The H3K36me2 writer-reader dependency in H3K27M-DIPG.

Histone H3K27M is a driving mutation in diffuse intrinsic pontine glioma (DIPG), a deadly pediatric brain tumor. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity and displacement of H3K27me2/3, promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation and tumorigenesis by disrupting tumor-promoting transcriptional programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers mediating the protumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG.

Active and Repressed Chromatin Domains Exhibit Distinct Nucleosome Segregation during DNA Replication.

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. However, 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 so that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through 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 of 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.

RNA Interactions Are Essential for CTCF-Mediated Genome Organization.

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.

Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.

Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.

Automethylation of PRC2 promotes H3K27 methylation and is impaired in H3K27M pediatric glioma.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.

Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma.

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.

Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2.

The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

Transcriptome-wide identification of in vivo interactions between RNAs and RNA-binding proteins by RIP and PAR-CLIP assays.

Comprehensive genomic and computational studies in the era of high-throughput sequencing revealed that the major proportion of the human genome is transcribed. This novel insight confronted the scientific community with new questions concerning the expanded role of RNA, especially noncoding RNA (ncRNA), in cellular pathways. In recent years, there has been mounting evidence that ncRNAs and RNA binding proteins (RBPs) are involved in a wide range of biological processes, such as developmental transitions, cell differentiation, stress response, genome organization, and regulation of gene expression. In particular, in the chromatin field long noncoding RNAs (lncRNAs) have drawn increasing attention to their function in epigenetic regulation due to the fact that they were found to interact with multiple chromatin regulators and modifiers. Recently, techniques to study the extent of RNA-protein interactions have been developed in many research laboratories. Here we describe protocols for RNA Immunoprecipitation-Sequencing (RIP-Seq) and Photoactivatable-Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation combined with deep sequencing (PAR-CLIP-Seq) to identify RNA targets of RNA-binding proteins (RBPs) on a transcriptome-wide level, discussing advantages and drawbacks.

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.

Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.

JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

Transcriptional and epigenetic regulation of the p53 tumor suppressor gene.

The p53 tumor suppressor is one of the most studied molecules in cancer research. Despite the fact that there is a detailed understanding involving multiple aspects of the p53-associated biology, many aspects of its transcriptional regulation are still not well clarified. Limited information is available on how the p53 gene is transcriptional and epigenetically regulated. The p53 gene expression is tightly controlled through a variety of transcription factors, miRNAs, its anti-sense RNA Wrap53, the insulator protein CTCF and very likely by other genetic and epigenetic mechanisms. It's the intent of this article to review in depth important aspects concerning the transcriptional regulation of the p53 gene and perhaps serve as a stepping-stone to begin a conceptual change on how future p53 research can be approached.

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron.

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.