Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells.

Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.

Identification of GA-Binding Protein Transcription Factor Alpha Subunit (GABPA) as a Novel Bookmarking Factor.

Mitotic bookmarking constitutes a mechanism for transmitting transcriptional patterns through cell division. Bookmarking factors, comprising a subset of transcription factors (TFs), and multiple histone modifications retained in mitotic chromatin facilitate reactivation of transcription in the early G1 phase. However, the specific TFs that act as bookmarking factors remain largely unknown. Previously, we identified the "early G1 genes" and screened TFs that were predicted to bind to the upstream region of these genes, then identified GA-binding protein transcription factor alpha subunit (GABPA) and Sp1 transcription factor (SP1) as candidate bookmarking factors. Here we show that GABPA and multiple histone acetylation marks such as H3K9/14AC, H3K27AC, and H4K5AC are maintained at specific genomic sites in mitosis. During the M/G1 transition, the levels of these histone acetylations at the upstream regions of genes bound by GABPA in mitosis are decreased. Upon depletion of GABPA, levels of histone acetylation, especially H4K5AC, at several gene regions are increased, along with transcriptional induction at 1 h after release. Therefore, we proposed that GABPA cooperates with the states of histone acetylation to act as a novel bookmarking factor which, may negatively regulate transcription during the early G1 phase.

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair.

The mammalian INO80 complex is recruited to DNA damage sites in an ARP8 dependent manner.

Dynamic changes in chromatin structure are essential for efficient DNA processing such as transcription, replication, and DNA repair. Histone modifications and ATP-dependent chromatin remodeling are important for the alteration of chromatin structure. The INO80 chromatin remodeling complex plays an important role in HR-mediated repair of DNA double-strand breaks (DSBs). In yeast, the INO80 complex is recruited to the sites of DSBs via direct interaction with phosphorylated histone H2A and facilitates the processing of DSB ends. However, the function of the mammalian INO80 complex in DNA repair is mostly unknown. Here, we show that the mammalian INO80 complex is recruited to the laser-induced DNA damage sites in a phosphorylated H2AX (γH2AX)-independent manner. We also found that an actin-related protein, ARP8, is an important subunit that is required for the recruitment of the mammalian INO80 complex to the DNA damage sites, although the recruitment of the yeast INO80 complex requires its Nhp10 or Arp4 subunits. These results suggest that the mammalian INO80 complex is also recruited to DNA damage sites similarly to the yeast INO80 complex, but the mechanism of this recruitment may be different from that of the yeast INO80 complex. These findings provide new insights into the mechanisms of DNA repair in mammalian cells.