Necessary and sufficient role for a mitosis skip in senescence induction.

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

The replication foci targeting sequence (RFTS) of DNMT1 functions as a potent histone H3 binding domain regulated by autoinhibition.

DNA methyltransferase 1 (DNMT1) plays an essential role in propagation of the DNA methylation pattern to daughter cells. The replication foci targeting sequence (RFTS) of DNMT1 is required for the recruitment of DNMT1 to DNA methylation sites through direct binding to ubiquitylated histone H3 mediated by UHRF1 (Ubiquitin-like containing PHD and RING finger domains 1). Recently, it has been reported that the RFTS plugs the catalytic pocket of DNMT1 in an intermediated manner and inhibits its DNA methyltransferase activity. However, it is unclear whether this binding affects RFTS function in terms of recruitment to DNA methylation sites. Using Xenopus egg extracts, we demonstrate here that abrogation of the interaction between the RFTS and the catalytic center of DNMT1, by deletion of the C-terminal portion or disruption of the hydrogen bond, results in non-ubiquitylated histone H3 binding and abnormal accumulation of DNMT1 on the chromatin. Interestingly, DNMT1 mutants identified in patients with a neurodegenerative disease, ADCA-DN, bound to non-ubiquitylated histone H3 and accumulated on chromatin during S phase in Xenopus egg extracts. These results suggest that the interaction between the RFTS and the catalytic center of DNMT1 serves as an autoinhibitory mechanism for suppressing the histone H3 binding of DNMT1 and ensuring the accurate recruitment of DNMT1 to sites of DNA methylation. The autoinhibitory mechanism may play an important role in the regulation of gene expression in neurogenesis.

Mammal-specific H2A variant, H2ABbd, is involved in apoptotic induction via activation of NF-κB signaling pathway.

Histone variants play specific roles in maintenance and regulation of chromatin structures. H2ABbd, an H2A variant, possesses a highly divergent structure compared with canonical H2A and is highly expressed in postmeiotic germ cells, but its functions in the regulation of gene expression are largely unknown. In the present study, we investigated the cellular phenotype associated with enforced H2ABbd expression. Among H2A variants, H2ABbd specifically caused growth defect in human cells and induced apoptosis. H2ABbd expression resulted in degradation of inhibitor of κB-α and translocation of NF-κB into nuclei, indicating the activation of NF-κB. Intriguingly, NF-κB activity was essential for H2ABbd-induced apoptosis. H2ABbd overexpression resulted in DNA damage after release from G1/S, progressed through the S phase slowly, and induced apoptosis. Furthermore, gene expression microarray analysis revealed that expression of H2ABbd activates groups of genes involved in apoptosis and postmeiotic germ cell development, suggesting that H2ABbd might influence transcription. Taken together, our data suggest that H2ABbd may contribute to specific chromatin structures and promote NF-κB activation, which could in turn induce apoptosis in mammalian cells.

Mitotic phosphorylation of MPP8 by cyclin-dependent kinases regulates chromatin dissociation.

Repressive epigenetic modifications, DNA methylation at CpG sites and histone H3 lysine 9 (H3K9) methylation, are enriched in heterochromatin, which undergoes drastic changes in structure during mitosis. MPP8 (M phase phosphoprotein 8) has been proposed to regulate positive association between these two repressive modifications, but actual involvement of this protein in changes in the heterochromatin structure during mitosis remains elusive. We demonstrate here that MPP8 predominantly localized to, but dissociated from, chromatin during interphase and early mitosis, respectively. Chromatin dissociation from MPP8 appeared to correlate with the phosphorylation status of MPP8. Experiments using inhibitors of various mitotic kinases demonstrated that the chromatin dissociation of MPP8 during metaphase to anaphase was specifically regulated by cyclin B1-Cdk1. Indeed, cyclin B1-Cdk1 effectively phosphorylated MPP8 in vitro and on STA mutant of MPP8 (all possible sites phosphorylated by Cdk were substituted by alanine) failed to dissociate from chromatin during early mitosis. Taken together, our results indicate that the chromatin association of MPP8 is regulated by Cdk-dependent phosphorylation.

Retinoic acid stimulates transcription of the rat SHARP-2 gene via multiple pathways.

Members of the enhancer of split- and hairy-related protein (SHARP) family, SHARP-1 and SHARP-2, are basic helix-loop-helix transcriptional repressors and belong to the clock genes. In this study, an effect of retinoic acid (RA) on the SHARP family gene expression in the differentiated cells was examined. RA rapidly and temporarily induced the SHARP-2 mRNA expression in hepatic H4IIE cells. Then, whether the SHARP-2 mRNA expression is altered by dexamethasone (Dex), insulin, and the combination of RA and Dex or RA and insulin was examined. Dex had different effects on the expression of SHARP-2 mRNA in the presence or absence of RA. Then, the molecular mechanisms were investigated using inhibitors of various signaling molecules. The RA-induction of SHARP-2 mRNA level was mainly inhibited by LY294002, staurosporine, and actinomycin D, respectively. Finally, whether RA acts on the transcriptional regulatory region of the SHARP-2 gene was analysed using luciferase reporter gene assay. At least two RA-responsive regions were mapped at the nucleotide sequences between -3,700 and -1,600 of the SHARP-2 gene. In addition, this effect was dependent on the RA receptor and retinoid X receptor. Thus, we conclude that RA stimulated transcription of the SHARP-2 gene via multiple pathways.

Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.