Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h (refs 2-6). During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. Here we show that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin.

Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR/Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by being a comprehensive pipeline that contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control, normalization, batch effect removal, copy number bias correction, gene hit identification, and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires approximately two hours on a desktop computer running Linux or Mac OS and with R support. The MAGeCKFlute package is available at http://www.bioconductor.org/packages/release/bioc/html/MAGeCKFlute.html.