SUMMARY Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 co-localizes with Tet1 and 5hmC in vivo, Mbd3 knockdown preferentially affects expression of 5hmC-marked genes, Mbd3 localization is Tet1-dependent, and Mbd3 preferentially binds to 5hmC relative to 5-methylcytosine in vitro. Finally, both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.

Summary The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in S. cerevisiae meiosis I and inhibiting merotelic attachment in S. pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned ∼10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, cross-linking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and cross-linking rDNA repeats to aid rDNA silencing.

During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5's central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern.P olo kinases are central regulators of chromosome segregation and control multiple mitotic events (1). Budding yeast contains a single Polo kinase, CDC5. Unlike in higher eukaryotes, budding yeast CDC5 primarily regulates postmetaphase events, its essential function being to trigger exit from mitosis (2). CDC5 also contributes to the efficient inactivation of cohesins, the protein complexes that hold sister chromatids together until the onset of chromosome segregation. Cdc5 phosphorylates the cohesin subunit Mcd1/Scc1 to facilitate its cleavage by the protease separase (3).CDC5 also regulates the specialized cell division that gives rise to gametes, known as meiosis (4). During meiosis, two consecutive rounds of chromosome segregation follow one round of DNA replication. During meiosis I, homologous chromosomes segregate; during meiosis II, sister chromatids separate (5). The chromosome segregation machinery is modified in three ways to facilitate the unusual meiosis I division. First, the combination of homologous recombination and cohesin complexes distal to the resulting cross-overs mediate the physical linkage of homologous chromosomes, which is essential for their accurate segregation during meiosis I. Second, sister chromatids of each homolog must be segregated to the same pole rather than to opposite poles, as they are during mitosis. The fusion of sister kinetochores by co-orientation factors (the monopolin complex in yeast) facilitates the attachment of microtubules emanating from one spindle pole. Third, cohesin complexes must be lost in a stepwise manner from chromosomes. During meiosis I cohesin complexes are lost from chromosome arms to bring about the segregation of homologous chromosomes (6). The residual cohesins at centromeres facilitate the accurate segregation of sister chromatids during meiosis II. Cdc5 has been implicated in the execution of all three meiosis I-specific events. CDC5 is required for the resolution of double Holliday junctions during homologous recombination (7,8). Cdc5 also controls the co-orientation of ...

In budding yeast, a signaling network known as the mitotic exit network (MEN) triggers exit from mitosis. We find that hypertonic stress allows MEN mutants to exit from mitosis in a manner dependent on the high osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase cascade. The HOG pathway drives exit from mitosis in MEN mutants by promoting the activation of the MEN effector, the protein phosphatase Cdc14. Activation of Cdc14 depends on the Cdc14 early anaphase release network, a group of proteins that functions in parallel to the MEN to promote Cdc14 function. Notably, exit from mitosis is promoted by the signaling branch defined by the Sho1 osmosensing system, but not by the Sln1 osmosensor of the HOG pathway. Our results suggest that the stress MAP kinase pathway mobilizes programs to promote completion of the cell cycle and entry into G 1 under unfavorable conditions.