Jun-ichi Nakayama

TLP1: A Gene Encoding a Protein Component of Mammalian Telomerase Is a Novel Member of WD Repeats Family

We have cloned and characterized the rat telomerase protein component 1 gene (TLP1), which is related to the gene for Tetrahymena p80. The cDNA encodes a 2629 amino acid sequence and produces the TLP1 proteins p240 and p230. The anti-TLP1 antibody specifically immunoprecipitated the telomerase activity. Moreover, p240 and p230 were copurified with telomerase activity in a series of extensive purification experiments. These results strongly suggest that the TLP1 proteins are components of, or are closely associated with, the rat telomerase. A pulse-chase experiment showed that p240 is modified to p230 in vivo. p230 was the dominant form in telomerase-positive cells, suggesting that modification of the TLP1 protein may regulate telomerase activity in vivo.

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.

A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions—centromeres, the mating‐type region, and telomeres—and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1+ gene causes centromere‐specific decreases in Swi6 localization and histone H3‐K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating‐type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1‐mediated establishment steps differently than do other heterochromatic regions.

Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections.

Human telomerase is expressed in germ tissues and in the majority of primary tumors. Cell renewal tissues and some pre-cancerous tissues also have weak telomerase activity. Yet, neither the exact location and frequency of telomerase-positive cells nor the changes in telomerase expression during differentiation or carcinogenesis of individual cells are known. This paper reports on the expression of hTERT (telomerase reverse transcriptase) protein in tumor and non-tumor colorectal tissues by Western blotting and tissue sections by immuno-histochemistry using antibodies raised against partial peptides of hTERT. Though telomerase activity and hTERT expression at both mRNA and protein levels were generally higher in tumor part than in non-tumor part, these two were not always correlated: expression of hTERT did not always give rise to high telomerase activity. Colonic carcinoma cell nuclei were stained with anti-hTERT antibodies but not with antigen-preabsorbed antibodies. In normal mucosa, hTERT protein was expressed, though weaker than in carcinoma, in all colonic crypt epithelial cells except those at the tip; the expressing-cell distribution was much wider than that of Ki-67 positive cells which were located at the bottom of the crypt. Isolated crypt contained a significant level of hTERT protein revealed by Western blotting, while having very weak telomerase activity. Telomerase activity was detected in epithelial cells only at the bottom half of the crypt. Specific hTERT-staining was positive in tissue lymphocytes but negative in almost all other stromal cells. It is of interest to see whether a significant level of hTERT expression with low telomerase activity is characteristic of physiologically regenerating tissues containing stem cells. In situ detection of the hTERT protein will permit further analysis of cancer diagnosis and stem cell differentiation.

A Chromodomain Protein, Swi6, Performs Imprinting Functions in Fission Yeast during Mitosis and Meiosis

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex.

The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus.

Heterochromatin is a structurally compacted region of chromosomes in which transcription and recombination are inactivated. DNA replication is temporally regulated in heterochromatin, but the molecular mechanism for regulation has not been elucidated. Among heterochromatin loci in Schizosaccharomyces pombe, the pericentromeric region and the silent mating-type (mat) locus replicate in early S phase, whereas the sub-telomeric region does not, suggesting complex mechanisms for regulation of replication in heterochromatic regions. Here, we show that Swi6, an S. pombe counterpart of heterochromatin protein 1 (HP1), is required for early replication of the pericentromeric region and the mat locus. Origin-loading of Sld3, which depends on Dfp1/Dbf4-dependent kinase Cdc7 (DDK), is stimulated by Swi6. An HP1-binding motif within Dfp1 is required for interaction with Swi6 in vitro and for early replication of the pericentromeric region and mat locus. Tethering of Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci. Our results show that a heterochromatic protein positively regulates initiation of replication in silenced chromatin by interacting with an essential kinase.

Presence of telomeric G-strand tails in the telomerase catalytic subunit TERT knockout mice

Telomerase consists of two essential subunits, the template RNA (TR; telomerase RNA) and the catalytic subunit TERT (telomerase reverse transcriptase). Knockout mice with a mTR (mouse TR) deletion have been described and well characterized. However, mice with a mTERT (mouse TERT) deletion have not been reported.

Two different Argonaute complexes are required for siRNA generation and heterochromatin assembly in fission yeast

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.

Physiological Roles of Class I HDAC Complex and Histone Demethylase

Epigenetic gene silencing is one of the fundamental mechanisms for ensuring proper gene expression patterns during cellular differentiation and development. Histone deacetylases (HDACs) are evolutionally conserved enzymes that remove acetyl modifications from histones and play a central role in epigenetic gene silencing. In cells, HDAC forms a multiprotein complex (HDAC complex) in which the associated proteins are believed to help HDAC carry out its cellular functions. Though each HDAC complex contains distinct components, the presence of isoforms for some of the components expands the variety of complexes and the diversity of their cellular roles. Recent studies have also revealed a functional link between HDAC complexes and specific histone demethylases. In this paper, we summarize the distinct and cooperative roles of four class I HDAC complexes, Sin3, NuRD, CoREST, and NCoR/SMRT, with respect to their component diversity and their relationship with specific histone demethylases.

The UUAG-specific RNA Binding Protein, Heterogeneous Nuclear Ribonucleoprotein D0 COMMON MODULAR STRUCTURE AND BINDING PROPERTIES OF THE 2xRBD-Gly FAMILY

Human cDNA clones encoding the UUAG-binding heterogeneous nuclear ribonucleoprotein (hnRNP) D0 protein have been isolated and expressed. The protein has two RNA-binding domains (RBDs) in the middle part of the protein and an RGG box, a region rich in glycine and arginine residues, in the C-terminal part (“2xRBD-Gly” structure). The hnRNP A1, A2/B1, and D0 proteins, all possess common features of the 2xRBD-Gly structure and binding specificity toward RNA. Together, they form a subfamily of RBD class RNA binding proteins (the 2xRBD-Gly family). One of the structural characteristics shared by these proteins is the presence of several isoforms presumably resulting from alternative splicing. Filter binding assays, using the recombinant hnRNP D0 proteins that have one of the two RBDs, indicated that one RBD specifically binds to the UUAG sequence. However, two isoforms with or without a 19-amino acid insertion at the N-terminal RBD showed different preference toward mutant RNA substrates. The 19-amino acid insertion is located in the N-terminal end of the first RBD. This result establishes the participation of the N terminus of RBD in determining the sequence specificity of binding. A similar insertion was also reported with the hnRNP A2/B1 proteins. Thus, it might be possible that this type of insertion with the 2xRBD-Gly type RNA binding proteins plays a role in “fine tuning” the specificity of RNA binding. RBD is supposed to bind with RNA in general and sequence-specific manners. These two discernible binding modes are proposed to be performed by different regions of the RBD. A structural model of these two binding sites is presented.