ASC proneural transcription factors mediate the timely initiation of the neural program during neuroectodermal to neuroblast transition ensuring progeny fidelity

Background: ASC/ASCL proneural transcription factors are oncogenic and exhibit impressive reprogramming and pioneer activities. In both Drosophila and mammals, these factors are central in the early specification of the neural fate, where they act in opposition to Notch signalling. However, the role of ASC on the chromatin during CNS neural stem cells birth remains elusive. Results: We investigated the chromatin changes accompanying neural commitment using an integrative genetics and genomics methodology. We found that ASC factors bind equally strongly to two distinct classes of cis-regulatory elements: open regions remodeled earlier during maternal to zygotic transition by Zelda and Zelda-independent, less accessible regions. Both classes cis-elements exhibit enhanced chromatin accessibility during neural specification and correlate with transcriptional regulation of genes involved in many biological processes necessary for neuroblast function. We identified an ASC-Notch regulated TF network that most likely act as the prime regulators of neuroblast function. Using a cohort of ASC target genes, we report that ASC null neuroblasts are defectively specified, remaining initially stalled, lacking expression of many proneural targets and unable to divide. When they eventually start proliferating, they produce compromised progeny. Generation of lacZ reporter lines driven by proneural-bound elements display enhancer activity within neuroblasts and proneural dependency. Therefore, the partial neuroblast identity seen in the absence of ASC genes is driven by other, proneural-independent, cis-elements. Neuroblast impairment and the late differentiation defects of ASC mutants are corrected by ectodermal induction of individual ASC genes but not by individual members of the TF network downstream of ASC. However, in wild type embryos induction of individual members of this network induces CNS hyperplasia, suggesting that they synergize with the activating function of ASC to establish the chromatin dynamics that promote neural specification. Conclusion: ASC factors bind a large number of enhancers to orchestrate the timely activation of the neural chromatin program during neuroectodermal to neuroblast transition. This early chromatin remodeling is crucial for both neuroblast homeostasis as well as future progeny fidelity.

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FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

Blood ◽

10.1182/blood-2019-126201 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1228-1228

Author(s):

Yanan Li ◽

Riddhi M Patel ◽

Emily Casey ◽

Jeffrey A. Magee

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Target Gene ◽

Target Genes ◽

Conflicts Of Interest ◽

Gene Activation ◽

Chromatin Accessibility ◽

Luciferase Reporter ◽

Enhancer Activity ◽

Target Gene Expression

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.

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Non-Coding Genome, Transcription Factors, and Sex Determination

Sexual Development ◽

10.1159/000519725 ◽

2021 ◽

pp. 1-13

Author(s):

Francis Poulat

Keyword(s):

Transcription Factors ◽

Sex Determination ◽

Target Genes ◽

Regulatory Elements ◽

Eukaryotic Cells ◽

Genomic Profiling ◽

Binding Motifs ◽

Male And Female ◽

Genome Transcription ◽

Control Complex

In vertebrates, gonadal sex determination is the process by which transcription factors drive the choice between the testicular and ovarian identity of undifferentiated somatic progenitors through activation of 2 different transcriptional programs. Studies in animal models suggest that sex determination always involves sex-specific transcription factors that activate or repress sex-specific genes. These transcription factors control their target genes by recognizing their regulatory elements in the non-coding genome and their binding motifs within their DNA sequence. In the last 20 years, the development of genomic approaches that allow identifying all the genomic targets of a transcription factor in eukaryotic cells gave the opportunity to globally understand the function of the nuclear proteins that control complex genetic programs. Here, the major transcription factors involved in male and female vertebrate sex determination and the genomic profiling data of mouse gonads that contributed to deciphering their transcriptional regulation role will be reviewed.

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An atlas of dynamic chromatin landscapes in mouse fetal development

Nature ◽

10.1038/s41586-020-2093-3 ◽

2020 ◽

Vol 583 (7818) ◽

pp. 744-751 ◽

Cited By ~ 13

Author(s):

David U. Gorkin ◽

Iros Barozzi ◽

Yuan Zhao ◽

Yanxiao Zhang ◽

Hui Huang ◽

...

Keyword(s):

Fetal Development ◽

Target Genes ◽

Developmental Stages ◽

Chromatin Accessibility ◽

Chromatin State ◽

Mammalian Development ◽

Mouse Fetus ◽

Chromatin Dynamics ◽

Mouse Tissues ◽

Genomic Resource

AbstractThe Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

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Integrative transcriptome and chromatin landscape analysis reveals distinct epigenetic regulations in human memory B cells

Nature Communications ◽

10.1038/s41467-020-19242-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Justin B. Moroney ◽

Anusha Vasudev ◽

Alexander Pertsemlidis ◽

Hong Zan ◽

Paolo Casali

Keyword(s):

B Cells ◽

Target Genes ◽

Human Memory ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Memory B Cells ◽

Transcriptional Signature ◽

Integrative Analyses ◽

Health And Disease ◽

Variable Gene

Abstract Memory B cells (MBCs) are long-lived and produce high-affinity, generally, class-switched antibodies. Here, we use a multiparameter approach involving CD27 to segregate naïve B cells (NBC), IgD+ unswitched (unsw)MBCs and IgG+ or IgA+ class-switched (sw)MBCs from humans of different age, sex and race. Conserved antibody variable gene expression indicates that MBCs emerge through unbiased selection from NBCs. Integrative analyses of mRNAs, miRNAs, lncRNAs, chromatin accessibility and cis-regulatory elements uncover a core mRNA-ncRNA transcriptional signature shared by IgG+ and IgA+ swMBCs and distinct from NBCs, while unswMBCs display a transitional transcriptome. Some swMBC transcriptional signature loci are accessible but not expressed in NBCs. Profiling miRNAs reveals downregulated MIR181, and concomitantly upregulated MIR181 target genes such as RASSF6, TOX, TRERF1, TRPV3 and RORα, in swMBCs. Finally, lncRNAs differentially expressed in swMBCs cluster proximal to the IgH chain locus on chromosome 14. Our findings thus provide new insights into MBC transcriptional programs and epigenetic regulation, opening new investigative avenues on these critical cell elements in human health and disease.

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Modeling gene regulation from paired expression and chromatin accessibility data

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704553114 ◽

2017 ◽

Vol 114 (25) ◽

pp. E4914-E4923 ◽

Cited By ~ 76

Author(s):

Zhana Duren ◽

Xi Chen ◽

Rui Jiang ◽

Yong Wang ◽

Wing Hung Wong

Keyword(s):

Gene Expression ◽

Target Genes ◽

Transcriptional Regulatory Network ◽

Joint Modeling ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Exciting Opportunity ◽

Transcriptional Regulatory ◽

Dna Elements ◽

Context Specific

The rapid increase of genome-wide datasets on gene expression, chromatin states, and transcription factor (TF) binding locations offers an exciting opportunity to interpret the information encoded in genomes and epigenomes. This task can be challenging as it requires joint modeling of context-specific activation of cis-regulatory elements (REs) and the effects on transcription of associated regulatory factors. To meet this challenge, we propose a statistical approach based on paired expression and chromatin accessibility (PECA) data across diverse cellular contexts. In our approach, we model (i) the localization to REs of chromatin regulators (CRs) based on their interaction with sequence-specific TFs, (ii) the activation of REs due to CRs that are localized to them, and (iii) the effect of TFs bound to activated REs on the transcription of target genes (TGs). The transcriptional regulatory network inferred by PECA provides a detailed view of how trans- and cis-regulatory elements work together to affect gene expression in a context-specific manner. We illustrate the feasibility of this approach by analyzing paired expression and accessibility data from the mouse Encyclopedia of DNA Elements (ENCODE) and explore various applications of the resulting model.

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Determination of the consensus DNA-binding sequence and a transcriptional activation domain for ESE-2

Biochemical Journal ◽

10.1042/bj20060375 ◽

2006 ◽

Vol 398 (3) ◽

pp. 497-507 ◽

Cited By ~ 19

Author(s):

Yeon Sook Choi ◽

Satrajit Sinha

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Transcriptional Activation ◽

Target Genes ◽

Regulatory Elements ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Ets Proteins ◽

Flanking Sequences ◽

Binding Sequence

The ESE (epithelium-specific Ets) subfamily of Ets transcription factors plays an important role in regulating gene expression in a variety of epithelial cell types. Although ESE proteins have been shown to bind to regulatory elements of some epithelial genes, the optimal DNA-binding sequence has not been experimentally ascertained for any member of the ESE subfamily of transcription factors. This has made the identification and validation of their targets difficult. We are studying ESE-2 (Elf5), which is highly expressed in epithelial cells of many tissues including skin keratinocytes. Here, we identify the preferred DNA-binding site of ESE-2 by performing CASTing (cyclic amplification and selection of targets) experiments. Our analysis shows that the optimal ESE-2 consensus motif consists of a GGA core and an AT-rich 5′- and 3′-flanking sequences. Mutational and competition experiments demonstrate that the flanking sequences that confer high DNA-binding affinity for ESE-2 show considerable differences from the known consensus DNA-binding sites of other Ets proteins, thus reinforcing the idea that the flanking sequences may impart recognition specificity for Ets proteins. In addition, we have identified a novel isoform of murine ESE-2, ESE-2L, that is generated by use of a hitherto unreported new exon and an alternate promoter. Interestingly, transient transfection assays with an optimal ESE-2 responsive reporter show that both ESE-2 and ESE-2L are weak transactivators. However, similar studies utilizing GAL4 chimaeras of ESE-2 demonstrate that while the DNA-binding ETS (E twenty-six) domain functions as a repressor, the PNT (pointed domain) of ESE-2 can act as a potent transcriptional activation domain. This novel transactivating property of PNT is also shared by ESE-3, another ESE family member. Identification of the ESE-2 consensus site and characterization of the transcriptional activation properties of ESE-2 shed new light on its potential as a regulator of target genes.

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Systematic dissection of genomic features determining transcription factor binding and enhancer function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621150114 ◽

2017 ◽

Vol 114 (7) ◽

pp. E1291-E1300 ◽

Cited By ~ 74

Author(s):

Sharon R. Grossman ◽

Xiaolan Zhang ◽

Li Wang ◽

Jesse Engreitz ◽

Alexandre Melnikov ◽

...

Keyword(s):

Regulatory Elements ◽

Chromatin Accessibility ◽

Binding Assays ◽

Genomic Locus ◽

Enhancer Activity ◽

Genomic Features ◽

Motif Site ◽

Reporter Assays ◽

Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

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Pancreatic islet chromatin accessibility and conformation defines distal enhancer networks of type 2 diabetes risk

10.1101/299388 ◽

2018 ◽

Cited By ~ 3

Author(s):

William W Greenwald ◽

Joshua Chiou ◽

Jian Yan ◽

Yunjiang Qiu ◽

Ning Dai ◽

...

Keyword(s):

Type 2 Diabetes ◽

Pancreatic Islets ◽

Regulatory Networks ◽

Complex Disease ◽

Protein Transport ◽

Target Genes ◽

Regulatory Elements ◽

Distal Enhancer ◽

Enhancer Activity

AbstractThe gene targets of enhancer activity in pancreatic islets are largely unknown, impeding discovery of islet regulatory networks involved in type 2 diabetes (T2D) risk. We mapped chromatin state, accessibility and conformation using ChIP-seq, ATAC-seq and Hi-C in human pancreatic islets, which we integrated with T2D genetic fine-mapping and islet expression QTL data. Active islet regulatory elements preferentially interacted with other active elements, often at distances over 1MB, and we identified target genes for thousands of distal islet enhancers. A third of T2D risk signals mapped in islet enhancers, and target genes regulated by these signals were specifically involved in processes related to protein transport and secretion. Among implicated target genes of T2D islet enhancer signals with no prior known role in islet function, we demonstrated that reduced IGF2BP2 activity in mouse islets leads to impaired glucose-stimulated insulin secretion. These results link distal islet enhancer regulation of protein secretion and transport to genetic risk of T2D, and highlight the utility of high-throughput chromatin conformation maps to uncover the gene regulatory networks of complex disease.

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Genome-wide reduction in chromatin accessibility and unique transcription factor footprints in endothelial cells and fibroblasts in scleroderma skin

10.1101/2020.06.24.20138040 ◽

2020 ◽

Author(s):

Pei-Suen Tsou ◽

Pamela J. Palisoc ◽

Mustafa Ali ◽

Dinesh Khanna ◽

Amr H Sawalha

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Critical Role ◽

Vascular Complications ◽

Chromatin Accessibility ◽

Unknown Etiology ◽

Healthy Controls ◽

Genome Wide

AbstractSystemic sclerosis (SSc) is a rare autoimmune disease of unknown etiology characterized by widespread fibrosis and vascular complications. We utilized an assay for genome-wide chromatin accessibility to examine the chromatin landscape and transcription factor footprints in both endothelial cells (ECs) and fibroblasts isolated from healthy controls and patients with diffuse cutaneous (dc) SSc. In both cell types, chromatin accessibility was significantly reduced in SSc patients compared to healthy controls. Genes annotated from differentially accessible chromatin regions were enriched in pathways and gene ontologies involved in the nervous system. In addition, our data revealed that chromatin binding of transcription factors SNAI2, ETV2, and ELF1 was significantly increased in dcSSc ECs, while recruitment of RUNX1 and RUNX2 was enriched in dcSSc fibroblasts. Significant elevation of SNAI2 and ETV2 levels in dcSSc ECs, and RUNX2 levels in dcSSc fibroblasts were confirmed. Further analysis of publicly available ETV2-target genes suggests that ETV2 may play a critical role in EC dysfunction in dcSSc. Our data, for the first time, uncovered the chromatin blueprint of dcSSc ECs and fibroblasts, and suggested that neural-related characteristics of SSc ECs and fibroblasts could be a culprit for dysregulated angiogenesis and enhanced fibrosis. Targeting these pathways and the key transcription factors identified might present novel therapeutic approaches for this disease.

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A Transcription Start Site Map in Human Pancreatic Islets Reveals Functional Regulatory Signatures

10.2337/figshare.14394707.v1 ◽

2021 ◽

Author(s):

Arushi Varshney ◽

Yasuhiro Kyono ◽

Venkateswaran Ramamoorthi Elangovan ◽

Collin Wang ◽

Michael R. Erdos ◽

...

Keyword(s):

Pancreatic Islets ◽

Transcription Initiation ◽

Genome Wide Association Study ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Transcriptional Enhancer ◽

Transcription Start ◽

Enhancer Activity ◽

Transcriptional Initiation ◽

Human Pancreatic Islets

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ~20% of which are islet-specific and occur mostly distal to known gene TSSs. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (~68%) tested islet CAGE elements (5% FDR). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

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