Summary The heterogeneous nature of mammalian PRC1 complexes has hindered our understanding of their biological functions. Here, we present a comprehensive proteomic and genomic analysis that uncovered six major groups of PRC1 complexes each containing a distinct PCGF subunit, a RING1A/B ubiquitin ligase, and a unique set of associated polypeptides. These PRC1 complexes differ in their genomic localization and only a small subset co-localize with H3K27me3. Further biochemical dissection revealed that the six PCGF-RING1A/B combinations form multiple complexes through association with RYBP or its homolog YAF2, which prevents the incorporation of other canonical PRC1 subunits such as CBX, PHC and SCM. Although both RYBP/YAF2- and CBX/PHC/SCM-containing complexes compact chromatin, only RYBP stimulates the activity of RING1B toward H2AK119ub1, suggesting a central role in PRC1 function. Knockdown of RYBP in ES cells compromised their ability to form embryoid bodies, likely because of defects in cell proliferation and maintenance of H2AK119ub1 level.

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We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.chip-Seq | chromatin modifications | muscle development | transcriptional regulation R egulation of the transcriptome through dynamic changes in chromatin plays an important role in lineage commitment and differentiation. Multiple histone modifications control gene expression through recruitment of factors that alter compaction of the chromatin fiber. Transient and long-term gene silencing is enforced through trimethylation of histone H3 on lysines 9 and 27 (hereafter H3K9me3 and H3K27me3) as well as H4K20, whereas gene activation is regulated by methylation of H3K4 and acetylation of the amino-terminal tails of H3 and H4 (reviewed in refs. 1 and 2). Chromatin modifications are often asymmetrically deposited with respect to the transcription start sites (TSS) of genes. Whereas H3K27me3 is found at promoters, throughout gene bodies, and in intergenic regions, histone tail acetylation and H3K4me3 are predominantly found at promoters and the 5′ ends of genes. On the other hand, H3K36 trimethylation marks gene bodies, signifying the passage of RNA polymerase II (PolII) on actively transcribed genes. Promoter acetylation and H3K4 trimethylation are often coordinated, whereas H3K27 and H3K4 trimethylation are largely anticorrelated, except within bivalent regions poised to adopt either active or repressed states at the appropriate developmental stage (3).Previous studies have shown that the pluripotent state of embryonic stem (ES) cells is in part governed by bivalent nucleosomes, characterized by simultaneous H3K4 and H3K27 trimethylation of nucleosomes in lineage commitment genes (3, 4). During ES cell differentiation, ...

While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2␣ phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumorpromoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis.During tumorigenesis, a disorganized vasculature leads to amino acid and glucose deprivation, cellular hypoxia, the accumulation of reactive oxygen species (ROS), and various other stresses (5, 12). Cellular adaptation to the hostile tumor microenvironment requires the regulation of stress-induced genes (reviewed in reference 16). For example, the transcription factor ATF-4, upregulated in human tumors due to the stress-induced phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF2␣), transactivates genes involved in amino acid metabolism, angiogenesis, and ROS attenuation (2,3,33). Cells that cannot phosphorylate eIF2␣ or that are deficient in ATF-4 and other stress-induced transcription factors do not form tumors in vivo (2, 13, 40), and therefore, a major goal in cancer biology has been to better understand and potentially target these adaptive mechanisms. However, while the translational and transcriptional responses that promote adaptation to the tumor microenvironment are well established, the role of mRNA stabilization in the cellular stress response has not been as thoroughly studied.Nonsense-mediated RNA decay (NMD) degrades up to 30% of all mutated protein-coding mRNAs, including those responsible for many genetic disorders, such as thalassemia, cystic fibrosis, and muscular dystrophy (11). During the processing of mammalian pre-mRNA, introns are excised and marked by an exon junction complex, which contains core NMD components. Newly synthesized mRNAs are thought to undergo a pioneering round of translation by a complex that includes eIF2␣ (6). When this translation complex pauses at a premature termination codon (PTC) upstream of an exon junction complex, the RNA helicase UPF1/Rent1, an essential component of the NMD process, is recruited and the...

BRAFV600E/K is a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAFV600E/K and BRAFWT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAFV600E/K, it activated the pathway in the resistant BRAFWT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAFWT melanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAFWT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

Summary Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5HI/SLC1A5/38A2LO expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.

Revealing the clonal composition of a single tumor is essential for identifying cell subpopulations with metastatic potential in primary tumors or with resistance to therapies in metastatic tumors. Sequencing technologies provide only an overview of the aggregate of numerous cells. Computational approaches to de-mix a collective signal composed of the aberrations of a mixed cell population of a tumor sample into its individual components are not available. We propose an evolutionary framework for deconvolving data from a single genome-wide experiment to infer the composition, abundance and evolutionary paths of the underlying cell subpopulations of a tumor. We have developed an algorithm (TrAp) for solving this mixture problem. In silico analyses show that TrAp correctly deconvolves mixed subpopulations when the number of subpopulations and the measurement errors are moderate. We demonstrate the applicability of the method using tumor karyotypes and somatic hypermutation data sets. We applied TrAp to Exome-Seq experiment of a renal cell carcinoma tumor sample and compared the mutational profile of the inferred subpopulations to the mutational profiles of single cells of the same tumor. Finally, we deconvolve sequencing data from eight acute myeloid leukemia patients and three distinct metastases of one melanoma patient to exhibit the evolutionary relationships of their subpopulations.

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