Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes—including Pbrm1, Arid2, and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex—sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T cells.

Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C 2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C 2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.development ͉ myogenesis ͉ transcription S mall ribonucleotide-based regulators of gene expression known as microRNAs play important roles in regulatory gene expression (see refs. 1 and 2 for reviews). Although initially identified through their role in the development of Caenorhabditis elegans larvae (3, 4), microRNA abundance (5-7) and conservation is suggestive of a much broader role within both the animal and plant kingdoms. Studies in vertebrates and invertebrates have confirmed this notion: microRNAs play a fundamental role in diverse biological and pathological processes including apoptosis (8), cell fate determination (9-11), metabolism (12, 13) and tumorigenesis (14-16). microRNAs are synthesized as longer primary microRNAs (17, 18) that are sequentially processed in the nucleus and cytoplasm by distinct multiprotein complexes containing and Dicer͞TRBP (24-26), respectively. The resulting duplex is unwound by a helicase activity, and the mature single-stranded RNA is loaded onto the RNA-induced silencing complex, wherein interactions between microRNAs and mRNAs occur. microRNAs repress gene expression by cleaving target mRNAs or by inhibiting the ability of the target mRNA to be translated into protein. Computational approaches have yielded hundreds of putative mRNA targets for individual microRNAs (27-31), but the number of verified targets with biological relevance is still very small.We are interested in studying the ability of microRNAs to regulate cell differentiation. Our work focuses on three microRNAs specifically and abundantly expressed in muscle tissue, miR-1, miR-133, and miR-206 (32-34). The overrepresentation of these and other microRNAs in various differentiated tissues implicates microRNAs in the determination or maintenance of the differentiated sta...

Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1-3. BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine recognition modules, leading to inhibition of oncogenic transcriptional programs7-9. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

RATIONALE Heart failure is a deadly and devastating disease that places immense costs on an aging society. In order to develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function. OBJECTIVE microRNAs are important regulators of gene expression and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity. METHODS AND RESULTS First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Secondly, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality. CONCLUSIONS These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs maybe ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.

DNase-seq is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize the performance of DNase-seq. We found that sequencing short 50-100bp fragments that accumulate in long inter-nucleosome linker regions is more efficient for identifying transcription factor binding sites than using longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy through the generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNaseI cutting bias we found a surprisingly strong effect that varied over more than two orders of magnitude. This confounds DNaseI footprint analysis to the extent that the nucleotide resolution cleavage patterns at most transcription factor binding sites are derived from intrinsic DNaseI cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNaseI hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations.

In the originally published version of this article, coauthor Andrew M. Intlekofer was listed incorrectly as Andrew M. Intlekoffer and coauthor Nicole R. LeBoeuf was listed incorrectly as Nicole LaBoeuf. These errors have now been corrected here and in the article online. The authors apologize for the errors and any inconvenience that may have resulted.

Highlights d ARfl and ARv7 genomic binding is interdependent and colocalized d ARv7, unlike ARfl, preferentially represses transcription d Expression of ARv7-repressed genes negatively correlates with recurrence d Re-expression of ARv7-repressed genes may serve as a biomarker of ARv7 inhibition