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Methyl CpG binding protein 2 (MeCP2) is a key component of constitutive heterochromatin, which is crucial for chromosome maintenance and transcriptional silencing1–3. Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome3–5, which is associated with severe mental disability and autism-like symptoms that affect girls during early childhood. Although previously thought to be a dense and relatively static structure1,2, heterochromatin is now understood to exhibit properties consistent with a liquid-like condensate6,7. Here we show that MeCP2 is a dynamic component of heterochromatin condensates in cells, and is stimulated by DNA to form liquid-like condensates. MeCP2 contains several domains that contribute to the formation of condensates, and mutations in MECP2 that lead to Rett syndrome disrupt the ability of MeCP2 to form condensates. Condensates formed by MeCP2 selectively incorporate and concentrate heterochromatin cofactors rather than components of euchromatic transcriptionally active condensates. We propose that MeCP2 enhances the separation of heterochromatin and euchromatin through its condensate partitioning properties, and that disruption of condensates may be a common consequence of mutations in MeCP2 that cause Rett syndrome. The chromatin protein MeCP2 is a component of dynamic, liquid-like heterochromatin condensates, and the ability of MeCP2 to form condensates is disrupted by mutations in the MECP2 gene that occur in the neurodevelopmental disorder Rett syndrome.

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.

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)

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.

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.