Masashige Bando

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (∼5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

Rapid discovery of highly potent and selective inhibitors of histone deacetylase 8 using click chemistry to generate candidate libraries.

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 μM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation

Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells

The Smc5/6 complex plays a critical role in processing DNA replication and is indispensable for sister chromatid assembly and faithful segregation in mitosis.

Esco1 Acetylates Cohesin via a Mechanism Different from That of Esco2

Sister chromatid cohesion is mediated by cohesin and is essential for accurate chromosome segregation. The cohesin subunits SMC1, SMC3, and Rad21 form a tripartite ring within which sister chromatids are thought to be entrapped. This event requires the acetylation of SMC3 and the association of sororin with cohesin by the acetyltransferases Esco1 and Esco2 in humans, but the functional mechanisms of these acetyltransferases remain elusive. Here, we showed that Esco1 requires Pds5, a cohesin regulatory subunit bound to Rad21, to form cohesion via SMC3 acetylation and the stabilization of the chromatin association of sororin, whereas Esco2 function was not affected by Pds5 depletion. Consistent with the functional link between Esco1 and Pds5, Pds5 interacted exclusively with Esco1, and this interaction was dependent on a unique and conserved Esco1 domain. Crucially, this interaction was essential for SMC3 acetylation and sister chromatid cohesion. Esco1 localized to cohesin localization sites on chromosomes throughout interphase in a manner that required the Esco1-Pds5 interaction, and it could acetylate SMC3 before and after DNA replication. These results indicate that Esco1 acetylates SMC3 via a mechanism different from that of Esco2. We propose that, by interacting with a unique domain of Esco1, Pds5 recruits Esco1 to chromatin-bound cohesin complexes to form cohesion. Furthermore, Esco1 acetylates SMC3 independently of DNA replication.