Laura Magnaghi-Jaulin

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.

Mps1 Is a Kinetochore-Associated Kinase Essential for the Vertebrate Mitotic Checkpoint

The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.

The APC is dispensable for first meiotic anaphase in Xenopus oocytes

Here we show that segregation of homologous chromosomes and that of sister chromatids are differentially regulated in Xenopus and possibly in other higher eukaryotes. Upon hormonal stimulation, Xenopus oocytes microinjected with antibodies against the anaphase-promoting complex (APC) activator Fizzy or the APC core subunit Cdc27, or with the checkpoint protein Mad2, a destruction-box peptide or methylated ubiquitin, readily progress through the first meiotic cell cycle and arrest at second meiotic metaphase. However, they fail to segregate sister chromatids and remain arrested at second meiotic metaphase when electrically stimulated or when treated with ionophore A34187, two treatments that mimic fertilization and readily induce chromatid segregation in control oocytes. Thus, APC is required for second meiotic anaphase but not for first meiotic anaphase.

Histone Deacetylase Inhibitors Induce Premature Sister Chromatid Separation and Override the Mitotic Spindle Assembly Checkpoint

Histone deacetylase inhibitors (HDACI) are powerful antiproliferative drugs, and are currently undergoing clinical trials as antitumor agents. It would be valuable for both cancer therapy and our knowledge of basic cellular processes to understand the mechanisms by which HDACIs block cell proliferation. Most current models postulate that HDACIs allow the reexpression of tumor suppressor genes silenced in cancer cells. However, other mechanisms, distinct from transcription regulation, may participate in HDACI antiproliferative properties. We report that HDACI treatment induces premature sister chromatid separation in cells in which the mitotic spindle assembly checkpoint (SAC) has already been activated. This effect was transcription-independent. In addition, HDACI-treated mitotic cells displayed SAC inactivation characteristics, including anaphase-promoting complex/cyclosome target degradation, cyclin-dependent kinase 1 inactivation, histone H3 dephosphorylation, and loss of the SAC component MAD2 from the kinetochore. Thus, HDAC inhibition renders the SAC ineffective. Our findings help elucidate the molecular mechanisms of proliferative cell death induced by HDACI treatment and may allow new HDACI-based preclinical and clinical trial protocols to be redesigned so as to target mitosis.

Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis

Chromosome condensation is thought to be an essential step for the faithful transmission of genetic information during cellular division or gamete formation. The folding of DNA into metaphase chromosomes and its partition during the cell cycle remains a fundamental cellular process that, at the molecular level, is poorly understood. Particularly, the role of histone deacetylase (HDAC) activities in establishing and maintaining meiotic metaphase chromosome condensation has been little documented. In order to better understand how metaphase chromosome condensation is achieved during meiosis, we explored, in vivo, the consequences of HDAC activities inhibition in a Xenopus oocyte model. Our results show that deacetylase activity plays a crucial role in chromosome condensation. This activity is necessary for correct chromosome condensation since the earlier stages of meiosis, but dispensable for meiosis progression, meiosis exit and mitosis entry. We show that HDAC activity correlates with chromosome condensation, being higher when chromosomes are fully condensed and lower during interphase, when chromosomes are decondensed. In addition, we show that, unlike histone H4, Xenopus maternal histone H3 is stored in the oocyte as a hypoacetylated form and is rapidly acetylated when the oocyte exits meiosis.

Activation of the c-fos SRE through SAP-1a

TCFs, which are members of the Ets family of transcription factors, are recruited to the Serum Response Element (SRE) in the c-fos promoter by SRF. These Ets proteins, which are substrates for the MAP kinases, are direct targets of the Ras/MAP kinase signal transduction pathway. In this paper, we demonstrate that one of the TCFs, SAP-1a, displays a significant level of autonomous binding to the SRE Ets box. In contrast to previous observations, deletion of the SRF binding domain did not modulate the autonomous binding of SAP-1a. Also, the autonomous binding was not modulated by the phosphorylation of SAP-1a by MAP kinases. The autonomous binding was also detected in live cells: transfected SAP-1a was able to restore the response of a CArG-less SRE in PC12 cells. The response occurred in the absence of SRF recruitment since a mutant of SAP-1a in which the B-box, a domain required for interaction with SRF, had been deleted was still able to transactivate the CArG-less SRE. The transactivation was repressed by a Ras transdominant negative mutant, indicating the involvement of the Ras/MAP kinase pathway. Taken together, these data demonstrate that SAP-1a is capable of binding to the c-fos SRE in the absence of SRF.

CDK11p58 kinase activity is required to protect sister chromatid cohesion at centromeres in mitosis

The cyclin-dependent kinase CDK11p58 is specifically expressed at G2/M phase. CDK11p58 depletion leads to different cell cycle defects such as mitotic arrest, failure in centriole duplication and centrosome maturation, and premature sister chromatid separation. We report that upon CDK11 depletion, loss of sister chromatid cohesion occurs during mitosis but not during G2 phase. CDK11p58 depletion prevents Bub1 and Shugoshin 1 recruitment but has no effect on the dimethylation of histone H3 lysine 4 at centromeres. We also report that a construct expressing a kinase dead version of CDK11p58 fails to prevent CDK11 depletion-induced sister chromatid separation, showing that CDK11p58 kinase activity is required for protection of sister chromatid cohesion at centromeres during mitosis. Thus, CDK11p58 kinase activity appears to be involved in early events in the establishment of the centromere protection machinery.

Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue

Sustained spindle tension applied to sister centromeres during mitosis eventually leads to uncoordinated loss of sister chromatid cohesion, a phenomenon known as “cohesion fatigue.” We report that Aurora A-dependent phosphorylation of serine 7 of the centromere histone variant CENP-A (p-CENP-AS7) protects bioriented chromosomes against cohesion fatigue. Expression of a non-phosphorylatable version of CENP-A (CENP-AS7A) weakens sister chromatid cohesion only when sister centromeres are under tension, providing the first evidence of a regulated mechanism involved in protection against passive cohesion loss. Consistent with this observation, p-CENP-AS7 is detected at the inner centromere where it forms a discrete domain. The depletion or inhibition of Aurora A phenocopies the expression of CENP-AS7A and we show that Aurora A is recruited to centromeres in a Bub1-dependent manner. We propose that Aurora A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules.Sustained spindle tension applied to sister centromeres during mitosis leads to loss of sister chromatid cohesion which is known as cohesion fatigue. Here the authors show that Aurora A-dependent phosphorylation of CENP-A at the inner centromeres protects bioriented chromosomes against cohesion fatigue.

Quand la dynamique chromosomique contrôle la division cellulaire

In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.