Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.

The hSNF5 chromatin-remodeling factor is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). A number of studies have shown that hSNF5 re-expression blocks MRT cell proliferation. However, the pathway through which hSNF5 acts remains unknown. To address this question, we generated MRT-derived cell lines in which restoration of hSNF5 expression leads to an accumulation in G 0 /G 1 , induces cellular senescence and increased apoptosis.

Nucleotide biosynthesis is fundamental to normal cell proliferation as well as to oncogenesis. Tumor suppressor p53, which prevents aberrant cell proliferation, is destabilized through ubiquitylation by MDM2. Ubiquitin-specific protease 7 (USP7) plays a dualistic role in p53 regulation and has been proposed to deubiquitylate either p53 or MDM2. Here, we show that guanosine 5 0 -monophosphate synthase (GMPS) is required for USP7-mediated stabilization of p53. Normally, most GMPS is sequestered in the cytoplasm, separated from nuclear USP7 and p53. In response to genotoxic stress or nucleotide deprivation, GMPS becomes nuclear and facilitates p53 stabilization by promoting its transfer from MDM2 to a GMPS-USP7 deubiquitylation complex. Intriguingly, cytoplasmic sequestration of GMPS requires ubiquitylation by TRIM21, a ubiquitin ligase associated with autoimmune disease. These results implicate a classic nucleotide biosynthetic enzyme and a ubiquitin ligase, better known for its role in autoimmune disease, in p53 control.

Polycomb group (PcG) proteins function through cis-acting DNA elements called PcG response elements (PREs) to stably silence developmental regulators, including the homeotic genes. However, the mechanism by which they are targeted to PREs remains largely unclear. Pleiohomeotic (PHO) is a sequence-specific DNAbinding PcG protein and therefore may function to tether other PcG proteins to the DNA. Here, we show that PHO can directly bind to a Polycomb (PC)-containing complex as well as the Brahma (BRM) chromatinremodeling complex. PHO contacts the BRM complex through its zinc finger DNA-binding domain and a short N-terminal region. A distinct domain of PHO containing a conserved motif contacts the PcG proteins PC and Polyhomeotic (PH). With mobility shift assays and DNA pulldown experiments, we demonstrated that PHO is able to link PC, which lacks sequence-specific DNA-binding activity, to the DNA. Importantly, we found that the PC-binding domain of PHO can mediate transcriptional repression in transfected Drosophila Schneider cells. Concomitant overexpression of PC resulted in stronger PHO-directed repression that was dependent on its PC-binding domain. Together, these results suggest that PHO can contribute to PRE-mediated silencing by direct recruitment of a PC complex to repress transcription.

Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.