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Regulation of biological processes typically incorporate mechanisms that both initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism wherein low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from both in vitro and in vivo experiments support the model that RNAs produced during early steps in transcription initiation stimulate condensate formation whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process.

Chromatin-bound LEDGF and HDGF2 proteins allow RNAPII to overcome the nucleosome-induced block to transcription., FACT (facilitates chromatin transcription) is a protein complex that allows RNA polymerase II (RNAPII) to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. We identified two proteins, lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2), each containing two high mobility group A (HMGA)–like AT-hooks and a methyl-lysine reading Pro-Trp-Trp-Pro (PWWP) domain that binds to H3K36me2 and H3K36me3.LEDGF and HDGF2 colocalize with H3K36me2/3 at genomic regions containing active genes. In myoblasts, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease, while HDGF2 levels are maintained. Moreover, HDGF2 is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation.

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained “write and read” activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-β, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity., National Science Foundation (U.S.). (Grant PHY1743900), National Institutes of Health (U.S.) (Grants GM123511, GM117370 and T32CA009172), German Research Foundation Research Fellowship (DE 3069/1-1)

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.

FACT (facilitates chromatin transcription) is a protein complex that allows RNAPII to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. Here we report the identification of two such proteins, LEDGF and HDGF2, each containing two HMGA-like AT-hooks and a PWWP methyl-lysine reading domain known to bind to H3K36me2 and H3K36me3. LEDGF and HDGF2 localize with H3K36me2/3 at genomic regions containing active genes, usually adjacent to H3K27me3 domains. In myoblasts, where FACT expression is low, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease across the genome while HDGF2 levels are maintained. Moreover, HDGF2 is recruited to the majority of myotube up-regulated genes and is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII proximal to the first nucleosome within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation. We propose that LEDGF and HDGF2 substitute for FACT in differentiated tissues and that their distribution on the genome helps maintain transcriptional programs unique to particular cell types. One Sentence Summary Chromatin bound LEDGF and HDGF2 proteins allow RNAPII to overcome the nucleosome-induced block to transcription.

H3K27M transiently recruits PRC2 to chromatin but persistently affects its activity, leading to an aberrant epigenome in DIPG., A methionine substitution at lysine-27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found that this interaction on chromatin is transient, with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-containing chromatin, suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to H3K27M, leading to the failure to spread H3K27me from PRC2 recruitment sites and consequently abrogating PRC2’s ability to establish H3K27me2-3 repressive chromatin domains. In turn, levels of polycomb antagonists such as H3K36me2 are elevated, suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

SummaryChromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.

SummaryPolycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting “nucleation sites”, creating H3K27me3-forming polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

SummaryErk1/2 activation contributes to mouse ES cell pluripotency. We found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation.

Chromatin immunoprecipitation (ChIP) is a technique used to determine the association of proteins or histone modifications with chromatin regions in living cells or tissues, and is used extensively in the chromatin biology field to study transcriptional and epigenetic mechanisms. Increasing evidence points to an epigenetic coordination of signaling cascades, such as ERK, that regulate key processes in development and disease, revealing novel principles of gene regulation. Here we describe a detailed protocol for performing chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) for probing histone modifications regulated by ERK signaling in mouse ESCs.

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.

SummaryGlioblastoma (GBM) is thought to be driven by a subpopulation of cancer stem cells (CSCs) that self-renew and recapitulate tumor heterogeneity yet remain poorly understood. Here, we present a comparative analysis of chromatin state in GBM CSCs that reveals widespread activation of genes normally held in check by Polycomb repressors. These activated targets include a large set of developmental transcription factors (TFs) whose coordinated activation is unique to the CSCs. We demonstrate that a critical factor in the set, ASCL1, activates Wnt signaling by repressing the negative regulator DKK1. We show that ASCL1 is essential for the maintenance and in vivo tumorigenicity of GBM CSCs. Genome-wide binding profiles for ASCL1 and the Wnt effector LEF-1 provide mechanistic insight and suggest widespread interactions between the TF module and the signaling pathway. Our findings demonstrate regulatory connections among ASCL1, Wnt signaling, and collaborating TFs that are essential for the maintenance and tumorigenicity of GBM CSCs.

SummaryThe aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.