Biomedical subjects

Jun-ichi Nakayama

Publications and source records attributed to Jun-ichi Nakayama.

5 recordsLinked to original sources

PubMed2006

Conserved ribonuclease, Eri1, negatively regulates heterochromatin assembly in fission yeast.

RNA interference (RNAi) is a conserved silencing mechanism that has widespread roles in RNA degradation, translational repression, and the epigenetic control of chromatin structure [1]. In fission yeast, heterochromatin assembly requires RNAi machinery and is initiated by small interference RNAs (siRNAs) derived from heterochromatic regions and by the RNA-induced transcriptional silencing (RITS) complex [2-7]. Although recent studies have been successful in uncovering the functions of effector complexes in the RNAi pathway [4, 5, 8-10], exactly how heterochromatic siRNAs are processed and function in assembling heterochromatin remains unclear. In this study we focused on a conserved ribonuclease, Eri1, which was originally identified as a negative regulator of RNAi in C. elegans [11], and show the importance of the Eri1 protein in RNAi-mediated heterochromatin assembly in fission yeast. Eri1 specifically degrades double-stranded siRNAs through two functional domains and represses the accumulation of cellular siRNAs in vivo. Deletion of eri1(+) causes an increase in siRNAs associated with the RITS complex and enhances heterochromatic silencing, which is accompanied by increased levels of histone H3-K9 methylation and the Swi6 protein. Our findings suggest that the fission yeast Eri1 controls the accumulation of heterochromatic siRNAs and negatively regulates the RNAi-mediated heterochromatin assembly.

Amino Acid Sequence↗

PubMed2006

Nuclear RanGAP is required for the heterochromatin assembly and is reciprocally regulated by histone H3 and Clr4 histone methyltransferase in Schizosaccharomyces pombe.

Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. We found that Schizosaccharomyces pombe RanGAP, SpRna1, bound the core of histone H3 (H3) and enhanced Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement was not observed for methylation of the H3-tail containing K9 and was independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionated with H3. Sprna1(ts) mutations caused decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bound SpRna1 and inhibited SpRna1-RanGAP activity. In contrast, Clr4 abolished the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affected the other histones. SpRna1 formed a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. We also found that SpRna1 formed a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3.

Base Sequence↗

PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗

PubMed2003

Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa.

Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.

DNA Methylation↗

PubMed2002

Stretch PCR assay.

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Autoradiography↗