bims-crepig 2023-06-04 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2023–06–04
25 papers selected by
Connor Rogerson, University of Cambridge

Multidimensional profiling reveals GATA1-modulated stage-specific chromatin states and functional associations during human erythropoiesis.
Multiomic analysis of cohesin reveals that ZBTB transcription factors contribute to chromatin interactions.
MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.
Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus.
Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis.
EWSR1 maintains centromere identity.
Inferring cell-type-specific causal gene regulatory networks during human neurogenesis.
ZBTB2 is recruited to a specific subset of HIF-1 target loci to facilitate full gene expression under hypoxia.
Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency.
macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.
PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.
Theory of chromatin organization maintained by active loop extrusion.
Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/β-catenin to induce exit from quiescence.
Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets.
Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions.
Light-switchable transcription factors obtained by direct screening in mammalian cells.
Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.
Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer.
The landscape of tolerated genetic variation in humans and primates.
LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.
SIMBA: single-cell embedding along with features.
Engineering of the endogenous HBD promoter increases HbA2.
A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila.
Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation.
Pyruvate metabolism controls chromatin remodeling during CD4+ T cell activation.

Nucleic Acids Res. 2023 May 31. pii: gkad468. [Epub ahead of print]

Multidimensional profiling reveals GATA1-modulated stage-specific chromatin states and functional associations during human erythropoiesis.

Dong Li, Xin-Ying Zhao, Shuo Zhou, Qi Hu, Fan Wu, Hsiang-Ying Lee.

Mammalian erythroid development can be divided into three stages: hematopoietic stem and progenitor cell (HSPC), erythroid progenitor (Ery-Pro), and erythroid precursor (Ery-Pre). However, the mechanisms by which the 3D genome changes to establish the stage-specific transcription programs that are critical for erythropoiesis remain unclear. Here, we analyze the chromatin landscape at multiple levels in defined populations from primary human erythroid culture. While compartments and topologically associating domains remain largely unchanged, ∼50% of H3K27Ac-marked enhancers are dynamic in HSPC versus Ery-Pre. The enhancer anchors of enhancer-promoter loops are enriched for occupancy of respective stage-specific transcription factors (TFs), indicating these TFs orchestrate the enhancer connectome rewiring. The master TF of erythropoiesis, GATA1, is found to occupy most erythroid gene promoters at the Ery-Pro stage, and mediate conspicuous local rewiring through acquiring binding at the distal regions in Ery-Pre, promoting productive erythroid transcription output. Knocking out GATA1 binding sites precisely abrogates local rewiring and corresponding gene expression. Interestingly, knocking down GATA1 can transiently revert the cell state to an earlier stage and prolong the window of progenitor state. This study reveals mechanistic insights underlying chromatin rearrangements during development by integrating multidimensional chromatin landscape analyses to associate with transcription output and cellular states.

DOI: https://doi.org/10.1093/nar/gkad468
Nucleic Acids Res. 2023 Jun 02. pii: gkad401. [Epub ahead of print]

Multiomic analysis of cohesin reveals that ZBTB transcription factors contribute to chromatin interactions.

Rui Wang, Qiqin Xu, Chenlu Wang, Kai Tian, Hui Wang, Xiong Ji.

One bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed proteomic profiling of the cohesin in chromatin and identified transcription factors, RNA-binding proteins and chromatin regulators associated with cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 contributes to cohesin chromatin binding, 3D chromatin interactions and transcriptional repression. Strikingly, multiomic analyses revealed that the other four ZBTB factors interacted with cohesin, and double degradation of ZBTB21 and ZBTB7B led to a further decrease in cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of cohesin to regulate chromatin interactions, and we provide a catalog of many additional proteins associated with cohesin that warrant further investigation.

DOI: https://doi.org/10.1093/nar/gkad401
Elife. 2023 Jun 01. pii: e80854. [Epub ahead of print]12

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.

Changyu Zhu, Yadira M Soto-Feliciano, John P Morris, Chun-Hao Huang, Richard P Koche, Yu-Jui Ho, Ana Banito, Chun-Wei David Chen, Aditya Shroff, Sha Tian, Geulah Livshits, Chi-Chao Chen, Myles Fennell, Scott A Armstrong, C David Allis, Darjus F Tschaharganeh, Scott W Lowe.

  Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

Keywords:  cancer biology; genetics; genomics; human; mouse

DOI:  https://doi.org/10.7554/eLife.80854
Cell Rep. 2023 May 30. pii: S2211-1247(23)00580-6. [Epub ahead of print]42(6): 112569

Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus.

Noa Gil, Rotem Ben-Tov Perry, Zohar Mukamel, Alex Tuck, Marc Bühler, Igor Ulitsky.

  Long non-coding RNAs (lncRNAs) are implicated in a plethora of cellular processes, but an in-depth understanding of their functional features or their mechanisms of action is currently lacking. Here we study Meteor, a lncRNA transcribed near the gene encoding EOMES, a pleiotropic transcription factor implicated in various processes throughout development and in adult tissues. Using a wide array of perturbation techniques, we show that transcription elongation through the Meteor locus is required for Eomes activation in mouse embryonic stem cells, with Meteor repression linked to a change in the subpopulation primed to differentiate to the mesoderm lineage. We further demonstrate that a distinct functional feature of the locus-namely, the underlying DNA element-is required for suppressing Eomes expression following neuronal differentiation. Our results demonstrate the complex regulation that can be conferred by a single locus and emphasize the importance of careful selection of perturbation techniques when studying lncRNA loci.

Keywords:  CP: Developmental biology; CP: Molecular biology; long non-coding RNAs, lncRNAs, embryonic stem cells, pluripotency, differentiation, EOMES

DOI:  https://doi.org/10.1016/j.celrep.2023.112569
Genome Biol. 2023 May 30. 24(1): 129

Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis.

Christian Kappel, Thomas Friedrich, Vicky Oberkofler, Li Jiang, Tim Crawford, Michael Lenhard, Isabel Bäurle.

   BACKGROUND: Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq.
RESULTS: HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes.
CONCLUSIONS: Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.

Keywords:  Arabidopsis thaliana; ChIP-seq; HSFA2; HSFA3; Heat stress; Histone H3K4 trimethylation; Priming; Transcriptional memory

DOI:  https://doi.org/10.1186/s13059-023-02970-5
Cell Rep. 2023 May 25. pii: S2211-1247(23)00579-X. [Epub ahead of print]42(6): 112568

EWSR1 maintains centromere identity.

Risa Kitagawa, Yohei Niikura, Argentina Becker, Peter J Houghton, Katsumi Kitagawa.

  The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.

Keywords:  CENP-A; CENP-A maintenance; CP: Molecular biology; EWSR1; EWSR1-FLI1; Ewing sarcoma; Ewing sarcoma breakpoint region 1; Ewing sarcoma oncogenic fusion protein; centromere; centromere identity; kinetochore; phase separation

DOI:  https://doi.org/10.1016/j.celrep.2023.112568
Genome Biol. 2023 May 30. 24(1): 130

Inferring cell-type-specific causal gene regulatory networks during human neurogenesis.

Nil Aygün, Dan Liang, Wesley L Crouse, Gregory R Keele, Michael I Love, Jason L Stein.

   BACKGROUND: Genetic variation influences both chromatin accessibility, assessed in chromatin accessibility quantitative trait loci (caQTL) studies, and gene expression, assessed in expression QTL (eQTL) studies. Genetic variants can impact either nearby genes (cis-eQTLs) or distal genes (trans-eQTLs). Colocalization between caQTL and eQTL, or cis- and trans-eQTLs suggests that they share causal variants. However, pairwise colocalization between these molecular QTLs does not guarantee a causal relationship. Mediation analysis can be applied to assess the evidence supporting causality versus independence between molecular QTLs. Given that the function of QTLs can be cell-type-specific, we performed mediation analyses to find epigenetic and distal regulatory causal pathways for genes within two major cell types of the developing human cortex, progenitors and neurons.
RESULTS: We find that the expression of 168 and 38 genes is mediated by chromatin accessibility in progenitors and neurons, respectively. We also find that the expression of 11 and 12 downstream genes is mediated by upstream genes in progenitors and neurons. Moreover, we discover that a genetic locus associated with inter-individual differences in brain structure shows evidence for mediation of SLC26A7 through chromatin accessibility, identifying molecular mechanisms of a common variant association to a brain trait.
CONCLUSIONS: In this study, we identify cell-type-specific causal gene regulatory networks whereby the impacts of variants on gene expression were mediated by chromatin accessibility or distal gene expression. Identification of these causal paths will enable identifying and prioritizing actionable regulatory targets perturbing these key processes during neurodevelopment.

Keywords:  Causal inference; Cis- and trans-regulation; Gene regulatory networks; Neurogenesis; Quantitative trait loci

DOI:  https://doi.org/10.1186/s13059-023-02959-0
J Mol Biol. 2023 May 29. pii: S0022-2836(23)00249-8. [Epub ahead of print] 168162

ZBTB2 is recruited to a specific subset of HIF-1 target loci to facilitate full gene expression under hypoxia.

Christalle Ct Chow, Minoru Kobayashi, Gouki Kambe, Hiroshi Harada.

  The cellular response to hypoxia is mainly governed by a transcription factor, hypoxia-inducible factor 1 (HIF-1). Although upregulation of HIF-1 target genes has been hypothesized to require interaction of HIF-1 with other coactivators, much remains to be elucidated regarding the underlying mechanisms. Here, we demonstrate that zinc finger and BTB domain-containing protein 2 (ZBTB2) enhances the expression of certain HIF-1 target genes under hypoxia. ChIP-Seq analysis showed that there is a subset of HIF-1 target genes with overlapping HIF-1 and ZBTB2 peaks. Examination of a representative gene, EGFR antisense RNA 1 (EGFR-AS1), showed that HIF-1 binding to the consensus hypoxia-responsive element (HRE) sequence resulted in the recruitment of ZBTB2 to the gene locus and increased p300-mediated histone acetylation, leading to enhanced gene expression under hypoxia. In contrast, expression of HIF-1 target genes lacking ZBTB2 peaks, such as carbonic anhydrase 9 (CA9), was not upregulated by ZBTB2. These findings demonstrate that ZBTB2 is a novel factor that can be recruited to the vicinity of HREs on a subset of HIF-1 target gene loci, and is required for their full expression under hypoxia.

Keywords:  ZBTB2; gene regulation; gene subset selection; hypoxia; hypoxia-inducible factor 1 (HIF-1)

DOI:  https://doi.org/10.1016/j.jmb.2023.168162
Cell Rep. 2023 May 30. pii: S2211-1247(23)00582-X. [Epub ahead of print]42(6): 112571

Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency.

Christopher B Mahony, Lucy Copper, Pavle Vrljicak, Boris Noyvert, Chrystala Constantinidou, Sofia Browne, Yi Pan, Claire Palles, Sascha Ott, Martin R Higgs, Rui Monteiro.

  Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.

Keywords:  CP: Molecular biology; CP: Stem cell research; DNA damage; GATA2 deficiency; hematopoietic stem cells; single-cell genomics; zebrafish

DOI:  https://doi.org/10.1016/j.celrep.2023.112571
Nat Commun. 2023 May 27. 14(1): 3062

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Ana Nikolic, Francesca Maule, Anna Bobyn, Katrina Ellestad, Seungil Paik, Sajid A Marhon, Parinaz Mehdipour, Xueqing Lun, Huey-Miin Chen, Claire Mallard, Alexander J Hay, Michael J Johnston, Christopher J Gafuik, Franz J Zemp, Yaoqing Shen, Nicoletta Ninkovic, Katalin Osz, Elodie Labit, N Daniel Berger, Duncan K Brownsey, John J Kelly, Jeff Biernaskie, Peter B Dirks, Darren J Derksen, Steven J M Jones, Donna L Senger, Jennifer A Chan, Douglas J Mahoney, Daniel D De Carvalho, Marco Gallo.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

DOI: https://doi.org/10.1038/s41467-023-38919-2
Proc Natl Acad Sci U S A. 2023 Jun 06. 120(23): e2220528120

PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

Wayne O Hemphill, Regan Fenske, Anne R Gooding, Thomas R Cech.

  The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, many in vitro studies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while some in vivo studies indicate that PRC2's RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2's RNA and DNA-binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between nucleic acid ligands without a free-enzyme intermediate. Direct transfer explains the variation in previously reported dissociation kinetics, allows reconciliation of prior in vitro and in vivo studies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.

Keywords:  displacement; exchange; methyltransferase; nucleosomes; polynucleotide

DOI:  https://doi.org/10.1073/pnas.2220528120
Proc Natl Acad Sci U S A. 2023 Jun 06. 120(23): e2222078120

Theory of chromatin organization maintained by active loop extrusion.

Brian Chan, Michael Rubinstein.

  The active loop extrusion hypothesis proposes that chromatin threads through the cohesin protein complex into progressively larger loops until reaching specific boundary elements. We build upon this hypothesis and develop an analytical theory for active loop extrusion which predicts that loop formation probability is a nonmonotonic function of loop length and describes chromatin contact probabilities. We validate our model with Monte Carlo and hybrid Molecular Dynamics-Monte Carlo simulations and demonstrate that our theory recapitulates experimental chromatin conformation capture data. Our results support active loop extrusion as a mechanism for chromatin organization and provide an analytical description of chromatin organization that may be used to specifically modify chromatin contact probabilities.

Keywords:  chromatin organization; loop extrusion; polymer physics

DOI:  https://doi.org/10.1073/pnas.2222078120
Cell Rep. 2023 May 26. pii: S2211-1247(23)00572-7. [Epub ahead of print]42(6): 112561

Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/β-catenin to induce exit from quiescence.

Faye L Robertson, Eoghan O'Duibhir, Ester Gangoso, Raul Bardini Bressan, Harry Bulstrode, Maria-Ángeles Marqués-Torrejón, Kirsty M Ferguson, Carla Blin, Vivien Grant, Neza Alfazema, Gillian M Morrison, Steven M Pollard.

  Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/β-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional β-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

Keywords:  CP: Cancer; CP: Stem cell research; FOXG1; GSK3 inhibitor; Wnt signaling; cell cycle; glioblastoma; neural stem cell; quiescence; β-catenin

DOI:  https://doi.org/10.1016/j.celrep.2023.112561
Nat Commun. 2023 May 27. 14(1): 3064

Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets.

Shilu Zhang, Saptarshi Pyne, Stefan Pietrzak, Spencer Halberg, Sunnie Grace McCalla, Alireza Fotuhi Siahpirani, Rupa Sridharan, Sushmita Roy.

Cell type-specific gene expression patterns are outputs of transcriptional gene regulatory networks (GRNs) that connect transcription factors and signaling proteins to target genes. Single-cell technologies such as single cell RNA-sequencing (scRNA-seq) and single cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), can examine cell-type specific gene regulation at unprecedented detail. However, current approaches to infer cell type-specific GRNs are limited in their ability to integrate scRNA-seq and scATAC-seq measurements and to model network dynamics on a cell lineage. To address this challenge, we have developed single-cell Multi-Task Network Inference (scMTNI), a multi-task learning framework to infer the GRN for each cell type on a lineage from scRNA-seq and scATAC-seq data. Using simulated and real datasets, we show that scMTNI is a broadly applicable framework for linear and branching lineages that accurately infers GRN dynamics and identifies key regulators of fate transitions for diverse processes such as cellular reprogramming and differentiation.

DOI: https://doi.org/10.1038/s41467-023-38637-9
EMBO Rep. 2023 May 31. e56404

Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions.

Emily H Zion, Daniel Ringwalt, Kristina Rinaldi, Elizabeth W Kahney, Yingying Li, Xin Chen.

  We report that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, we expressed a mutant H3T3A to compromise asymmetric histone inheritance. Under this condition, we observe an increase in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, our results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells.

Keywords:  asymmetric cell division; differentiation; epigenetic inheritance; histone; stem cells

DOI:  https://doi.org/10.15252/embr.202256404
Nat Commun. 2023 Jun 02. 14(1): 3185

Light-switchable transcription factors obtained by direct screening in mammalian cells.

Liyuan Zhu, Harold M McNamara, Jared E Toettcher.

Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

DOI: https://doi.org/10.1038/s41467-023-38993-6
Nat Commun. 2023 May 29. 14(1): 3076

Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Jose M Ramos-Pittol, Isabel Fernandes-Freitas, Alexandra Milona, Stephen M Manchishi, Kara Rainbow, Brian Y H Lam, John A Tadross, Anthony Beucher, William H Colledge, Inês Cebola, Kevin G Murphy, Irene Miguel-Aliaga, Giles S H Yeo, Waljit S Dhillo, Bryn M Owen.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

DOI: https://doi.org/10.1038/s41467-023-38618-y
EMBO J. 2023 Jun 01. e112559

Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer.

Joana Pinto Couto, Milica Vulin, Charly Jehanno, Marie-May Coissieux, Baptiste Hamelin, Alexander Schmidt, Robert Ivanek, Atul Sethi, Konstantin Bräutigam, Anja L Frei, Carolina Hager, Madhuri Manivannan, Jorge Gómez-Miragaya, Milan Ms Obradović, Zsuzsanna Varga, Viktor H Koelzer, Kirsten D Mertz, Mohamed Bentires-Alj.

  Metastatic colonization of distant organs accounts for over 90% of deaths related to solid cancers, yet the molecular determinants of metastasis remain poorly understood. Here, we unveil a mechanism of colonization in the aggressive basal-like subtype of breast cancer that is driven by the NAD+ metabolic enzyme nicotinamide N-methyltransferase (NNMT). We demonstrate that NNMT imprints a basal genetic program into cancer cells, enhancing their plasticity. In line, NNMT expression is associated with poor clinical outcomes in patients with breast cancer. Accordingly, ablation of NNMT dramatically suppresses metastasis formation in pre-clinical mouse models. Mechanistically, NNMT depletion results in a methyl overflow that increases histone H3K9 trimethylation (H3K9me3) and DNA methylation at the promoters of PR/SET Domain-5 (PRDM5) and extracellular matrix-related genes. PRDM5 emerged in this study as a pro-metastatic gene acting via induction of cancer-cell intrinsic transcription of collagens. Depletion of PRDM5 in tumor cells decreases COL1A1 deposition and impairs metastatic colonization of the lungs. These findings reveal a critical activity of the NNMT-PRDM5-COL1A1 axis for cancer cell plasticity and metastasis in basal-like breast cancer.

Keywords:  NNMT; breast cancer; collagen; colonization; metastasis

DOI:  https://doi.org/10.15252/embj.2022112559
Science. 2023 Jun 02. 380(6648): eabn8153

The landscape of tolerated genetic variation in humans and primates.

Hong Gao, Tobias Hamp, Jeffrey Ede, Joshua G Schraiber, Jeremy McRae, Moriel Singer-Berk, Yanshen Yang, Anastasia S D Dietrich, Petko P Fiziev, Lukas F K Kuderna, Laksshman Sundaram, Yibing Wu, Aashish Adhikari, Yair Field, Chen Chen, Serafim Batzoglou, Francois Aguet, Gabrielle Lemire, Rebecca Reimers, Daniel Balick, Mareike C Janiak, Martin Kuhlwilm, Joseph D Orkin, Shivakumara Manu, Alejandro Valenzuela, Juraj Bergman, Marjolaine Rousselle, Felipe Ennes Silva, Lidia Agueda, Julie Blanc, Marta Gut, Dorien de Vries, Ian Goodhead, R Alan Harris, Muthuswamy Raveendran, Axel Jensen, Idriss S Chuma, Julie E Horvath, Christina Hvilsom, David Juan, Peter Frandsen, Fabiano R de Melo, Fabrício Bertuol, Hazel Byrne, Iracilda Sampaio, Izeni Farias, João Valsecchi do Amaral, Mariluce Messias, Maria N F da Silva, Mihir Trivedi, Rogerio Rossi, Tomas Hrbek, Nicole Andriaholinirina, Clément J Rabarivola, Alphonse Zaramody, Clifford J Jolly, Jane Phillips-Conroy, Gregory Wilkerson, Christian Abee, Joe H Simmons, Eduardo Fernandez-Duque, Sree Kanthaswamy, Fekadu Shiferaw, Dongdong Wu, Long Zhou, Yong Shao, Guojie Zhang, Julius D Keyyu, Sascha Knauf, Minh D Le, Esther Lizano, Stefan Merker, Arcadi Navarro, Thomas Bataillon, Tilo Nadler, Chiea Chuen Khor, Jessica Lee, Patrick Tan, Weng Khong Lim, Andrew C Kitchener, Dietmar Zinner, Ivo Gut, Amanda Melin, Katerina Guschanski, Mikkel Heide Schierup, Robin M D Beck, Govindhaswamy Umapathy, Christian Roos, Jean P Boubli, Monkol Lek, Shamil Sunyaev, Anne O'Donnell-Luria, Heidi L Rehm, Jinbo Xu, Jeffrey Rogers, Tomas Marques-Bonet, Kyle Kai-How Farh.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole-genome sequencing data for 809 individuals from 233 primate species and identified 4.3 million common protein-altering variants with orthologs in humans. We show that these variants can be inferred to have nondeleterious effects in humans based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases.

DOI: https://doi.org/10.1126/science.abn8197
iScience. 2023 Jun 16. 26(6): 106880

LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.

Ulla Dolde, Fernando Muzzopappa, Charlotte Delesalle, Julie Neveu, Fabian Erdel, Grégory Vert.

  The transcription factor LEAFY (LFY) plays crucial roles in flower development by activating floral homeotic genes. Activation of LFY targets requires the combined action of LFY and the E3 ubiquitin ligase UFO, although the precise underlying mechanism remains unclear. Here, we show that LFY accumulates in biomolecular condensates within the cytoplasm, while recombinant LFY forms condensates with similar properties in vitro. UFO interacts with LFY within these condensates and marks it for degradation. LFY levels in the nucleus are buffered against changes in total LFY levels induced by proteasome inhibition, UFO overexpression, or mutation of lysine residues in a disordered region of LFY. Perturbation of cytoplasmic LFY condensates by 1,6-hexanediol treatment induces the relocalization of LFY to the nucleus and the subsequent activation of the LFY target AP3 in flowers. Our data suggest that nucleocytoplasmic partitioning, condensation, and ubiquitin-dependent degradation regulate LFY levels in the nucleus to control its activity.

Keywords:  Cell biology; Plant biochemistry; Plant development

DOI:  https://doi.org/10.1016/j.isci.2023.106880
Nat Methods. 2023 May 29.

SIMBA: single-cell embedding along with features.

Huidong Chen, Jayoung Ryu, Michael E Vinyard, Adam Lerer, Luca Pinello.

Most current single-cell analysis pipelines are limited to cell embeddings and rely heavily on clustering, while lacking the ability to explicitly model interactions between different feature types. Furthermore, these methods are tailored to specific tasks, as distinct single-cell problems are formulated differently. To address these shortcomings, here we present SIMBA, a graph embedding method that jointly embeds single cells and their defining features, such as genes, chromatin-accessible regions and DNA sequences, into a common latent space. By leveraging the co-embedding of cells and features, SIMBA allows for the study of cellular heterogeneity, clustering-free marker discovery, gene regulation inference, batch effect removal and omics data integration. We show that SIMBA provides a single framework that allows diverse single-cell problems to be formulated in a unified way and thus simplifies the development of new analyses and extension to new single-cell modalities. SIMBA is implemented as a comprehensive Python library ( https://simba-bio.readthedocs.io ).

DOI: https://doi.org/10.1038/s41592-023-01899-8
Elife. 2023 Jun 02. pii: e85258. [Epub ahead of print]12

Engineering of the endogenous HBD promoter increases HbA2.

Mandy Y Boontanrart, Elia Mächler, Simone Ponta, Jan C Nelis, Viviana G Preiano, Jacob E Corn.

  The b-hemoglobinopathies, such as sickle cell disease and b-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.

Keywords:  genetics; genomics; human

DOI:  https://doi.org/10.7554/eLife.85258
PLoS Genet. 2023 Jun 02. 19(6): e1010792

A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila.

Alexander G Teague, Maria Quintero, Fateme Karimi Dermani, Ross L Cagan, Erdem Bangi.

Experimental models that capture the genetic complexity of human disease and allow mechanistic explorations of the underlying cell, tissue, and organ interactions are crucial to furthering our understanding of disease biology. Such models require combinatorial manipulations of multiple genes, often in more than one tissue at once. The ability to perform complex genetic manipulations in vivo is a key strength of Drosophila, where many tools for sophisticated and orthogonal genetic perturbations exist. However, combining the large number of transgenes required to establish more representative disease models and conducting mechanistic studies in these already complex genetic backgrounds is challenging. Here we present a design that pushes the limits of Drosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene. The polycistronic transcript encoded by this transgene includes a synthetic short hairpin cluster cloned within an intron placed at the 5' end of the transcript, followed by two protein-coding sequences separated by the T2A sequence that mediates ribosome skipping. This technology is particularly useful for modeling genetically complex diseases like cancer, which typically involve concurrent activation of multiple oncogenes and loss of multiple tumor suppressors. Furthermore, consolidating multiple genetic perturbations into a single transgene further streamlines the ability to perform combinatorial genetic manipulations and makes it readily adaptable to a broad palette of transgenic systems. This flexible design for combinatorial genetic perturbations will also be a valuable tool for functionally exploring multigenic gene signatures identified from omics studies of human disease and creating humanized Drosophila models to characterize disease-associated variants in human genes. It can also be adapted for studying biological processes underlying normal tissue homeostasis and development that require simultaneous manipulation of many genes.

DOI: https://doi.org/10.1371/journal.pgen.1010792
Cell Rep. 2023 Jun 01. pii: S2211-1247(23)00589-2. [Epub ahead of print]42(6): 112578

Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation.

Sinthu Pathmanapan, Raymond Poon, Tomasa Barrientos De Renshaw, Puviindran Nadesan, Makoto Nakagawa, Gireesh A Seesankar, Adrian Kwan Ho Loe, Hongyuan H Zhang, Joan J Guinovart, Jordi Duran, Christopher B Newgard, Jay S Wunder, Benjamin A Alman.

  Chondrosarcomas are the most common malignancy of cartilage and are associated with somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes. Somatic IDH mutations are also found in its benign precursor lesion, enchondromas, suggesting that IDH mutations are early events in malignant transformation. Human mutant IDH chondrosarcomas and mutant Idh mice that develop enchondromas investigated in our studies display glycogen deposition exclusively in mutant cells from IDH mutant chondrosarcomas and Idh1 mutant murine growth plates. Pharmacologic blockade of glycogen utilization induces changes in tumor cell behavior, downstream energetic pathways, and tumor burden in vitro and in vivo. Mutant IDH1 interacts with hypoxia-inducible factor 1α (HIF1α) to regulate expression of key enzymes in glycogen metabolism. Here, we show a critical role for glycogen in enchondromas and chondrosarcomas, which is likely mediated through an interaction with mutant IDH1 and HIF1α.

Keywords:  CP: Cancer; CP: Metabolism; cancer; chondrosarcoma; development; genetic mutation; glycogen; metabolism; mutant IDH

DOI:  https://doi.org/10.1016/j.celrep.2023.112578
Cell Rep. 2023 Jun 01. pii: S2211-1247(23)00594-6. [Epub ahead of print]42(6): 112583

Pyruvate metabolism controls chromatin remodeling during CD4+ T cell activation.

Enric Mocholi, Laura Russo, Keshav Gopal, Andrew G Ramstead, Sophia M Hochrein, Harmjan R Vos, Geert Geeven, Adeolu O Adegoke, Anna Hoekstra, Robert M van Es, Jose Ramos Pittol, Sebastian Vastert, Jared Rutter, Timothy Radstake, Jorg van Loosdregt, Celia Berkers, Michal Mokry, Colin C Anderson, Ryan M O'Connell, Martin Vaeth, John Ussher, Boudewijn M T Burgering, Paul J Coffer.

  Upon antigen-specific T cell receptor (TCR) engagement, human CD4+ T cells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient T cells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, T cell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ T cell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven T cell activation.

Keywords:  CP: Metabolism; T cell; citrate; epigenetics; epigenome remodeling; glucose metabolism; glycolysis; histone acetylation; nuclear metabolism; pyruvate; pyruvate dehydrogenase

DOI:  https://doi.org/10.1016/j.celrep.2023.112583