No abstract

Summary Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8 driven cancers.

Summary Polycomb Group RING finger homologs (PCGF1, 2, 3, 4, 5 and 6) are critical components in the assembly of distinct Polycomb Repression Complex 1 (PRC1) related complexes. Here we identify a protein interaction domain in BCL6 co-repressor, BCOR, which binds the ubiquitin-like RAWUL domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold Discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals 1. that PUFD binds to the same surfaces as observed for a different Polycomb Group RAWUL domain and 2. the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data are the first to show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

The polycomb group proteins are required for the stable maintenance of gene repression patterns established during development. They function as part of large multiprotein complexes created via a multitude of proteinprotein interaction domains. Here we examine the interaction between the SAM domains of the polycomb group proteins polyhomeotic (Ph) and Sex-comb-on-midleg (Scm). Previously we showed that Ph-SAM polymerizes as a helical structure. We find that Scm-SAM also polymerizes, and a crystal structure reveals an architecture similar to the Ph-SAM polymer. These results suggest that Ph-SAM and Scm-SAM form a copolymer. Binding affinity measurements between Scm-SAM and Ph-SAM subunits in different orientations indicate a preference for the formation of a single junction copolymer. To provide a model of the copolymer, we determined the structure of the Ph-SAM/Scm-SAM junction. Similar binding modes are observed in both homo-and heterocomplex formation with minimal change in helix axis direction at the polymer joint. The copolymer model suggests that polymeric Scm complexes could extend beyond the local domains of polymeric Ph complexes on chromatin, possibly playing a role in long range repression.The PcG 1 proteins form large multiprotein complexes that repress transcription over long distances and maintain repressed states in a stable and heritable manner during embryogenesis, development, and into adulthood (1, 2). PcG mutations result in both developmental defects and cancer (3-5). The formation of such large complex structures requires the precise organization of many proteins of the PcG. Indeed, protein-protein interaction domains are the most common functional motifs that have been identified in the PcG family. Nevertheless, few of these domains have been characterized in detail, and the structural architecture of the resultant protein complexes is largely unknown.One protein-protein interaction domain present in the PcG family is the SAM (sterile alpha motif) domain (often called SPM in PcG literature) found in polyhomeotic (Ph) and Sexcomb-on-midleg (Scm). SAM domains are small, helical protein modules found in proteins with diverse functional roles ranging from receptor tyrosine kinases to transcription factors (6). Unlike many other protein-protein interaction modules that have a common, well defined function such as SH2 domains, SAM domains can play diverse roles. To date, SAM domains are known to form homo-and heterooligomers that can be polymeric (7-9) or have a discrete oligomeric state (10), they can bind to other proteins (11, 12), and they can even bind RNA (13,14).Ph and Scm proteins co-localize on polytene chromosomes (15) and can interact via their SAM domains (15-17). The interaction between Ph and Scm appears to be complex. In addition to binding to each other, the SAM domains of Ph and Scm can also self-associate and thus exist in a higher oligomeric state of their own (8,15,16). Although they appear to work at the same sites, isolation of the soluble form of one PcG protein complex, PR...