dandekar, Olof Idevall-Hagren.
      The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.
    DOI:  https://doi.org/10.1083/jcb.202108101
  88. Nat Commun. 2022 Nov 09. 13(1): 6784
    An allosteric modulator activates BK channels by perturbing coupling between Ca2+ binding and pore opening.
    Guohui Zhang, Xianjin Xu, Zhiguang Jia, Yanyan Geng, Hongwu Liang, Jingyi Shi, Martina Marras, Carlota Abella, Karl L Magleby, Jonathan R Silva, Jianhan Chen, Xiaoqin Zou, Jianmin Cui.
      BK type Ca2+-activated K+ channels activate in response to both voltage and Ca2+. The membrane-spanning voltage sensor domain (VSD) activation and Ca2+ binding to the cytosolic tail domain (CTD) open the pore across the membrane, but the mechanisms that couple VSD activation and Ca2+ binding to pore opening  are not clear. Here we show that a compound, BC5, identified from in silico screening, interacts with the CTD-VSD interface and specifically modulates the Ca2+ dependent activation mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs a pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is further supported by atomistic simulations and mutagenesis. The results suggest that the CTD-VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.
    DOI:  https://doi.org/10.1038/s41467-022-34359-6
  89. Nat Commun. 2022 Nov 09. 13(1): 6762
    METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury.
    Lijun Wang, Jiaqi Wang, Pujiao Yu, Jingyi Feng, Gui-E Xu, Xuan Zhao, Tianhui Wang, H Immo Lehmann, Guoping Li, Joost P G Sluijter, Junjie Xiao.
      RNA m6A modification is the most widely distributed RNA methylation and is closely related to various pathophysiological processes. Although the benefit of regular exercise on the heart has been well recognized, the role of RNA m6A in exercise training and exercise-induced physiological cardiac hypertrophy remains largely unknown. Here, we show that endurance exercise training leads to reduced cardiac mRNA m6A levels. METTL14 is downregulated by exercise, both at the level of RNA m6A and at the protein level. In vivo, wild-type METTL14 overexpression, but not MTase inactive mutant METTL14, blocks exercise-induced physiological cardiac hypertrophy. Cardiac-specific METTL14 knockdown attenuates acute ischemia-reperfusion injury as well as cardiac dysfunction in ischemia-reperfusion remodeling. Mechanistically, silencing METTL14 suppresses Phlpp2 mRNA m6A modifications and activates Akt-S473, in turn regulating cardiomyocyte growth and apoptosis. Our data indicates that METTL14 plays an important role in maintaining cardiac homeostasis. METTL14 downregulation represents a promising therapeutic strategy to attenuate cardiac remodeling.
    DOI:  https://doi.org/10.1038/s41467-022-34434-y
  90. Nat Biotechnol. 2022 Nov;40(11): 1575
    CAR-T cells tackle autoimmunity.
    João H Duarte.
      
    DOI:  https://doi.org/10.1038/s41587-022-01576-9
  91. Sci Adv. 2022 Nov 11. 8(45): eadd3686
    DNA replication initiation shapes the mutational landscape and expression of the human genome.
    Pierre Murat, Consuelo Perez, Alastair Crisp, Patrick van Eijk, Simon H Reed, Guillaume Guilbaud, Julian E Sale.
      The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity. We demonstrate that these replication initiation-dependent mutational processes exert an influence on phenotypic diversity in humans that is disproportionate to the origins' genomic size: By increasing mutational loads at gene promoters and splice junctions, the presence of an origin significantly influences both gene expression and mRNA isoform usage. Last, we show that mutagenesis at origins not only drives the evolution of origin sequences but also contributes to sculpting regulatory domains of the human genome.
    DOI:  https://doi.org/10.1126/sciadv.add3686
  92. Nat Genet. 2022 Nov;54(11): 1585-1586
    Chromatin oxygen sensing by histone H3 prolyl hydroxylation.
    James A Nathan.
      
    DOI:  https://doi.org/10.1038/s41588-022-01201-0
  93. JAMA Intern Med. 2022 Nov 07.
    Ensuring Progress Toward Racial Equity in Pulse Oximetry-Reply.
    Eric Raphael Gottlieb, Jennifer Ziegler, Barret Rush.
      
    DOI:  https://doi.org/10.1001/jamainternmed.2022.4857
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