Kiyoshi Ohtani

Phosphorylation of MCM4 by Cdc7 Kinase Facilitates Its Interaction with Cdc45 on the Chromatin

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Molecular mechanism of cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I

The trans-activator protein Tax of human T-cell leukemia virus type I (HTLV-I) plays an important role in the development of adult T-cell leukemia through, at least in part, its ability to stimulate cell growth. We previously reported that Tax induced cell cycle progression from G0/G1 phase to S and G2/M phases in human T-cell line Kit 225 cells. To elucidate molecular mechanism of Tax-induced cell cycle progression, we systematically examined the effects of Tax on biochemical events associated with cell cycle progression. Introduction of Tax into resting Kit 225 cells induced activation of the G1/S transition regulation cascade consisting of activation of cyclin dependent kinase 2 (CDK2) and CDK4, phosphorylation of the Rb family proteins and an increase in free E2F. The kinase activation was found to result from Tax-induced expression of genes for cell cycle regulatory molecules including cyclin D2, cyclin E, E2F1, CDK2, CDK4 and CDK6, and Tax-induced reduction of CDK inhibitors p19INK4d and p27Kip1. These modulations by Tax always paralleled the ability of Tax to activate the NF-κB transcription pathway. These results indicate the important role of Tax-mediated trans-activation of the genes for cell cycle regulatory molecules in Tax-induced cell cycle progression.

Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors.

ABSTRACT Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWISTand FGFR3 genes.

Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F

Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.

Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F

CDC6 of Saccharomyces cerevisiae regulates the DNA replication initiation through the origin recognition complex (ORC). Identification of a human homolog of the CDC6 gene (HsCdc6) suggests a universal role of the gene product in DNA replication. Expression of HsCdc6 is growth-regulated. We investigated the molecular basis of growth-regulated expression of mammalian Cdc6. The promoter activity of isolated HsCdc6 upstream region was activated at late G1 and G1/S boundary in the cell cycle of rat embryonic fibroblast REF52 cells by the addition of serum. The isolated promoter was activated by exogenous expression of E2F without serum stimulation. However a mutant promoter lacking the E2F recognition sites failed to respond to serum stimulation and exogenous expression of E2F. Expression of endogenous Cdc6 was induced by exogenous expression of E2F. Therefore, we concluded that the growth-regulated expression of mammalian Cdc6 was mediated by E2F. Moreover, we demonstrated that exogenous overexpression of either HsCdc6 or HsOrc1 failed to induce DNA synthesis unlike overexpression of E2F1, even though E2F1 induced both Cdc6 and Orc1, suggesting that E2F may regulate the expression of another gene(s), besides Cdc6 and Orc1, required for induction of cellular DNA synthesis in mammalian cells.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19INK4d, a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-κB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-κB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of −444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Distinct E2F-mediated transcriptional program regulates p14ARF gene expression

The tumor suppressor p14ARF gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14ARF gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14ARF gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F‐responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin‐dependent kinase‐4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14ARF function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.

Identification of novel E2F1 target genes regulated in cell cycle-dependent and independent manners

The transcription factor E2F mediates cell cycle-dependent expression of genes important for cell proliferation in response to growth stimulation. To further understand the role of E2F, we utilized a sensitive subtraction method to explore new E2F1 targets, which are expressed at low levels and might have been unrecognized in previous studies. We identified 33 new E2F1-inducible genes, including checkpoint genes Claspin and Rad51ap1, and four genes with unknown function required for cell cycle progression. Moreover, we found three groups of E2F1-inducible genes that were not induced by growth stimulation. At least, two groups of genes were directly induced by E2F1, indicating that E2F1 can regulate expression of genes not induced during the cell cycle. One included Neogenin, WASF1 and SGEF genes, which may have a role in differentiation or development. The other was the cyclin-dependent kinase inhibitor p27Kip1, which was involved in suppression of inappropriate cell cycle progression induced by deregulated E2F. E2F1-responsive regions of these genes were located more upstream than those of typical E2F targets and did not have typical E2F sites. These results indicate that there are groups of E2F1 targets, which are regulated in a distinct manner from that of typical E2F targets.

Identification of two distinct elements in the long terminal repeat of HTLV-I responsible for maximum gene expression.

Human T‐cell leukemia virus type I has a unique sequence, pX, between env and the 3′ long terminal repeat (LTR). One of its products, p40, activates gene expression directed by the LTR in a trans‐acting manner. We have analysed the mechanism of this trans‐activation mediated by p40 in human T cells co‐transfected with a plasmid expressing p40 using the transient CAT gene expression. We identified two distinct elements in the LTR which are involved in maximum gene expression. The first was present in a 230‐bp fragment upstream from TATA box in the U3 region and behaved as a classical enhancer. This region was also shown to be responsible for trans‐activation by p40. This element alone together with functional p40 could direct the gene expression at only approximately 10% of the level achieved by the complete LTR and p40. The second element was present within a 300‐bp fragment downstream from the RNA start site and profoundly enhanced the gene expression in a way independent from trans‐activation mechanism. This enhancement was observed only when the element was located immediately downstream from the RNA start site without orientation preference. These two elements participate independently in the enhancement of gene expression.

Differential regulation of expression of the mammalian DNA repair genes by growth stimulation

During DNA replication, DNA becomes more vulnerable to certain DNA damages. DNA repair genes involved in repair of the damages may be induced by growth stimulation. However, regulation of DNA repair genes by growth stimulation has not been analysed in detail. In this report, we analysed the regulation of expression of mammalian MSH2, MSH3 and MLH1 genes involved in mismatch repair, and Rad51 and Rad50 genes involved in homologous recombination repair, in relation to cell growth. Unexpectedly, we found a clear difference in regulation of these repair gene expression by growth stimulation even in the same repair system. The expression of MSH2, MLH1 and Rad51 genes was clearly growth regulated, whereas MSH3 and Rad50 genes were constitutively expressed, suggesting differential requirement of the repair gene products for cell proliferation. MSH3 gene is located in a bidirectionally divergent manner with DHFR gene that is regulated by growth stimulation, indicating that bidirectionally divergent promoters are not necessarily coordinately regulated. Promoter analysis showed that the growth-regulated expression of MLH1 and Rad51 genes was mainly mediated by E2F that plays crucial roles in regulation of DNA replication, suggesting close relation between some of the repair genes and DNA replication.