bims-crepig 2022-03-20 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2022–03–20
27 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells.
A network of transcription factors governs the dynamics of NODAL/Activin transcriptional responses.
DeepCAGE: Incorporating transcription factors in genome-wide prediction of chromatin accessibility.
Hemogen /BRG1 cooperativity modulates promoter and enhancer activation during erythropoiesis.
Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia.
The PAF1 complex promotes 3' processing of pervasive transcripts.
The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.
Ten-Eleven Translocation Ablation Impairs Cardiac Differentiation of Mouse Embryonic Stem Cells.
A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia.
Arid1a regulates bladder urothelium formation and maintenance.
Systematic reconstruction of cellular trajectories across mouse embryogenesis.
m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome.
HMGA1 Chromatin Regulators Induce Transcriptional Networks Involved in GATA2 and Proliferation During MPN Progression.
Nup98-dependent transcriptional memory is established independently of transcription.
Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor.
Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.
A shared ancient enhancer element differentially regulates the bric-a-brac tandem gene duplicates in the developing Drosophila leg.
The TFIIH complex is required to establish and maintain mitotic chromosome structure.
Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting.
High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.
Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains.
The Oligodendrocyte Transcription Factor 2 OLIG2 regulates transcriptional repression during myelinogenesis in rodents.
HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.
Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease.
PCR-based profiling of transcription factor activity in vivo by a virus-based reporter battery.
FOXO1 represses Sprouty2 and Sprouty4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.
LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Genome Biol. 2022 Mar 17. 23(1): 81

CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells.

Derek H Janssens, Dominik J Otto, Michael P Meers, Manu Setty, Kami Ahmad, Steven Henikoff.

  Cleavage Under Targets and Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers, and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

Keywords:  Chromatin profiling; Epigenomic signal deconvolution; Multi-omics; Single-cell genomics

DOI:  https://doi.org/10.1186/s13059-022-02642-w
J Cell Sci. 2022 Mar 18. pii: jcs.259972. [Epub ahead of print]

A network of transcription factors governs the dynamics of NODAL/Activin transcriptional responses.

Davide M Coda, Harshil Patel, Ilaria Gori, Tessa E Gaarenstroom, Ok-Ryul Song, Michael Howell, Caroline S Hill.

  SMAD2, an effector of the NODAL/Activin signalling pathway, regulates developmental processes by sensing distinct chromatin states and interacting with different transcriptional partners. However, the network of factors that controls SMAD2 chromatin binding and shapes its transcriptional programme over time is poorly characterised. Here we combine ATAC-seq with computational footprinting to identify temporal changes in chromatin accessibility and transcription factor activity upon NODAL/Activin signalling. We show that SMAD2 binding induces chromatin opening genome wide. We discover footprints for FOXI3/FOXO3 and ZIC3 at the SMAD2-bound enhancers of the early response genes, Pmepa1 and Wnt3 respectively, and demonstrate their functionality. Finally, we determine a mechanism by which NODAL/Activin signalling induces delayed gene expression, by uncovering a self-enabling transcriptional cascade whereby activated SMADs, together with ZIC3, induce the expression of Wnt3. The resultant activated WNT pathway then acts together with the NODAL/Activin pathway to regulate expression of delayed target genes in prolonged NODAL/Activin signalling conditions.

Keywords:  ATAC-seq; FOXI3; NODAL/Activin; SMAD; Transcription; ZIC3

DOI:  https://doi.org/10.1242/jcs.259972
Genomics Proteomics Bioinformatics. 2022 Mar 12. pii: S1672-0229(22)00024-9. [Epub ahead of print]

DeepCAGE: Incorporating transcription factors in genome-wide prediction of chromatin accessibility.

Qiao Liu, Kui Hua, Xuegong Zhang, Wing Hung Wong, Rui Jiang.

  Although computational approaches have been complementing high-throughput biological experiments for the identification of functional regions in the human genome, it remains a great challenge to systematically decipher interactions between transcription factors and regulatory elements to achieve interpretable annotations of chromatin accessibility across diverse cellular contexts. To solve this problem, we propose DeepCAGE, a deep learning framework that integrates sequence information and binding status of transcription factors, for the accurate prediction of chromatin accessible regions at a genome-wide scale in a variety of cell types. DeepCAGE takes advantage of a densely connected deep convolutional neural network architecture to automatically learn sequence signatures of known chromatin accessible regions and then incorporates such features with expression levels and binding activities of human core transcription factors to predict novel chromatin accessible regions. In a series of systematic comparisons with existing methods, DeepCAGE exhibits superior performance in not only the classification but also the regression of chromatin accessibility signals. In a detailed analysis of transcription factor activities, DeepCAGE successfully extracts novel binding motifs and measures the contribution of a transcription factor to the regulation with respect to a specific locus in a certain cell type. When applied to whole-genome sequencing data analysis, our method successfully prioritizes putative deleterious variants underlying a human complex trait and thus provides insights into the understanding of disease-associated genetic variants. DeepCAGE can be downloaded from https://github.com/kimmo1019/DeepCAGE.

Keywords:  Chromatin accessibility; Deep learning; Gene expression; Transcription factor

DOI:  https://doi.org/10.1016/j.gpb.2021.08.015
Blood. 2022 Mar 16. pii: blood.2021014308. [Epub ahead of print]

Hemogen /BRG1 cooperativity modulates promoter and enhancer activation during erythropoiesis.

Xiang Guo, Ann Dean.

Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.

DOI: https://doi.org/10.1182/blood.2021014308
Nat Commun. 2022 Mar 17. 13(1): 1434

Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia.

Moritz Binder, Ryan M Carr, Terra L Lasho, Christy M Finke, Abhishek A Mangaonkar, Christopher L Pin, Kurt R Berger, Amelia Mazzone, Sandeep Potluri, Tamas Ordog, Keith D Robertson, David L Marks, Martin E Fernandez-Zapico, Alexandre Gaspar-Maia, Mrinal M Patnaik.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

DOI: https://doi.org/10.1038/s41467-022-29142-6
Cell Rep. 2022 Mar 15. pii: S2211-1247(22)00255-8. [Epub ahead of print]38(11): 110519

The PAF1 complex promotes 3' processing of pervasive transcripts.

Xinhong Liu, Ziwei Guo, Jing Han, Bo Peng, Bin Zhang, Haitao Li, Xiaoyu Hu, Charles J David, Mo Chen.

  The PAF1 complex (PAF1C) functions in multiple transcriptional processes involving RNA polymerase II (RNA Pol II). Enhancer RNAs (eRNAs) and promoter upstream transcripts (PROMPTs) are pervasive transcripts transcribed by RNA Pol II and degraded rapidly by the nuclear exosome complex after 3' endonucleolytic cleavage by the Integrator complex (Integrator). Here we show that PAF1C has a role in termination of eRNAs and PROMPTs that are cleaved 1-3 kb downstream of the transcription start site. Mechanistically, PAF1C facilitates recruitment of Integrator to sites of pervasive transcript cleavage, promoting timely cleavage and transcription termination. We also show that PAF1C recruits Integrator to coding genes, where PAF1C then dissociates from Integrator upon entry into processive elongation. Our results demonstrate a function of PAF1C in limiting the length and accumulation of pervasive transcripts that result from non-productive transcription.

Keywords:  3′ end processing; PAF1 complex; enhancer RNA; integrator complex; pervasive transcription; transcription regulation; transcription termination

DOI:  https://doi.org/10.1016/j.celrep.2022.110519
Cell Rep. 2022 Mar 15. pii: S2211-1247(22)00260-1. [Epub ahead of print]38(11): 110524

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.

Görkem Garipler, Congyi Lu, Alexis Morrissey, Lorena S Lopez-Zepeda, Yingzhen Pei, Simon E Vidal, Ana Paula Zen Petisco Fiore, Begüm Aydin, Matthias Stadtfeld, Uwe Ohler, Shaun Mahony, Neville E Sanjana, Esteban O Mazzoni.

  In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

Keywords:  CRISPR screen; differentiation; embryonic stem cells; pluripotency; poised genes; transcription factors

DOI:  https://doi.org/10.1016/j.celrep.2022.110524
Stem Cells. 2022 Mar 03. pii: sxab017. [Epub ahead of print]

Ten-Eleven Translocation Ablation Impairs Cardiac Differentiation of Mouse Embryonic Stem Cells.

Shaohai Fang, Dan Cui, Tingting Hong, Lei Guo, Yi-Tsang Lee, Minjung Lee, Sevinj Isgandarova, Margarita Martinez-Moczygemba, Yubin Zhou, Jia Li, Yun Huang.

Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.

DOI: https://doi.org/10.1093/stmcls/sxab017
Genes Dev. 2022 Mar 17.

A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia.

Taku Harada, Yaser Heshmati, Jérémie Kalfon, Monika W Perez, Juliana Xavier Ferrucio, Jazmin Ewers, Benjamin Hubbell Engler, Andrew Kossenkov, Jana M Ellegast, Joanna S Yi, Allyson Bowker, Qian Zhu, Kenneth Eagle, Tianxin Liu, Yan Kai, Joshua M Dempster, Guillaume Kugener, Jayamanna Wickramasinghe, Zachary T Herbert, Charles H Li, Jošt Vrabič Koren, David M Weinstock, Vikram R Paralkar, Behnam Nabet, Charles Y Lin, Neekesh V Dharia, Kimberly Stegmaier, Stuart H Orkin, Maxim Pimkin.

  Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.

Keywords:  IRF8; KMT2A-rearranged AML; MEF2D; transcriptional addiction

DOI:  https://doi.org/10.1101/gad.349284.121
Dev Biol. 2022 Mar 10. pii: S0012-1606(22)00030-6. [Epub ahead of print]485 61-69

Arid1a regulates bladder urothelium formation and maintenance.

Chunming Guo, Yingsheng Zhang, Ruirong Tan, Zonghao Tang, Christa M Lam, Xing Ye, Zhong Wang, Xue Li.

  Epigenetic regulation of gene expression plays a central role in bladder urothelium development and maintenance. ATPase-dependent chromatin remodeling is a major epigenetic regulatory mechanism, but its role in the bladder has not been explored. Here, we show the functions of Arid1a, the largest subunit of the SWI/SNF or BAF chromatin remodeling ATPase complex, in embryonic and adult bladder urothelium. Knockout of Arid1a in urothelial progenitor cells significantly increases cell proliferation during bladder development. Deletion of Arid1a causes ectopic cell proliferation in the terminally differentiated superficial cells in adult mice. Consistently, gene-set enrichment analysis of differentially expressed genes demonstrates that the cell cycle-related pathways are significantly enriched in Arid1a knockouts. Gene-set of the polycomb repression complex 2 (PRC2) pathway is also enriched, suggesting that Arid1a antagonizes the PRC2-dependent epigenetic gene silencing program in the bladder. During acute cyclophosphamide-induced bladder injury, Arid1a knockouts develop hyperproliferative and hyperinflammatory phenotypes and exhibit a severe loss of urothelial cells. A Hallmark gene-set of the oxidative phosphorylation pathway is significantly reduced in Aria1a mutants before injury and is unexpectedly enriched during injury response. Together, this study uncovers functions of Arid1a in both bladder progenitor cells and the mature urothelium, suggesting its critical roles in urothelial development and regeneration.

Keywords:  ARID1A; Bladder; Epigenetics; PRC2; Regeneration; SWI/SNF; Urothelium

DOI:  https://doi.org/10.1016/j.ydbio.2022.02.008
Nat Genet. 2022 Mar;54(3): 328-341

Systematic reconstruction of cellular trajectories across mouse embryogenesis.

Chengxiang Qiu, Junyue Cao, Beth K Martin, Tony Li, Ian C Welsh, Sanjay Srivatsan, Xingfan Huang, Diego Calderon, William Stafford Noble, Christine M Disteche, Stephen A Murray, Malte Spielmann, Cecilia B Moens, Cole Trapnell, Jay Shendure.

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type's specification, as well as 'cell-type homologs' across vertebrate evolution.

DOI: https://doi.org/10.1038/s41588-022-01018-x
Nat Biotechnol. 2022 Mar 14.

m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome.

Lulu Hu, Shun Liu, Yong Peng, Ruiqi Ge, Rui Su, Chamara Senevirathne, Bryan T Harada, Qing Dai, Jiangbo Wei, Lisheng Zhang, Ziyang Hao, Liangzhi Luo, Huanyu Wang, Yuru Wang, Minkui Luo, Mengjie Chen, Jianjun Chen, Chuan He.

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.

DOI: https://doi.org/10.1038/s41587-022-01243-z
Blood. 2022 Mar 14. pii: blood.2021013925. [Epub ahead of print]

HMGA1 Chromatin Regulators Induce Transcriptional Networks Involved in GATA2 and Proliferation During MPN Progression.

Liping Li, Jung-Hyun Kim, Wenyan Lu, Donna M Williams, Joseph Kim, Lesile Cope, Raajit K Rampal, Richard Patrick Koche, Lingling Xian, Li Z Luo, Marija Vasiljevic, Daniel Rusch Matson, Zhizhuang Joe Zhao, Ophelia Rogers, Matthew C Stubbs, Karen Reddy, Antonio Rodriguez-Romera, Bethan Psaila, Jerry L Spivak, Alison R Moliterno, Linda M S Resar.

Myeloproliferative neoplasms (MPN) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the High Mobility Group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is up-regulated in MPN with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and bone marrow. RNA- and chromatin immunoprecipitation-sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and promising therapeutic target to treat or prevent disease progression.

DOI: https://doi.org/10.1182/blood.2021013925
Elife. 2022 Mar 15. pii: e63404. [Epub ahead of print]11

Nup98-dependent transcriptional memory is established independently of transcription.

Pau Pascual-Garcia, Shawn C Little, Maya Capelson.

  Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. We combined smFISH with mathematical modeling and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. We confirmed this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.

Keywords:  D. melanogaster; Nup98; chromosomes; epigenetic; gene expression; nuclear pore; transcription; transcriptional memory

DOI:  https://doi.org/10.7554/eLife.63404
Nat Commun. 2022 Mar 17. 13(1): 1426

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor.

Federico Miozzo, Eva P Valencia-Alarcón, Luca Stickley, Michaëla Majcin Dorcikova, Francesco Petrelli, Damla Tas, Nicolas Loncle, Irina Nikonenko, Peter Bou Dib, Emi Nagoshi.

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

DOI: https://doi.org/10.1038/s41467-022-29075-0
Dev Cell. 2022 Mar 10. pii: S1534-5807(22)00120-4. [Epub ahead of print]

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.

Eric D Thomas, Andrew E Timms, Sarah Giles, Sarah Harkins-Perry, Pin Lyu, Thanh Hoang, Jiang Qian, Victoria E Jackson, Melanie Bahlo, Seth Blackshaw, Martin Friedlander, Kevin Eade, Timothy J Cherry.

  Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Keywords:  MIR-9; cis-regulatory element; development; enhancer; macular telangiectasia type 2; neurogenesis; retina; retinal organoid; single-cell ATAC-seq; single-cell RNA-seq

DOI:  https://doi.org/10.1016/j.devcel.2022.02.018
PLoS Genet. 2022 Mar 16. 18(3): e1010083

A shared ancient enhancer element differentially regulates the bric-a-brac tandem gene duplicates in the developing Drosophila leg.

Henri-Marc G Bourbon, Mikhail H Benetah, Emmanuelle Guillou, Luis Humberto Mojica-Vazquez, Aissette Baanannou, Sandra Bernat-Fabre, Vincent Loubiere, Frédéric Bantignies, Giacomo Cavalli, Muriel Boube.

Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. We previously characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. We show here that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.

DOI: https://doi.org/10.1371/journal.pgen.1010083
Elife. 2022 Mar 16. pii: e75475. [Epub ahead of print]11

The TFIIH complex is required to establish and maintain mitotic chromosome structure.

Julian Haase, Richard Chen, Wesley M Parker, Mary Kate Bonner, Lisa M Jenkins, Alexander E Kelly.

  Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB rapidly and reversibly induces a complete loss of chromosome structure and prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, inhibiting TFIIH prevents condensation of both mouse and Xenopus nuclei in Xenopus egg extracts, which suggests an evolutionarily conserved mechanism of TFIIH action. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering the chromatin environment to facilitate condensin loading and condensin-dependent loop extrusion.

Keywords:  cell biology; chromosomes; gene expression; mouse; xenopus

DOI:  https://doi.org/10.7554/eLife.75475
Cell Rep. 2022 Mar 15. pii: S2211-1247(22)00246-7. [Epub ahead of print]38(11): 110510

Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting.

Ace E Lewis, Akela Kuwahara, Jacqueline Franzosi, Jeffrey O Bush.

  The mechanisms coupling fate specification of distinct tissues to their physical separation remain to be understood. The trachea and esophagus differentiate from a single tube of definitive endoderm, requiring the transcription factors SOX2 and NKX2-1, but how the dorsoventral site of tissue separation is defined to allocate tracheal and esophageal cell types is unknown. Here, we show that the EPH/EPHRIN signaling gene Efnb2 regulates tracheoesophageal separation by controlling the dorsoventral allocation of tracheal-fated cells. Ventral loss of NKX2-1 results in disruption of separation and expansion of Efnb2 expression in the trachea independent of SOX2. Through chromatin immunoprecipitation and reporter assays, we find that NKX2-1 likely represses Efnb2 directly. Lineage tracing shows that loss of NKX2-1 results in misallocation of ventral foregut cells into the esophagus, while mosaicism for Nkx2-1 generates ectopic NKX2-1/EPHRIN-B2 boundaries that organize ectopic tracheal separation. Together, these data demonstrate that NKX2-1 coordinates tracheal specification with tissue separation through the regulation of EPHRIN-B2 and tracheoesophageal cell sorting.

Keywords:  Eph/ephrin; Nkx2-1; Sox2; cell sorting; endoderm; ephrin-B2; esophagus; foregut; trachea; tracheoesophageal fistula

DOI:  https://doi.org/10.1016/j.celrep.2022.110510
Elife. 2022 Mar 15. pii: e73943. [Epub ahead of print]11

High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.

Mingrui Duan, Smitha Sivapragasam, Jacob S Antony, Jenna Ulibarri, John M Hinz, Gregory M K Poon, John J Wyrick, Peng Mao.

  DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of UV damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

Keywords:  S. cerevisiae; cancer biology; genetics; genomics

DOI:  https://doi.org/10.7554/eLife.73943
Nat Commun. 2022 Mar 18. 13(1): 1483

Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains.

Cristiana Bersaglieri, Jelena Kresoja-Rakic, Shivani Gupta, Dominik Bär, Rostyslav Kuzyakiv, Martina Panatta, Raffaella Santoro.

Eukaryotic chromosomes are folded into hierarchical domains, forming functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks contributing to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for the identification of nucleolar associated domains (NADs). Here we have established DamID- and HiC-based methodologies to generate accurate genome-wide maps of NADs in embryonic stem cells (ESCs) and neural progenitor cells (NPCs), revealing layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs show higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation into NPCs, chromosomes around the nucleolus acquire a more compact, rigid architecture with neural genes moving away from nucleoli and becoming unlocked for later activation. Further, histone modifications and the interaction strength within A and B compartments of NADs and LADs in ESCs set the choice to associate with NL or nucleoli upon dissociation from their respective compartments during differentiation. The methodologies here developed will make possible to include the nucleolar contribution in nuclear space and genome function in diverse biological systems.

DOI: https://doi.org/10.1038/s41467-022-29146-2
Nat Commun. 2022 Mar 17. 13(1): 1423

The Oligodendrocyte Transcription Factor 2 OLIG2 regulates transcriptional repression during myelinogenesis in rodents.

Kunkun Zhang, Shaoxuan Chen, Qihua Yang, Shuanghui Guo, Qiang Chen, Zhixiong Liu, Li Li, Mengyun Jiang, Hongda Li, Jin Hu, Xu Pan, Wenbo Deng, Naian Xiao, Bo Wang, Zhan-Xiang Wang, Liang Zhang, Wei Mo.

OLIG2 is a transcription factor that activates the expression of myelin-associated genes in the oligodendrocyte-lineage cells. However, the mechanisms of myelin gene inactivation are unclear. Here, we uncover a non-canonical function of OLIG2 in transcriptional repression to modulate myelinogenesis by functionally interacting with tri-methyltransferase SETDB1. Immunoprecipitation and chromatin-immunoprecipitation assays show that OLIG2 recruits SETDB1 for H3K9me3 modification on the Sox11 gene, which leads to the inhibition of Sox11 expression during the differentiation of oligodendrocytes progenitor cells (OPCs) into immature oligodendrocytes (iOLs). Tissue-specific depletion of Setdb1 in mice results in the hypomyelination during development and remyelination defects in the injured rodents. Knockdown of Sox11 by siRNA in rat primary OPCs or depletion of Sox11 in the oligodendrocyte lineage in mice could rescue the hypomyelination phenotype caused by the loss of OLIG2. In summary, our work demonstrates that the OLIG2-SETDB1 complex can mediate transcriptional repression in OPCs, affecting myelination.

DOI: https://doi.org/10.1038/s41467-022-29068-z
Sci Adv. 2022 Mar 18. 8(11): eabj6526

HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state.

Roger S Smith, Seesha R Takagishi, David R Amici, Kyle Metz, Sitaram Gayatri, Milad J Alasady, Yaqi Wu, Sonia Brockway, Stephanie L Taiberg, Natalia Khalatyan, Mikko Taipale, Sandro Santagata, Luke Whitesell, Susan Lindquist, Jeffrey N Savas, Marc L Mendillo.

Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find that HSF2, an HSF1 paralog with no substantial role in the HSR, physically and functionally interacts with HSF1 across diverse types of cancer. HSF1 and HSF2 have notably similar chromatin occupancy and regulate a common set of genes that include both HSPs and noncanonical transcriptional targets with roles critical in supporting malignancy. Loss of either HSF1 or HSF2 results in a dysregulated response to nutrient stresses in vitro and reduced tumor progression in cancer cell line xenografts. Together, these findings establish HSF2 as a critical cofactor of HSF1 in driving a cancer cell transcriptional program to support the anabolic malignant state.

DOI: https://doi.org/10.1126/sciadv.abj6526
Nucleic Acids Res. 2022 Mar 16. pii: gkac165. [Epub ahead of print]

Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease.

Andrew M Jobbins, Nejc Haberman, Natalia Artigas, Christopher Amourda, Helen A B Paterson, Sijia Yu, Samuel J I Blackford, Alex Montoya, Marian Dore, Yi-Fang Wang, Alessandro Sardini, Inês Cebola, Johannes Zuber, Sheikh Tamir Rashid, Boris Lenhard, Santiago Vernia.

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.

DOI: https://doi.org/10.1093/nar/gkac165
iScience. 2022 Mar 18. 25(3): 103927

PCR-based profiling of transcription factor activity in vivo by a virus-based reporter battery.

Hitomi Abe, Kentaro Abe.

  Understanding the molecular mechanisms of gene regulation is pivotal for understanding how cells establish and modify their identities and functions. Multiple transcription factors (TFs) coordinate to alter gene expression in cells; however, a method to quantitatively analyze the activity of each TF is lacking, particularly in vivo. Here, we introduce a viral-vector-based TF reporter battery that can be used to simultaneously analyze the activity of multiple TFs, visualized as the TF activity profile (TFAP) obtained by qPCR. We show that the cells possess distinct TFAPs that dynamically change according to experimental manipulation or physiological activity. We report a practical method to obtain the TFAP of a defined cell population and their experience-dependent changes in the mouse brain in vivo. The TFAP obtained by our method will help bridge the information gap between the genome and transcriptome and aid the multi-omics view of understanding the gene regulation system.

Keywords:  Biological sciences; Biological sciences research methodologies; Biology experimental methods; Cell biology; Methodology in biological sciences; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2022.103927
Development. 2022 Mar 17. pii: dev.200131. [Epub ahead of print]

FOXO1 represses Sprouty2 and Sprouty4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Nanbing Li-Villarreal, Rebecca Lee Yean Wong, Monica D Garcia, Ryan S Udan, Ross A Poché, Tara L Rasmussen, Alexander M Rhyner, Joshua D Wythe, Mary E Dickinson.

  Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of FoxO1 in Tie2-cre expressing cells resulted in increased Sprouty2 and Sprouty4, reduced arterial genes, and reduced Flk1/Vegfr2 transcripts without affecting overall endothelial cell identity, survival, or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, Delta like 4, is significantly reduced in FoxO1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Sprouty2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Sprouty4 GREs to repress their expression. Furthermore, overexpression of Sprouty4 in transient transgenic embryos largely recapitulates reduced arterial genes seen in conditional FoxO1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Keywords:  Arterial specification; Dll4; FoxO1; Sprouty

DOI:  https://doi.org/10.1242/dev.200131
Nat Cancer. 2022 Mar 17.

LCOR mediates interferon-independent tumor immunogenicity and responsiveness to immune-checkpoint blockade in triple-negative breast cancer.

Iván Pérez-Núñez, Catalina Rozalén, José Ángel Palomeque, Irene Sangrador, Mariona Dalmau, Laura Comerma, Anna Hernández-Prat, David Casadevall, Silvia Menendez, Daniel Dan Liu, Minhong Shen, Jordi Berenguer, Irene Rius Ruiz, Raul Peña, José Carlos Montañés, M Mar Albà, Sarah Bonnin, Julia Ponomarenko, Roger R Gomis, Juan Miguel Cejalvo, Sonia Servitja, Diego M Marzese, Lluis Morey, Leonie Voorwerk, Joaquín Arribas, Begoña Bermejo, Marleen Kok, Lajos Pusztai, Yibin Kang, Joan Albanell, Toni Celià-Terrassa.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN.

DOI: https://doi.org/10.1038/s43018-022-00339-4