Oct4 primarily controls enhancer activity rather than accessibility

The transcription factor Oct4 is essential for maintaining stem cell pluripotency and for efficient cell reprogramming, but its functional roles are far from being understood. Here, we investigate the functions of Oct4 by rapidly depleting Oct4 from mouse embryonic stem cells and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to putative enhancers that are accessible in chromatin. Loss of Oct4 is accompanied by a concomitant decrease in mRNA synthesis from putative target genes that are part of the transcriptional network that maintains pluripotency. Oct4 binding to enhancers does not correlate with chromatin accessibility, whereas Sox2 can apparently retain accessibility after Oct4 depletion even in the absence of eRNA synthesis. These results are consistent with the model that Sox2 primarily acts as a pioneer factor that renders enhancers accessible, whereas Oct4 acts primarily as a transcriptional activator that stimulates transcription of pluripotency enhancers and their target genes

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The Transcription Factor Zfp281 Controls Embryonic Stem Cell Pluripotency by Direct Activation and Repression of Target Genes

Stem Cells ◽

10.1634/stemcells.2008-0443 ◽

2008 ◽

Vol 26 (11) ◽

pp. 2791-2799 ◽

Cited By ~ 48

Author(s):

Zheng-Xu Wang ◽

Christina Hui-Leng Teh ◽

Caroline Man-Yee Chan ◽

Ci Chu ◽

Michael Rossbach ◽

...

Keyword(s):

Transcription Factor ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Target Genes ◽

Embryonic Stem ◽

Direct Activation ◽

Stem Cell Pluripotency ◽

Embryonic Stem Cell Pluripotency

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

10.1101/2021.11.02.466869 ◽

2021 ◽

Author(s):

Vasiliki Theodorou ◽

Aikaterini Stefanaki ◽

Minas Drakos ◽

Dafne Triantafyllou ◽

Christos Delidakis

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Cis Elements ◽

Enhancer Activity ◽

Timely Initiation ◽

Chromatin Dynamics ◽

Neural Fate ◽

Neural Specification

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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LncRNA Platr22 promotes super-enhancer activity and stem cell pluripotency

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa056 ◽

2020 ◽

Author(s):

Pixi Yan ◽

J Yuyang Lu ◽

Jing Niu ◽

Juntao Gao ◽

Michael Q Zhang ◽

...

Keyword(s):

Cell Fate ◽

Noncoding Rna ◽

Embryonic Stem ◽

Cell Fate Determination ◽

Enhancer Activity ◽

Lncrna Gene ◽

Fate Determination ◽

Super Enhancer ◽

Stem Cell Pluripotency ◽

Active Transcription

Abstract Super-enhancers (SEs) comprise large clusters of enhancers, which are co-occupied by multiple lineage-specific and master transcription factors, and play pivotal roles in regulating gene expression and cell fate determination. However, it is still largely unknown whether and how SEs are regulated by the non-coding portion of the genome. Here, through genome-wide analysis, we found that long noncoding RNA (lncRNA) genes preferentially lie next to SEs. In mouse embryonic stem cells (mESCs), depletion of SE-associated lncRNA transcripts dysregulated the activity of their nearby SEs. Specifically, we revealed a critical regulatory role of the lncRNA gene Platr22 in modulating the activity of a nearby SE and the expression of the nearby pluripotency regulator ZFP281. Through these regulatory events, Platr22 contributes to pluripotency maintenance and proper differentiation of mESCs. Mechanistically, Platr22 transcripts coat chromatin near the SE region and interact with DDX5 and hnRNP-L. DDX5 further recruits p300 and other factors related to active transcription. We propose that these factors assemble into a transcription hub, thus promoting an open and active epigenetic chromatin state. Our study highlights an unanticipated role for a class of lncRNAs in epigenetically controlling the activity and vulnerability to perturbation of nearby SEs for cell fate determination.

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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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LSD1/KDM1A Maintains Genome-wide Homeostasis of Transcriptional Enhancers

10.1101/146357 ◽

2017 ◽

Cited By ~ 4

Author(s):

Saurabh Agarwal ◽

Patricia Marie Garay ◽

Robert Scott Porter ◽

Emily Brookes ◽

Yumie Murata-Nakamura ◽

...

Keyword(s):

Target Genes ◽

Large Fraction ◽

Embryonic Stem ◽

Gene Promoters ◽

Enhancer Activity ◽

Control Of Gene Expression ◽

Chromatin Signature ◽

Transcriptional Enhancers ◽

Lysine Specific Demethylase ◽

Genome Wide

AbstractTranscriptional enhancers enable exquisite spatiotemporal control of gene expression in metazoans. Enrichment of mono-methylation of histone H3 lysine 4 (H3K4me1) is a major chromatin signature that distinguishes enhancers from gene promoters. Lysine Specific Demethylase 1 (LSD1, aka KDM1A), an enzyme specific for demethylating H3K4me2/me1, has been shown to “decommission” stem cell enhancers during the differentiation of mouse embryonic stem cells (mESC). However, the roles of LSD1 in undifferentiated mESC remain obscure. Here, we show that LSD1 occupies a large fraction of enhancers (63%) that are primed with binding of transcription factors (TFs) and H3K4me1 in mESC. In contrast, LSD1 is largely absent at latent enhancers, which are not yet primed by TF binding. Unexpectedly, LSD1 levels at enhancers exhibited a clear positive correlation with its substrate, H3K4me2 and enhancer activity. These enhancers gain additional H3K4 methylation upon the loss of LSD1 in mESC. The aberrant increase in H3K4me at enhancers was accompanied with increases in enhancer H3K27 acetylation and expression of enhancer RNAs (eRNAs) and their target genes. In post-mitotic neurons, loss of LSD1 resulted in premature activation of enhancers and genes that are normally induced after neuronal activation. These results demonstrate that LSD1 is a versatile suppressor of primed enhancers, and is involved in homeostasis of enhancer activity.

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Transcriptional network dynamics during the progression of pluripotency revealed by integrative statistical learning

Nucleic Acids Research ◽

10.1093/nar/gkz1179 ◽

2019 ◽

Vol 48 (4) ◽

pp. 1828-1842 ◽

Cited By ~ 2

Author(s):

Hani Jieun Kim ◽

Pierre Osteil ◽

Sean J Humphrey ◽

Senthilkumar Cinghu ◽

Andrew J Oldfield ◽

...

Keyword(s):

Statistical Learning ◽

Binding Sites ◽

Target Genes ◽

Embryonic Stem ◽

Transcriptional Networks ◽

Time Resolved ◽

Developmental Potential ◽

Spatiotemporal Mapping ◽

Primed State

Abstract The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved ‘trans-omic’ datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos.

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Polycomb repressive complex 1 defines the nucleosome landscape but not accessibility at target genes

10.1101/280305 ◽

2018 ◽

Author(s):

Hamish W King ◽

Robert J Klose

Keyword(s):

Target Genes ◽

Nucleosome Occupancy ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Polycomb Group ◽

Gene Promoters ◽

Polycomb Repressive Complex ◽

Chromatin Domains ◽

Genetic Ablation ◽

Polycomb Repressive Complex 1

ABSTRACTPolycomb group (PcG) proteins are transcriptional repressors that play important roles regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin limiting the activity of chromatin remodelling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promotors. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we measured chromatin accessibility and examined the nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins and Polycomb chromatin domains do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape and PcG-mediated transcriptional repression.

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KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

Nature Communications ◽

10.1038/s41467-021-27502-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alexandra D’Oto ◽

Jie Fang ◽

Hongjian Jin ◽

Beisi Xu ◽

Shivendra Singh ◽

...

Keyword(s):

High Risk ◽

Binding Sites ◽

Target Genes ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Chromatin Accessibility ◽

Histone Demethylase ◽

Chromatin Interaction ◽

Cell Resistance ◽

Enhancer Activity

AbstractThe H3K27me2/me3 histone demethylase KDM6B is essential to neuroblastoma cell survival. However, the mechanism of KDM6B action remains poorly defined. We demonstrate that inhibition of KDM6B activity 1) reduces the chromatin accessibility of E2F target genes and MYCN, 2) selectively leads to an increase of H3K27me3 but a decrease of the enhancer mark H3K4me1 at the CTCF and BORIS binding sites, which may, consequently, disrupt the long-range chromatin interaction of MYCN and E2F target genes, and 3) phenocopies the transcriptome induced by the specific CDK4/6 inhibitor palbociclib. Overexpression of CDK4/6 or Rb1 knockout confers neuroblastoma cell resistance to both palbociclib and the KDM6 inhibitor GSK-J4. These data indicate that KDM6B promotes an oncogenic CDK4/6-pRB-E2F pathway in neuroblastoma cells via H3K27me3-dependent enhancer-promoter interactions, providing a rationale to target KDM6B for high-risk neuroblastoma.

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Chromatin accessibility combined with enhancer clusters activation mediates heterogeneous response to dexamethasone in myeloma cells

10.1101/2021.09.06.459068 ◽

2021 ◽

Author(s):

Victor GABORIT ◽

Jonathan CRUARD ◽

Catherine Guerin-Charbonnel ◽

Jennifer Derrien ◽

Jean-Baptiste Alberge ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Interaction Network ◽

Chromatin Accessibility ◽

Enhancer Activity ◽

Myeloma Cells ◽

Combinatorial Assembly ◽

Induced Apoptosis ◽

Combination Regimens

Glucocorticoids (GC) effects occur through binding to the GC receptor (GR) which, once translocated to the nucleus, binds to GC response elements (GREs) to activate or repress target genes. Among GCs, dexamethasone (Dex) is widely used in treatment of multiple myeloma (MM), mainly in combination regimens. However, despite a definite benefit, all patients relapse. Moreover, while GC efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive. To determine the functional role of GR binding in myeloma cells, we generated bulk and single cell multi-omic data and high-resolution contact maps of active enhancers and target genes. We show that a minority (6%) of GR binding sites are associated with enhancer activity gains and increased interaction loops. We find that enhancers contribute to regulate gene activity through combinatorial assembly of large stretches of enhancers and/or enhancer cliques. Furthermore, one enhancer, proximal to GR-responsive genes, is predominantly associated with increased chromatin accessibility and higher H3K27ac occupancy. Finally, we show that Dex exposure leads to co-accessibility changes between predominant enhancer and other regulatory regions of the interaction network. Notably, these epigenomic changes are associated with cell-to-cell transcriptional heterogeneity. As consequences, BIM critical for GR-induced apoptosis and CXCR4 protective from chemotherapy-induced apoptosis are rather upregulated in different cells. In summary, our work provides new insights into the molecular mechanisms involved in Dex escape.

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SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

10.1101/2020.08.21.261131 ◽

2020 ◽

Author(s):

Bieke Decaesteker ◽

Amber Louwagie ◽

Siebe Loontiens ◽

Fanny De Vloed ◽

Juliette Roels ◽

...

Keyword(s):

Target Genes ◽

Epigenetic Modification ◽

Chromatin Accessibility ◽

Copy Number Alterations ◽

Specific Expression ◽

Genome Wide ◽

Mutation Burden ◽

Epigenetic Regulator ◽

Pioneer Factor

ABSTRACTThe pediatric extra-cranial tumor neuroblastoma (NB) is characterised by a low mutation burden while copy number alterations are present in most high-risk cases. We identified SOX11 as a strong lineage dependency transcription factor in adrenergic NB based on recurrent chromosome 2p focal gains and amplifications, its specific expression in the normal sympatho-adrenal lineage and adrenergic NBs and its regulation by multiple adrenergic specific cis-interacting (super-)enhancers. Adrenergic NBs are strongly dependent on high SOX11 expression levels for growth and proliferation. Through genome-wide DNA-binding and transcriptome analysis, we identified and validated functional SOX11 target genes, several of which implicated in chromatin remodeling and epigenetic modification. SOX11 controls chromatin accessibility predominantly affecting distal adrenergic lineage-specific enhancers marked by binding sites of the adrenergic core regulatory circuitry. During normal sympathoblast differentiation we find expression of SOX11 prior to members of the adrenergic core regulatory circuitry. Given the broad control of SOX11 of multiple epigenetic regulatory complexes and its presumed pioneer factor function, we propose that adrenergic NB cells have co-opted the normal role of SOX11 as a crucial regulator of chromatin accessibility and cell identity.

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