bims-nimamd Biomed News
on Neuroimmunity and neuroinflammation in ageing and metabolic disease
Issue of 2022–05–08
forty papers selected by
Fawaz Alzaïd, Sorbonne Université
  1. Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells.
  2. TH17 cells promote CNS inflammation by sensing danger signals via Mincle.
  3. Pantothenate kinase 2 interacts with PINK1 to regulate mitochondrial quality control via acetyl-CoA metabolism.
  4. Activation and allosteric regulation of the orphan GPR88-Gi1 signaling complex.
  5. Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.
  6. Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin.
  7. CDC7-independent G1/S transition revealed by targeted protein degradation.
  8. Recall of B cell memory depends on relative locations of prime and boost immunization.
  9. IRF4 drives clonal evolution and lineage choice in a zebrafish model of T-cell lymphoma.
  10. Multivalent interactions essential for lentiviral integrase function.
  11. Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
  12. Placental multi-omics integration identifies candidate functional genes for birthweight.
  13. Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.
  14. High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control.
  15. CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids.
  16. Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.
  17. Genome and transcriptome mechanisms driving cephalopod evolution.
  18. The representation of context in mouse hippocampus is preserved despite neural drift.
  19. Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.
  20. Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus.
  21. Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.
  22. Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.
  23. Unique binding pattern for a lineage of human antibodies with broad reactivity against influenza A virus.
  24. ON/OFF domains shape receptive field structure in mouse visual cortex.
  25. The deacetylase SIRT2 contributes to autoimmune disease pathogenesis by modulating IL-17A and IL-2 transcription.
  26. The dopamine transporter antiports potassium to increase the uptake of dopamine.
  27. Multivariate genome-wide association study on tissue-sensitive diffusion metrics highlights pathways that shape the human brain.
  28. Name that animal: my DNA detector.
  29. Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.
  30. Preventing erosion of X-chromosome inactivation in human embryonic stem cells.
  31. Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.
  32. NEAT-seq: simultaneous profiling of intra-nuclear proteins, chromatin accessibility and gene expression in single cells.
  33. Regulation of A-to-I RNA editing and stop codon recoding to control selenoprotein expression during skeletal myogenesis.
  34. Comparative optimization of combinatorial CRISPR screens.
  35. Chat-ty B cells talk down hematopoiesis.
  36. Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer.
  37. Universal non-Hermitian skin effect in two and higher dimensions.
  38. A reversible mitochondrial complex I thiol switch mediates hypoxic avoidance behavior in C. elegans.
  39. Adiponectin receptors sustain haematopoietic stem cells throughout adulthood by protecting them from inflammation.
  40. Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.
  1. Nat Genet. 2022 May 05.
    Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells.
    Kousuke Mouri, Michael H Guo, Carl G de Boer, Michelle M Lissner, Ingrid A Harten, Gregory A Newby, Hannah A DeBerg, Winona F Platt, Matteo Gentili, David R Liu, Daniel J Campbell, Nir Hacohen, Ryan Tewhey, John P Ray.
      Genome-wide association studies (GWASs) have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variants within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naive CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness and, upon acute viral infection, displayed greater propensity to become effector T cells. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.
    DOI:  https://doi.org/10.1038/s41588-022-01056-5
  2. Nat Commun. 2022 May 03. 13(1): 2406
    TH17 cells promote CNS inflammation by sensing danger signals via Mincle.
    Quanri Zhang, Weiwei Liu, Han Wang, Hao Zhou, Katarzyna Bulek, Xing Chen, Cun-Jin Zhang, Junjie Zhao, Renliang Zhang, Caini Liu, Zizhen Kang, Robert A Bermel, George Dubyak, Derek W Abbott, Tsan Sam Xiao, Laura E Nagy, Xiaoxia Li.
      The C-type lectin receptor Mincle is known for its important role in innate immune cells in recognizing pathogen and damage associated molecular patterns. Here we report a T cell-intrinsic role for Mincle in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Genomic deletion of Mincle in T cells impairs TH17, but not TH1 cell-mediated EAE, in alignment with significantly higher expression of Mincle in TH17 cells than in TH1 cells. Mechanistically, dying cells release β-glucosylceramide during inflammation, which serves as natural ligand for Mincle. Ligand engagement induces activation of the ASC-NLRP3 inflammasome, which leads to Caspase8-dependent IL-1β production and consequentially TH17 cell proliferation via an autocrine regulatory loop. Chemical inhibition of β-glucosylceramide synthesis greatly reduces inflammatory CD4+ T cells in the central nervous system and inhibits EAE progression in mice. Taken together, this study indicates that sensing of danger signals by Mincle on TH17 cells plays a critical role in promoting CNS inflammation.
    DOI:  https://doi.org/10.1038/s41467-022-30174-1
  3. Nat Commun. 2022 May 03. 13(1): 2412
    Pantothenate kinase 2 interacts with PINK1 to regulate mitochondrial quality control via acetyl-CoA metabolism.
    Yunpeng Huang, Zhihui Wan, Yinglu Tang, Junxuan Xu, Bretton Laboret, Sree Nallamothu, Chenyu Yang, Boxiang Liu, Rongze Olivia Lu, Bingwei Lu, Juan Feng, Jing Cao, Susan Hayflick, Zhihao Wu, Bing Zhou.
      Human neurodegenerative disorders often exhibit similar pathologies, suggesting a shared aetiology. Key pathological features of Parkinson's disease (PD) are also observed in other neurodegenerative diseases. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is caused by mutations in the human PANK2 gene, which catalyzes the initial step of de novo CoA synthesis. Here, we show that fumble (fbl), the human PANK2 homolog in Drosophila, interacts with PINK1 genetically. fbl and PINK1 mutants display similar mitochondrial abnormalities, and overexpression of mitochondrial Fbl rescues PINK1 loss-of-function (LOF) defects. Dietary vitamin B5 derivatives effectively rescue CoA/acetyl-CoA levels and mitochondrial function, reversing the PINK1 deficiency phenotype. Mechanistically, Fbl regulates Ref(2)P (p62/SQSTM1 homolog) by acetylation to promote mitophagy, whereas PINK1 regulates fbl translation by anchoring mRNA molecules to the outer mitochondrial membrane. In conclusion, Fbl (or PANK2) acts downstream of PINK1, regulating CoA/acetyl-CoA metabolism to promote mitophagy, uncovering a potential therapeutic intervention strategy in PD treatment.
    DOI:  https://doi.org/10.1038/s41467-022-30178-x
  4. Nat Commun. 2022 May 02. 13(1): 2375
    Activation and allosteric regulation of the orphan GPR88-Gi1 signaling complex.
    Geng Chen, Jun Xu, Asuka Inoue, Maximilian F Schmidt, Chen Bai, Qiuyuan Lu, Peter Gmeiner, Zheng Liu, Yang Du.
      GPR88 is an orphan class A G-protein-coupled receptor that is highly expressed in the striatum and regulates diverse brain and behavioral functions. Here we present cryo-EM structures of the human GPR88-Gi1 signaling complex with or without a synthetic agonist (1R, 2R)-2-PCCA. We show that (1R, 2R)-2-PCCA is an allosteric modulator binding to a herein identified pocket formed by the cytoplasmic ends of transmembrane segments 5, 6, and the extreme C terminus of the α5 helix of Gi1. We also identify an electron density in the extracellular orthosteric site that may represent a putative endogenous ligand of GPR88. These structures, together with mutagenesis studies and an inactive state model obtained from metadynamics simulations, reveal a unique activation mechanism for GPR88 with a set of distinctive structure features and a water-mediated polar network. Overall, our results provide a structural framework for understanding the ligand binding, activation and signaling mechanism of GPR88, and will facilitate the innovative drug discovery for neuropsychiatric disorders and for deorphanization of this receptor.
    DOI:  https://doi.org/10.1038/s41467-022-30081-5
  5. Nat Commun. 2022 May 04. 13(1): 2454
    Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.
    Michela Milani, Cesare Canepari, Tongyao Liu, Mauro Biffi, Fabio Russo, Tiziana Plati, Rosalia Curto, Susannah Patarroyo-White, Douglas Drager, Ilaria Visigalli, Chiara Brombin, Paola Albertini, Antonia Follenzi, Eduard Ayuso, Christian Mueller, Andrea Annoni, Luigi Naldini, Alessio Cantore.
      Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.
    DOI:  https://doi.org/10.1038/s41467-022-30102-3
  6. Nat Commun. 2022 May 02. 13(1): 2380
    Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin.
    Nils Marechal, Banyuhay P Serrano, Xinyan Zhang, Charles J Weitz.
      Thyroid hormones are essential regulators of metabolism, development, and growth. They are formed from pairs of iodinated tyrosine residues within the precursor thyroglobulin (TG), a 660-kDa homodimer of the thyroid gland, by an oxidative coupling reaction. Tyrosine pairs that give rise to thyroid hormones have been assigned within the structure of human TG, but the process of hormone formation is poorly understood. Here we report a ~3.3-Å cryo-EM structure of native bovine TG with nascent thyroid hormone formed at one of the predicted hormonogenic sites. Local structural rearrangements provide insight into mechanisms underlying thyroid hormone formation and stabilization.
    DOI:  https://doi.org/10.1038/s41467-022-30082-4
  7. Nature. 2022 May 04.
    CDC7-independent G1/S transition revealed by targeted protein degradation.
    Jan M Suski, Nalin Ratnayeke, Marcin Braun, Tian Zhang, Vladislav Strmiska, Wojciech Michowski, Geylani Can, Antoine Simoneau, Konrad Snioch, Mikolaj Cup, Caitlin M Sullivan, Xiaoji Wu, Joanna Nowacka, Timothy B Branigan, Lindsey R Pack, James A DeCaprio, Yan Geng, Lee Zou, Steven P Gygi, Johannes C Walter, Tobias Meyer, Piotr Sicinski.
      The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division1. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication.
    DOI:  https://doi.org/10.1038/s41586-022-04698-x
  8. Sci Immunol. 2022 May 06. 7(71): eabn5311
    Recall of B cell memory depends on relative locations of prime and boost immunization.
    Masayuki Kuraoka, Chen-Hao Yeh, Goran Bajic, Ryutaro Kotaki, Shengli Song, Ian Windsor, Stephen C Harrison, Garnett Kelsoe.
      Immunization or microbial infection can establish long-term B cell memory not only systemically but also locally. Evidence has suggested that local B cell memory contributes to early local plasmacytic responses after secondary challenge. However, it is unclear whether locality of immunization plays any role in memory B cell participation in recall germinal centers (GCs), which is essential for updating their B cell antigen receptors (BCRs). Using single B cell culture and fate mapping, we have characterized BCR repertoires in recall GCs after boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit the progeny of primary GC B cells to recall GCs more efficiently than do distal boosts. Recall GCs elicited by local boosts contain significantly more B cells with elevated levels of immunoglobulin (Ig) mutation and higher avidity BCRs. This local preference is unaffected by blocking CD40:CD154 interaction to terminate active, GC responses. Local boosts with heterologous antigens elicit secondary GCs with B cell populations enriched for cross-reactivity to the prime and boost antigens; in contrast, cross-reactive GC B cells are rare after distal boosts. Our results suggest that local B cell memory is retained in the form of memory B cells, GC B cells, and GC phenotype B cells that are independent of organized GC structures and that these persistent "primed B cells" contribute to recall GC responses at local sites. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.
    DOI:  https://doi.org/10.1126/sciimmunol.abn5311
  9. Nat Commun. 2022 May 03. 13(1): 2420
    IRF4 drives clonal evolution and lineage choice in a zebrafish model of T-cell lymphoma.
    Stella Amanda, Tze King Tan, Jolynn Zu Lin Ong, Madelaine Skolastika Theardy, Regina Wan Ju Wong, Xiao Zi Huang, Muhammad Zulfaqar Ali, Yan Li, Zhiyuan Gong, Hiroshi Inagaki, Ee Yong Foo, Brendan Pang, Soo Yong Tan, Shinsuke Iida, Takaomi Sanda.
      IRF4 is a master regulator of immunity and is also frequently overexpressed in mature lymphoid neoplasms. Here, we demonstrate the oncogenicity of IRF4 in vivo, its potential effects on T-cell development and clonal evolution using a zebrafish model. IRF4-transgenic zebrafish develop aggressive tumors with massive infiltration of abnormal lymphocytes that spread to distal organs. Many late-stage tumors are mono- or oligoclonal, and tumor cells can expand in recipient animals after transplantation, demonstrating their malignancy. Mutation of p53 accelerates tumor onset, increases penetrance, and results in tumor heterogeneity. Surprisingly, single-cell RNA-sequencing reveals that the majority of tumor cells are double-negative T-cells, many of which express tcr-γ that became dominant as the tumors progress, whereas double-positive T-cells are largely diminished. Gene expression and epigenetic profiling demonstrates that gata3, mycb, lrrn1, patl1 and psip1 are specifically activated in tumors, while genes responsible for T-cell differentiation including id3 are repressed. IRF4-driven tumors are sensitive to the BRD inhibitor.
    DOI:  https://doi.org/10.1038/s41467-022-30053-9
  10. Nat Commun. 2022 May 03. 13(1): 2416
    Multivalent interactions essential for lentiviral integrase function.
    Allison Ballandras-Colas, Vidya Chivukula, Dominika T Gruszka, Zelin Shan, Parmit K Singh, Valerie E Pye, Rebecca K McLean, Gregory J Bedwell, Wen Li, Andrea Nans, Nicola J Cook, Hind J Fadel, Eric M Poeschla, David J Griffiths, Javier Vargas, Ian A Taylor, Dmitry Lyumkis, Hasan Yardimci, Alan N Engelman, Peter Cherepanov.
      A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.
    DOI:  https://doi.org/10.1038/s41467-022-29928-8
  11. Nat Commun. 2022 May 05. 13(1): 2483
    Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.
    Xiaojian Shi, Bryn Reinstadler, Hardik Shah, Tsz-Leung To, Katie Byrne, Luanna Summer, Sarah E Calvo, Olga Goldberger, John G Doench, Vamsi K Mootha, Hongying Shen.
      The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.
    DOI:  https://doi.org/10.1038/s41467-022-30126-9
  12. Nat Commun. 2022 May 02. 13(1): 2384
    Placental multi-omics integration identifies candidate functional genes for birthweight.
    Fasil Tekola-Ayele, Xuehuo Zeng, Suvo Chatterjee, Marion Ouidir, Corina Lesseur, Ke Hao, Jia Chen, Markos Tesfaye, Carmen J Marsit, Tsegaselassie Workalemahu, Ronald Wapner.
      Abnormal birthweight is associated with increased risk for cardiometabolic diseases in later life. Although the placenta is critical to fetal development and later life health, it has not been integrated into largescale functional genomics initiatives, and mechanisms of birthweight-associated variants identified by genome wide association studies (GWAS) are unclear. The goal of this study is to provide functional mechanistic insight into the causal pathway from a genetic variant to birthweight by integrating placental methylation and gene expression with established GWAS loci for birthweight. We identify placental DNA methylation and gene expression targets for several birthweight GWAS loci. The target genes are broadly enriched in cardiometabolic, immune response, and hormonal pathways. We find that methylation causally influences WNT3A, CTDNEP1, and RANBP2 expression in placenta. Multi-trait colocalization identifies PLEKHA1, FES, CTDNEP1, and PRMT7 as likely functional effector genes. These findings reveal candidate functional pathways that underpin the genetic regulation of birthweight via placental epigenetic and transcriptomic mechanisms. Clinical trial registration; ClinicalTrials.gov, NCT00912132.
    DOI:  https://doi.org/10.1038/s41467-022-30007-1
  13. Nat Commun. 2022 May 05. 13(1): 2460
    Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.
    Yang Zhang, Laura Garcia-Ibanez, Carolin Ulbricht, Laurence S C Lok, Jeremy A Pike, Jennifer Mueller-Winkler, Thomas W Dennison, John R Ferdinand, Cameron J M Burnett, Juan C Yam-Puc, Lingling Zhang, Raul Maqueda Alfaro, Yousuke Takahama, Izumi Ohigashi, Geoffrey Brown, Tomohiro Kurosaki, Victor L J Tybulewicz, Antal Rot, Anja E Hauser, Menna R Clatworthy, Kai-Michael Toellner.
      Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift.
    DOI:  https://doi.org/10.1038/s41467-022-29978-y
  14. Nat Commun. 2022 May 03. 13(1): 2402
    High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control.
    Dashiell J Massey, Amnon Koren.
      DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.
    DOI:  https://doi.org/10.1038/s41467-022-30212-y
  15. Nat Commun. 2022 May 03. 13(1): 2407
    CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids.
    Megan Lo, Amnon Sharir, Michael D Paul, Hayarpi Torosyan, Christopher Agnew, Amy Li, Cynthia Neben, Pauline Marangoni, Libin Xu, David R Raleigh, Natalia Jura, Ophir D Klein.
      The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can lead to congenital malformations and diseases including cancer. Although cholesterol and several oxysterol lipids have been shown to play crucial roles in HH activation, the molecular mechanisms governing their regulation remain unresolved. Here, we identify Canopy4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that modulates levels of membrane sterol lipids. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway in vitro and elevates membrane levels of accessible sterol lipids, such as cholesterol, an endogenous ligand involved in HH activation. Our data demonstrate that CNPY4 is a negative regulator that fine-tunes HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.
    DOI:  https://doi.org/10.1038/s41467-022-30186-x
  16. Nature. 2022 May 04.
    Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.
    Giacomo Oliveira, Kari Stromhaug, Nicoletta Cieri, J Bryan Iorgulescu, Susan Klaeger, Jacquelyn O Wolff, Suzanna Rachimi, Vipheaviny Chea, Kate Krause, Samuel S Freeman, Wandi Zhang, Shuqiang Li, David A Braun, Donna Neuberg, Steven A Carr, Kenneth J Livak, Dennie T Frederick, Edward F Fritsch, Megan Wind-Rotolo, Nir Hacohen, Moshe Sade-Feldman, Charles H Yoon, Derin B Keskin, Patrick A Ott, Scott J Rodig, Genevieve M Boland, Catherine J Wu.
      Within the tumour microenvironment, CD4+ T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules1,2, but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4+ T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4+ T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4+ T regulatory (TReg) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4+ TReg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4+ T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4+ TReg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.
    DOI:  https://doi.org/10.1038/s41586-022-04682-5
  17. Nat Commun. 2022 May 04. 13(1): 2427
    Genome and transcriptome mechanisms driving cephalopod evolution.
    Caroline B Albertin, Sofia Medina-Ruiz, Therese Mitros, Hannah Schmidbaur, Gustavo Sanchez, Z Yan Wang, Jane Grimwood, Joshua J C Rosenthal, Clifton W Ragsdale, Oleg Simakov, Daniel S Rokhsar.
      Cephalopods are known for their large nervous systems, complex behaviors and morphological innovations. To investigate the genomic underpinnings of these features, we assembled the chromosomes of the Boston market squid, Doryteuthis (Loligo) pealeii, and the California two-spot octopus, Octopus bimaculoides, and compared them with those of the Hawaiian bobtail squid, Euprymna scolopes. The genomes of the soft-bodied (coleoid) cephalopods are highly rearranged relative to other extant molluscs, indicating an intense, early burst of genome restructuring. The coleoid genomes feature multi-megabase, tandem arrays of genes associated with brain development and cephalopod-specific innovations. We find that a known coleoid hallmark, extensive A-to-I mRNA editing, displays two fundamentally distinct patterns: one exclusive to the nervous system and concentrated in genic sequences, the other widespread and directed toward repetitive elements. We conclude that coleoid novelty is mediated in part by substantial genome reorganization, gene family expansion, and tissue-dependent mRNA editing.
    DOI:  https://doi.org/10.1038/s41467-022-29748-w
  18. Nat Commun. 2022 May 03. 13(1): 2415
    The representation of context in mouse hippocampus is preserved despite neural drift.
    Alexandra T Keinath, Coralie-Anne Mosser, Mark P Brandon.
      The hippocampus is thought to mediate episodic memory through the instantiation and reinstatement of context-specific cognitive maps. However, recent longitudinal experiments have challenged this view, reporting that most hippocampal cells change their tuning properties over days even in the same environment. Often referred to as neural or representational drift, these dynamics raise questions about the capacity and content of the hippocampal code. One such question is whether and how these long-term dynamics impact the hippocampal code for context. To address this, we image large CA1 populations over more than a month of daily experience as freely behaving mice participate in an extended geometric morph paradigm. We find that long-timescale changes in population activity occur orthogonally to the representation of context in network space, allowing for consistent readout of contextual information across weeks. This population-level structure is supported by heterogeneous patterns of activity at the level of individual cells, where we observe evidence of a positive relationship between interpretable contextual coding and long-term stability. Together, these results demonstrate that long-timescale changes to the CA1 spatial code preserve the relative structure of contextual representation.
    DOI:  https://doi.org/10.1038/s41467-022-30198-7
  19. Nat Commun. 2022 May 02. 13(1): 2387
    Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content.
    Fabio Papes, Antonio P Camargo, Janaina S de Souza, Vinicius M A Carvalho, Ryan A Szeto, Erin LaMontagne, José R Teixeira, Simoni H Avansini, Sandra M Sánchez-Sánchez, Thiago S Nakahara, Carolina N Santo, Wei Wu, Hang Yao, Barbara M P Araújo, Paulo E N F Velho, Gabriel G Haddad, Alysson R Muotri.
      Transcription Factor 4 (TCF4) has been associated with autism, schizophrenia, and other neuropsychiatric disorders. However, how pathological TCF4 mutations affect the human neural tissue is poorly understood. Here, we derive neural progenitor cells, neurons, and brain organoids from skin fibroblasts obtained from children with Pitt-Hopkins Syndrome carrying clinically relevant mutations in TCF4. We show that neural progenitors bearing these mutations have reduced proliferation and impaired capacity to differentiate into neurons. We identify a mechanism through which TCF4 loss-of-function leads to decreased Wnt signaling and then to diminished expression of SOX genes, culminating in reduced progenitor proliferation in vitro. Moreover, we show reduced cortical neuron content and impaired electrical activity in the patient-derived organoids, phenotypes that were rescued after correction of TCF4 expression or by pharmacological modulation of Wnt signaling. This work delineates pathological mechanisms in neural cells harboring TCF4 mutations and provides a potential target for therapeutic strategies for genetic disorders associated with this gene.
    DOI:  https://doi.org/10.1038/s41467-022-29942-w
  20. Nat Immunol. 2022 Apr 29.
    Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus.
    Miho Shinzawa, E Ashley Moseman, Selamawit Gossa, Yasuko Mano, Abhisek Bhattacharya, Terry Guinter, Amala Alag, Xiongfong Chen, Maggie Cam, Dorian B McGavern, Batu Erman, Alfred Singer.
      T cell specificity and function are linked during development, as MHC-II-specific TCR signals generate CD4 helper T cells and MHC-I-specific TCR signals generate CD8 cytotoxic T cells, but the basis remains uncertain. We now report that switching coreceptor proteins encoded by Cd4 and Cd8 gene loci functionally reverses the T cell immune system, generating CD4 cytotoxic and CD8 helper T cells. Such functional reversal reveals that coreceptor proteins promote the helper-lineage fate when encoded by Cd4, but promote the cytotoxic-lineage fate when encoded in Cd8-regardless of the coreceptor proteins each locus encodes. Thus, T cell lineage fate is determined by cis-regulatory elements in coreceptor gene loci and is not determined by the coreceptor proteins they encode, invalidating coreceptor signal strength as the basis of lineage fate determination. Moreover, we consider that evolution selected the particular coreceptor proteins that Cd4 and Cd8 gene loci encode to avoid generating functionally reversed T cells because they fail to promote protective immunity against environmental pathogens.
    DOI:  https://doi.org/10.1038/s41590-022-01187-1
  21. Nat Commun. 2022 May 05. 13(1): 2480
    Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.
    Chinnu Rose Joseph, Sabrina Dusi, Michele Giannattasio, Dana Branzei.
      DNA damage tolerance (DDT), activated by replication stress during genome replication, is mediated by translesion synthesis and homologous recombination (HR). Here we uncover that DDK kinase, essential for replication initiation, is critical for replication-associated recombination-mediated DDT. DDK relies on its multi-monoSUMOylation to facilitate HR-mediated DDT and optimal retention of Rad51 recombinase at replication damage sites. Impairment of DDK kinase activity, reduced monoSUMOylation and mutations in the putative SUMO Interacting Motifs (SIMs) of Rad51 impair replication-associated recombination and cause fork uncoupling with accumulation of large single-stranded DNA regions at fork branching points. Notably, genetic activation of salvage recombination rescues the uncoupled fork phenotype but not the recombination-dependent gap-filling defect of DDK mutants, revealing that the salvage recombination pathway operates preferentially proximal to fork junctions at stalled replication forks. Overall, we uncover that monoSUMOylated DDK acts with Rad51 in an axis that prevents replication fork uncoupling and mediates recombination-dependent gap-filling.
    DOI:  https://doi.org/10.1038/s41467-022-30215-9
  22. Nat Commun. 2022 May 06. 13(1): 2492
    Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.
    Franziska Lehne, Thomas Pokrant, Sabnam Parbin, Gabriela Salinas, Jörg Großhans, Katja Rust, Jan Faix, Sven Bogdan.
      Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.
    DOI:  https://doi.org/10.1038/s41467-022-30167-0
  23. Nat Commun. 2022 May 02. 13(1): 2378
    Unique binding pattern for a lineage of human antibodies with broad reactivity against influenza A virus.
    Xiaoyu Sun, Caixuan Liu, Xiao Lu, Zhiyang Ling, Chunyan Yi, Zhen Zhang, Zi Li, Mingliang Jin, Wenshuai Wang, Shubing Tang, Fangfang Wang, Fang Wang, Sonam Wangmo, Shuangfeng Chen, Li Li, Liyan Ma, Yaguang Zhang, Zhuo Yang, Xiaoping Dong, Zhikang Qian, Jianping Ding, Dayan Wang, Yao Cong, Bing Sun.
      Most structurally characterized broadly neutralizing antibodies (bnAbs) against influenza A viruses (IAVs) target the conserved conformational epitopes of hemagglutinin (HA). Here, we report a lineage of naturally occurring human antibodies sharing the same germline gene, VH3-48/VK1-12. These antibodies broadly neutralize the major circulating strains of IAV in vitro and in vivo mainly by binding a contiguous epitope of H3N2 HA, but a conformational epitope of H1N1 HA, respectively. Our structural and functional studies of antibody 28-12 revealed that the continuous amino acids in helix A, particularly N49HA2 of H3 HA, are critical to determine the binding feature with 28-12. In contrast, the conformational epitope feature is dependent on the discontinuous segments involving helix A, the fusion peptide, and several HA1 residues within H1N1 HA. We report that this antibody was initially selected by H3 (group 2) viruses and evolved via somatic hypermutation to enhance the reactivity to H3 and acquire cross-neutralization to H1 (group 1) virus. These findings enrich our understanding of different antigenic determinants of heterosubtypic influenza viruses for the recognition of bnAbs and provide a reference for the design of influenza vaccines and more effective antiviral drugs.
    DOI:  https://doi.org/10.1038/s41467-022-29950-w
  24. Nat Commun. 2022 May 05. 13(1): 2466
    ON/OFF domains shape receptive field structure in mouse visual cortex.
    Elaine Tring, Konnie K Duan, Dario L Ringach.
      In higher mammals, thalamic afferents to primary visual cortex (area V1) segregate according to their responses to increases (ON) or decreases (OFF) in luminance. This organization induces columnar, ON/OFF domains postulated to provide a scaffold for the emergence of orientation tuning. To further test this idea, we asked whether ON/OFF domains exist in mouse V1. Here we show that mouse V1 is indeed parceled into ON/OFF domains. Interestingly, fluctuations in the relative density of ON/OFF neurons on the cortical surface mirror fluctuations in the relative density of ON/OFF receptive field centers on the visual field. Moreover, the local diversity of cortical receptive fields is explained by a model in which neurons linearly combine a small number of ON and OFF signals available in their cortical neighborhoods. These findings suggest that ON/OFF domains originate in fluctuations of the balance between ON/OFF responses across the visual field which, in turn, shapes the structure of cortical receptive fields.
    DOI:  https://doi.org/10.1038/s41467-022-29999-7
  25. Cell Mol Immunol. 2022 May 06.
    The deacetylase SIRT2 contributes to autoimmune disease pathogenesis by modulating IL-17A and IL-2 transcription.
    Ryo Hisada, Nobuya Yoshida, Masataka Umeda, Catalina Burbano, Rhea Bhargava, Marc Scherlinger, Michihito Kono, Vasileios C Kyttaris, Suzanne Krishfield, George C Tsokos.
      Aberrant IL-17A expression together with reduced IL-2 production by effector CD4+ T cells contributes to the pathogenesis of systemic lupus erythematosus (SLE). Here, we report that Sirtuin 2 (SIRT2), a member of the family of NAD+-dependent histone deacetylases, suppresses IL-2 production by CD4+ T cells while promoting their differentiation into Th17 cells. Mechanistically, we show that SIRT2 is responsible for the deacetylation of p70S6K, activation of the mTORC1/HIF-1α/RORγt pathway and induction of Th17-cell differentiation. Additionally, SIRT2 was shown to be responsible for the deacetylation of c-Jun and histones at the Il-2 gene, resulting in decreased IL-2 production. We found that the transcription factor inducible cAMP early repressor (ICER), which is overexpressed in T cells from people with SLE and lupus-prone mice, bound directly to the Sirt2 promoter and promoted its transcription. AK-7, a SIRT2 inhibitor, limited the ability of adoptively transferred antigen-specific CD4+ T cells to cause autoimmune encephalomyelitis in mice and limited disease in lupus-prone MRL/lpr mice. Finally, CD4+ T cells from SLE patients exhibited increased expression of SIRT2, and pharmacological inhibition of SIRT2 in primary CD4+ T cells from patients with SLE attenuated the ability of these cells to differentiate into Th17 cells and promoted the generation of IL-2-producing T cells. Collectively, these results suggest that SIRT2-mediated deacetylation is essential in the aberrant expression of IL-17A and IL-2 and that SIRT2 may be a promising molecular target for new SLE therapies.
    Keywords:  IL-17A; IL-2; SIRT2; Systemic lupus erythematosus
    DOI:  https://doi.org/10.1038/s41423-022-00874-1
  26. Nat Commun. 2022 May 04. 13(1): 2446
    The dopamine transporter antiports potassium to increase the uptake of dopamine.
    Solveig G Schmidt, Mette Galsgaard Malle, Anne Kathrine Nielsen, Søren S-R Bohr, Ciara F Pugh, Jeppe C Nielsen, Ida H Poulsen, Kasper D Rand, Nikos S Hatzakis, Claus J Loland.
      The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the Drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we find that K+ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.
    DOI:  https://doi.org/10.1038/s41467-022-30154-5
  27. Nat Commun. 2022 May 03. 13(1): 2423
    Multivariate genome-wide association study on tissue-sensitive diffusion metrics highlights pathways that shape the human brain.
    Chun Chieh Fan, Robert Loughnan, Carolina Makowski, Diliana Pecheva, Chi-Hua Chen, Donald J Hagler, Wesley K Thompson, Nadine Parker, Dennis van der Meer, Oleksandr Frei, Ole A Andreassen, Anders M Dale.
      The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discover 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% are validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development are found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders.
    DOI:  https://doi.org/10.1038/s41467-022-30110-3
  28. Nature. 2022 May;605(7908): 186
    Name that animal: my DNA detector.
    Chris Woolston.
      
    Keywords:  Careers; Ecology; Genetics
    DOI:  https://doi.org/10.1038/d41586-022-01206-z
  29. Nat Commun. 2022 May 04. 13(1): 2442
    Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen.
    Alice Mac Kain, Ghizlane Maarifi, Sophie-Marie Aicher, Nathalie Arhel, Artem Baidaliuk, Sandie Munier, Flora Donati, Thomas Vallet, Quang Dinh Tran, Alexandra Hardy, Maxime Chazal, Françoise Porrot, Molly OhAinle, Jared Carlson-Stevermer, Jennifer Oki, Kevin Holden, Gert Zimmer, Etienne Simon-Lorière, Timothée Bruel, Olivier Schwartz, Sylvie van der Werf, Nolwenn Jouvenet, Sébastien Nisole, Marco Vignuzzi, Ferdinand Roesch.
      Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.
    DOI:  https://doi.org/10.1038/s41467-022-30134-9
  30. Nat Commun. 2022 May 06. 13(1): 2516
    Preventing erosion of X-chromosome inactivation in human embryonic stem cells.
    Marissa Cloutier, Surinder Kumar, Emily Buttigieg, Laura Keller, Brandon Lee, Aaron Williams, Sandra Mojica-Perez, Indri Erliandri, Andre Monteiro Da Rocha, Kenneth Cadigan, Gary D Smith, Sundeep Kalantry.
      X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.
    DOI:  https://doi.org/10.1038/s41467-022-30259-x
  31. Cell. 2022 Apr 29. pii: S0092-8674(22)00443-3. [Epub ahead of print]
    Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.
    Tzu-Tang Wei, Mark Chandy, Masataka Nishiga, Angela Zhang, Kaavya Krishna Kumar, Dilip Thomas, Amit Manhas, Siyeon Rhee, Johanne Marie Justesen, Ian Y Chen, Hung-Ta Wo, Saereh Khanamiri, Johnson Y Yang, Frederick J Seidl, Noah Z Burns, Chun Liu, Nazish Sayed, Jiun-Jie Shie, Chih-Fan Yeh, Kai-Chien Yang, Edward Lau, Kara L Lynch, Manuel Rivas, Brian K Kobilka, Joseph C Wu.
      Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.
    Keywords:  Billy Martin tetrad; G protein-coupled receptor; GPCR; UK Biobank; atherosclerosis; cardiovascular disease; human-induced pluripotent stem cell; in silico drug screening; in vivo ligand binding; marijuana
    DOI:  https://doi.org/10.1016/j.cell.2022.04.005
  32. Nat Methods. 2022 May 02.
    NEAT-seq: simultaneous profiling of intra-nuclear proteins, chromatin accessibility and gene expression in single cells.
    Amy F Chen, Benjamin Parks, Arwa S Kathiria, Benjamin Ober-Reynolds, Jorg J Goronzy, William J Greenleaf.
      In this work, we describe NEAT-seq (sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells), enabling interrogation of regulatory mechanisms spanning the central dogma. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive T cell subsets and identify examples of TFs with regulatory activity gated by transcription, translation and regulation of chromatin binding. We also link a noncoding genome-wide association study single-nucleotide polymorphism (SNP) within a GATA motif to a putative target gene, using NEAT-seq data to internally validate SNP impact on GATA3 regulation.
    DOI:  https://doi.org/10.1038/s41592-022-01461-y
  33. Nat Commun. 2022 May 06. 13(1): 2503
    Regulation of A-to-I RNA editing and stop codon recoding to control selenoprotein expression during skeletal myogenesis.
    Yuta Noda, Shunpei Okada, Tsutomu Suzuki.
      Selenoprotein N (SELENON), a selenocysteine (Sec)-containing protein with high reductive activity, maintains redox homeostasis, thereby contributing to skeletal muscle differentiation and function. Loss-of-function mutations in SELENON cause severe neuromuscular disorders. In the early-to-middle stage of myoblast differentiation, SELENON maintains redox homeostasis and modulates endoplasmic reticulum (ER) Ca2+ concentration, resulting in a gradual reduction from the middle-to-late stages due to unknown mechanisms. The present study describes post-transcriptional mechanisms that regulate SELENON expression during myoblast differentiation. Part of an Alu element in the second intron of SELENON pre-mRNA is frequently exonized during splicing, resulting in an aberrant mRNA that is degraded by nonsense-mediated mRNA decay (NMD). In the middle stage of myoblast differentiation, ADAR1-mediated A-to-I RNA editing occurs in the U1 snRNA binding site at 5' splice site, preventing Alu exonization and producing mature mRNA. In the middle-to-late stage of myoblast differentiation, the level of Sec-charged tRNASec decreases due to downregulation of essential recoding factors for Sec insertion, thereby generating a premature termination codon in SELENON mRNA, which is targeted by NMD.
    DOI:  https://doi.org/10.1038/s41467-022-30181-2
  34. Nat Commun. 2022 May 05. 13(1): 2469
    Comparative optimization of combinatorial CRISPR screens.
    Ruitong Li, Olaf Klingbeil, Davide Monducci, Michael J Young, Diego J Rodriguez, Zaid Bayyat, Joshua M Dempster, Devishi Kesar, Xiaoping Yang, Mahdi Zamanighomi, Christopher R Vakoc, Takahiro Ito, William R Sellers.
      Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.
    DOI:  https://doi.org/10.1038/s41467-022-30196-9
  35. Sci Immunol. 2022 May 06. 7(71): eabq7244
    Chat-ty B cells talk down hematopoiesis.
    Sapheya H Elhadi, Emily R Siniscalco, Stephanie C Eisenbarth.
      B cells produce acetylcholine that is sensed by bone marrow stromal cells and reduces hematopoiesis.
    DOI:  https://doi.org/10.1126/sciimmunol.abq7244
  36. Nat Commun. 2022 May 06. 13(1): 2493
    Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer.
    Aitor Almanza, Katarzyna Mnich, Arnaud Blomme, Claire M Robinson, Giovanny Rodriguez-Blanco, Sylwia Kierszniowska, Eoghan P McGrath, Matthieu Le Gallo, Eleftherios Pilalis, Johannes V Swinnen, Aristotelis Chatziioannou, Eric Chevet, Adrienne M Gorman, Afshin Samali.
      IRE1α is constitutively active in several cancers and can contribute to cancer progression. Activated IRE1α cleaves XBP1 mRNA, a key step in production of the transcription factor XBP1s. In addition, IRE1α cleaves select mRNAs through regulated IRE1α-dependent decay (RIDD). Accumulating evidence implicates IRE1α in the regulation of lipid metabolism. However, the roles of XBP1s and RIDD in this process remain ill-defined. In this study, transcriptome and lipidome profiling of triple negative breast cancer cells subjected to pharmacological inhibition of IRE1α reveals changes in lipid metabolism genes associated with accumulation of triacylglycerols (TAGs). We identify DGAT2 mRNA, encoding the rate-limiting enzyme in TAG biosynthesis, as a RIDD target. Inhibition of IRE1α, leads to DGAT2-dependent accumulation of TAGs in lipid droplets and sensitizes cells to nutritional stress, which is rescued by treatment with the DGAT2 inhibitor PF-06424439. Our results highlight the importance of IRE1α RIDD activity in reprograming cellular lipid metabolism.
    DOI:  https://doi.org/10.1038/s41467-022-30159-0
  37. Nat Commun. 2022 May 06. 13(1): 2496
    Universal non-Hermitian skin effect in two and higher dimensions.
    Kai Zhang, Zhesen Yang, Chen Fang.
      Skin effect, experimentally discovered in one dimension, describes the physical phenomenon that on an open chain, an extensive number of eigenstates of a non-Hermitian Hamiltonian are localized at the end(s) of the chain. Here in two and higher dimensions, we establish a theorem that the skin effect exists, if and only if periodic-boundary spectrum of the Hamiltonian covers a finite area on the complex plane. This theorem establishes the universality of the effect, because the above condition is satisfied in almost every generic non-Hermitian Hamiltonian, and, unlike in one dimension, is compatible with all point-group symmetries. We propose two new types of skin effect in two and higher dimensions: the corner-skin effect where all eigenstates are localized at corners of the system, and the geometry-dependent-skin effect where skin modes disappear for systems of a particular shape, but appear on generic polygons. An immediate corollary of our theorem is that any non-Hermitian system having exceptional points (lines) in two (three) dimensions exhibits skin effect, making this phenomenon accessible to experiments in photonic crystals, Weyl semimetals, and Kondo insulators.
    DOI:  https://doi.org/10.1038/s41467-022-30161-6
  38. Nat Commun. 2022 May 03. 13(1): 2403
    A reversible mitochondrial complex I thiol switch mediates hypoxic avoidance behavior in C. elegans.
    John O Onukwufor, M Arsalan Farooqi, Anežka Vodičková, Shon A Koren, Aksana Baldzizhar, Brandon J Berry, Gisela Beutner, George A Porter, Vsevolod Belousov, Alan Grossfield, Andrew P Wojtovich.
      C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.
    DOI:  https://doi.org/10.1038/s41467-022-30169-y
  39. Nat Cell Biol. 2022 May 05.
    Adiponectin receptors sustain haematopoietic stem cells throughout adulthood by protecting them from inflammation.
    Corbin E Meacham, Elise C Jeffery, Rebecca J Burgess, Charukesi D Sivakumar, Madison A Arora, Anne Marie Stanley, Emily M Colby, Genevieve M Crane, Zhiyu Zhao, Sean J Morrison.
      How are haematopoietic stem cells (HSCs) protected from inflammation, which increases with age and can deplete HSCs? Adiponectin, an anti-inflammatory factor that is not required for HSC function or haematopoiesis, promotes stem/progenitor cell proliferation after bacterial infection and myeloablation. Adiponectin binds two receptors, AdipoR1 and AdipoR2, which have ceramidase activity that increases upon adiponectin binding. Here we found that adiponectin receptors are non-cell-autonomously required in haematopoietic cells to promote HSC quiescence and self-renewal. Adiponectin receptor signalling suppresses inflammatory cytokine expression by myeloid cells and T cells, including interferon-γ and tumour necrosis factor. Without adiponectin receptors, the levels of these factors increase, chronically activating HSCs, reducing their self-renewal potential and depleting them during ageing. Pathogen infection accelerates this loss of HSC self-renewal potential. Blocking interferon-γ or tumour necrosis factor signalling partially rescues these effects. Adiponectin receptors are thus required in immune cells to sustain HSC quiescence and to prevent premature HSC depletion by reducing inflammation.
    DOI:  https://doi.org/10.1038/s41556-022-00909-9
  40. Science. 2022 May 05. e
    Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.
    Victoria Acosta-Rodríguez, Filipa Rijo-Ferreira, Mariko Izumo, Pin Xu, Mary Wight-Carter, Carla B Green, Joseph S Takahashi.
      Caloric restriction (CR) prolongs lifespan, yet the mechanisms by which it does so remain poorly understood. Under CR, mice self-impose chronic cycles of 2-hour-feeding and 22-hour-fasting, raising the question whether calories, fasting, or time of day are causal. We show that 30%-CR is sufficient to extend lifespan 10%; however, a daily fasting interval and circadian-alignment of feeding act together to extend lifespan 35% in male C57BL/6J mice. These effects are independent of body weight. Aging induces widespread increases in gene expression associated with inflammation and decreases in expression of genes encoding components of metabolic pathways in liver from ad lib fed mice. CR at night ameliorates these aging-related changes. Thus, circadian interventions promote longevity and provide a perspective to further explore mechanisms of aging.
    DOI:  https://doi.org/10.1126/science.abk0297
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