PRC2 is high maintenance.

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.

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.

Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.

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.

TGF-ß/Smad signaling through DOCK4 facilitates lung adenocarcinoma metastasis.

The mechanisms by which TGF-ß promotes lung adenocarcinoma (ADC) metastasis are largely unknown. Here, we report that in lung ADC cells, TGF-ß potently induces expression of DOCK4, but not other DOCK family members, via the Smad pathway and that DOCK4 induction mediates TGF-ß's prometastatic effects by enhancing tumor cell extravasation. TGF-ß-induced DOCK4 stimulates lung ADC cell protrusion, motility, and invasion without affecting epithelial-to-mesenchymal transition. These processes, which are fundamental to tumor cell extravasation, are driven by DOCK4-mediated Rac1 activation, unveiling a novel link between TGF-ß and Rac1. Thus, our findings uncover the atypical Rac1 activator DOCK4 as a key component of the TGF-ß/Smad pathway that promotes lung ADC cell extravasation and metastasis.

The Dearth of Representation in FDA Approved Drug Trials.

The generalizability of data derived from randomized controlled trials is of paramount importance given their utility in the Food & Drug Administration (FDA) drug approval process. An essential part of this process is the inclusion of reliably reported gender, race and ethnicity data in trials that lead to FDA drug approval. Despite previous mandates by the FDA and Clinicaltrials.gov, gender and race-specific data remains under reported. We reviewed 100 most recently approved FDA medications, and abstracted the clinical trial data from Clinicaltrials.gov that supported their approval. We then compared these FDA approved trials to non-FDA approved trials from the same year and of similar size. We found that 40% of the FDA trials were missing race/ethnicity information, while 24% of these trials did not include gender information. We demonstrate that there remains a significant amount of missing gender and racial/ethnic data in trials that lead to FDA-approved medications.

Inheritance of repressed chromatin domains during S phase requires the histone chaperone NPM1.

The epigenetic process safeguards cell identity during cell division through the inheritance of appropriate gene expression profiles. We demonstrated previously that parental nucleosomes are inherited by the same chromatin domains during DNA replication only in the case of repressed chromatin. We now show that this specificity is conveyed by NPM1, a histone H3/H4 chaperone. Proteomic analyses of late S-phase chromatin revealed NPM1 in association with both H3K27me3, an integral component of facultative heterochromatin, and MCM2, an integral component of the DNA replication machinery; moreover, NPM1 interacts directly with PRC2 and with MCM2. Given that NPM1 is essential, the inheritance of repressed chromatin domains was examined anew using mESCs expressing an auxin-degradable version of endogenous NPM1. Upon NPM1 degradation, cells accumulated in the G1-S phase of the cell cycle and parental nucleosome inheritance from repressed chromatin domains was markedly compromised. NPM1 chaperone activity may contribute to the integrity of this process as appropriate inheritance required the NPM1 acidic patches.

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.

Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-Associated L1CAM.

Among the diverse interneuron subtypes in the neocortex, chandelier cells (ChCs) are the only population that selectively innervate pyramidal neurons (PyNs) at their axon initial segment (AIS), the site of action potential initiation, allowing them to exert powerful control over PyN output. Yet, mechanisms underlying their subcellular innervation of PyN AISs are unknown. To identify molecular determinants of ChC/PyN AIS innervation, we performed an in vivo RNAi screen of PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, we found the L1 family member L1CAM to be the only molecule required for ChC/PyN AIS innervation. Further, we show that L1CAM is required during both the establishment and maintenance of innervation, and that selective innervation of PyN AISs by ChCs requires AIS anchoring of L1CAM by the cytoskeletal ankyrin-G/ßIV-spectrin complex. Thus, our findings identify PyN-expressed L1CAM as a critical CAM required for innervation of neocortical PyN AISs by ChCs. VIDEO ABSTRACT.

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells.

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.

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.

Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain.

Throughout life, stem cells in the ventricular-subventricular zone generate neuroblasts that migrate via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into local interneurons. Although progress has been made toward identifying extracellular factors that guide the migration of these cells, little is known about the intracellular mechanisms that govern the dynamic reshaping of the neuroblasts' morphology required for their migration along the RMS. In this study, we identify DOCK7, a member of the DOCK180-family, as a molecule essential for tangential neuroblast migration in the postnatal mouse forebrain. DOCK7 regulates the migration of these cells by controlling both leading process (LP) extension and somal translocation via distinct pathways. It controls LP stability/growth via a Rac-dependent pathway, likely by modulating microtubule networks while also regulating F-actin remodeling at the cell rear to promote somal translocation via a previously unrecognized myosin phosphatase-RhoA-interacting protein-dependent pathway. The coordinated action of both pathways is required to ensure efficient neuroblast migration along the RMS.