Toshiyuki Umata

JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation

The rate‐limiting step in ribosome biogenesis is the transcription of ribosomal RNA, which is controlled by environmental conditions. The JmjC enzyme KDM2A/JHDM1A/FbxL11 demethylates mono‐ and dimethylated Lys 36 of histone H3, but its function is unclear. Here, we show that KDM2A represses the transcription of ribosomal RNA. KDM2A was localized in nucleoli and bound to the ribosomal RNA gene promoter. Overexpression of KDM2A repressed the transcription of ribosomal RNA in a demethylase activity‐dependent manner. When ribosomal RNA transcription was reduced under starvation, a cell‐permeable succinate that inhibited the demethylase activity of KDM2A prevented the reduction of ribosomal RNA transcription. Starvation reduced the levels of mono‐ and dimethylated Lys 36 of histone H3 marks on the rDNA promoter, and treatment with the cell‐permeable succinate suppressed the reduction of the marks during starvation. The knockdown of KDM2A increased mono‐ and dimethylated Lys 36 of histone H3 marks, and suppressed the reduction of ribosomal RNA transcription under starvation. These results show a novel mechanism by which KDM2A activity is stimulated by starvation to reduce ribosomal RNA transcription.

Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

A comparison of the mutagenic and apoptotic effects of tritiated water and acute or chronic caesium-137 gamma exposure on spleen T lymphocytes on normal and p53-deficient mice

Purpose: This study was carried out to compare the mutagenic effects on spleen T lymphocytes of mice exposed to tritiated water (HTO) and chronic or acute 137Cs γ irradiation. Materials and methods: p53 wild type (p53+/+) and p53 null type (p53−/−) mice were exposed to a total dose of 3 Gy of HTO, chronic 137Cs and acute 137Cs. Results: In spontaneous T-cell receptor (TCR) variant fractions and fractions following exposure to HTO, chronic 137Cs and acute 137Cs, TCR variant fractions in p53+/+ mice were 5.9 × 10–4, 9.8 × 10–4, 6.4 × 10–4 and 20.1 × 10–4, respectively. In contrast, those fractions were increased in p53−/− mice to 11.2 × 10–4, 18.8 × 10–4, 15.7 × 10–4 and 31.3 × 10–4, respectively. The frequency of apoptotic cells of the spleen 12 h after HTO injection increased to 5.0% in p53+/+ mice, but did not increase at all in p53−/− mice. Conclusions: When compared on the basis of induced TCR variant fractions in p53−/− mice, HTO (7.6 × 10−4) was 1.7 times more mutagenic than chronic 137Cs (4.5 × 10−4), but 2.6 times less mutagenic than acute 137Cs γ irradiation (20.1 × 10−4).

Analysis of the Mutagenic and Apoptotic Effects of Tritiated Water on Spleen T Lymphocytes of Wild Mice and p53 -Deficient Mice

Abstract A large amount of tritium is required as the fuel source for the nuclear fusion reaction. As a result, during the routine operation or in case of accidents, one of the major issues is the assessment of the biological effects of tritium released from nuclear fusion power plants. In this study, the mutagenic effects of tritiated water (HTO) were compared to those of 137Cs γ irradiation on spleen T lymphocytes of wild (p53+/+) mice and p53-deficient (p53-/-) mice. In both mice, TCR variant fractions induced by HTO was higher than those by simulation-irradiation of 137Cs γ rays. When compared on the basis of the induced TCR variant fractions in p53-/- mice at 3 Gy, tritium β rays appear to be 1.7 times more mutagenic than γ rays. On the other hand, in p53+/+ mice, HTO injection increased induced TCR variant fractions significantly, whereas simulation-irradiation did not increase those at all. In order to elucidate the reason responsible for this difference in p53+/+ mice, we investigated the apoptotic ability of spleen T lymphocytes. As a result, the apoptotic ability of spleen T lymphocytes from p53+/+ mice exposed to HTO was reduced significantly compared to that from p53+/+ mice not exposed.