Motoaki Ohtsubo

CRL4^Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4^Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C‐terminal half region of Cdt2 (Cdt2‐18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2‐18A was colocalized with PCNA foci during S phase in comparison with Cdt2‐WT. Poly‐ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2‐18A. Other CRL4^Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2‐18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK‐mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Geminin exerts two distinct molecular roles. Geminin negatively regulates DNA replication licensing through the direct interaction with Cdt1 to prevent re-replication in proliferating cells. Geminin also regulates chromatin remodeling through the direct interaction with Brahma/Brg1 to maintain undifferentiated states of stem cells. We previously uncovered that Polycomb-group complex 1 and Hoxb4/Hoxa9, well-known intrinsic factors that are essential for maintaining the hematopoietic stem cell (HSC) activity, alternatively act as ubiquitin–proteasome systems for Geminin protein to reduce the protein expression level, and sustain the HSC activity. Thus, Geminin is presumed to play an important role in determining cell fate, i.e., turning on and off cellular quiescence and proliferation/differentiation, in HSCs. We recently generated recombinant cell-penetrating Geminin (CP-Geminin), enabling rapid incorporation and withdraw of Geminin protein in cells. CP-Geminin may be useful in regulating the cell cycle and chromatin configuration. In this article, we summarize current information on the molecular functions of Geminin and the regulatory system for Geminin protein expression, and argue for the molecular role of Geminin in cell fate determination of HSCs, and future perspective of a new technology for manipulating the activities of HSCs and cancer stem cells (CSCs).

Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced tri­methylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling.

The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections.

CIZ1 is a nuclear protein involved in DNA replication and is also implicated in human diseases including cancers. To gain an insight into its function in vivo, we generated mice lacking Ciz1. Ciz1‐deficient (Ciz1 ^−/−) mice grew without any obvious abnormalities, and Ciz1 ^−/− mouse embryonic fibroblasts (MEFs) did not show any defects in cell cycle status, cell growth, and DNA damage response. However, Ciz1 ^−/− MEFs were sensitive to hydroxyurea‐mediated replication stress and susceptible to oncogene‐induced cellular transformation. In addition, Ciz1 ^−/− mice developed various types of leukemias by retroviral insertional mutagenesis. These results indicate that CIZ1 functions as a tumor suppressor in vivo.

Polycomb-group (PcG) complex 1 acts as an E3 ubiquitin ligase both for histone H2A to silence transcription and for geminin to regulate its stability. Scmh1 is a substoichiometric component of PcG complex 1 that provides the complex with an interaction domain for geminin. Scmh1 is unstable and regulated through the ubiquitin-proteasome system, but its molecular roles are unknown, so we generated Scmh1-deficient mice to elucidate its function. Loss of Scmh1 caused derepression of Hoxb4 and Hoxa9, direct targets of PcG complex 1-mediated transcriptional silencing in hematopoietic cells. Double knockdown of Hoxb4 and Hoxa9 or transduction of a dominant-negative Hoxb4N→A mutant caused geminin accumulation. Age-related transcriptional downregulation of derepressed Hoxa9 also leads to geminin accumulation. Transduction of Scmh1 lacking a geminin-binding domain restored derepressed expression of Hoxb4 and Hoxa9 but did not downregulate geminin like full-length Scmh1. Each of Hoxb4 and Hoxa9 can form a complex with Roc1-Ddb1-Cul4a to act as an E3 ubiquitin ligase for geminin. We suggest that geminin dysregulation may be restored by derepressed Hoxb4 and Hoxa9 in Scmh1-deficient mice. These findings suggest that PcG and a subset of Hox genes compose a homeostatic regulatory system for determining expression level of geminin.

Hoxb4, a 3′-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5′-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5′-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.

Patients carrying two loss‐of‐function (or hypomorphic) alleles of STAT1 are vulnerable to intracellular bacterial and viral diseases. Heterozygosity for loss‐of‐function dominant‐negative mutations in STAT1 is responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD), whereas heterozygosity for gain‐of‐function loss‐of‐dephosphorylation mutations causes AD chronic mucocutaneous candidiasis (CMC). The two previously reported types of AD MSMD‐causing STAT1 mutations are located in the tail segment domain (p.L706S) or in the DNA‐binding domain (p.E320Q and p.Q463H), whereas the AD CMC‐causing mutations are located in the coiled‐coil domain. We identified two cases with AD‐STAT1 deficiency in two unrelated patients from Japan and Saudi Arabia carrying heterozygous missense mutations affecting the SH2 domain (p.K637E and p.K673R). p.K673R is a hypomorphic mutation that impairs STAT1 tyrosine phosphorylation, whereas the p.K637E mutation is null and affects both STAT1 phosphorylation and DNA‐binding activity. Both alleles are dominant negative and result in impaired STAT1‐mediated cellular responses to interferon (IFN)‐γ and IL‐27. In contrast, STAT1‐mediated cellular responses against IFN‐α and IFN‐λ1 were preserved at normal levels in patients' cells. We describe here the first dominant mutations in the SH2 domain of STAT1, revealing the importance of this domain for tyrosine phosphorylation and DNA binding, as well as for antimycobacterial immunity. Hum Mutat 33:1377–1387, 2012.