RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast.Petrie VJ, Wuitschick JD, Givens CD, Kosinski AM, Partridge JF. Department of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA. The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway. PMID: 15743828 [PubMed - indexed for MEDLINE] A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast.Sadaie M, Iida T, Urano T, Nakayama J. PRESTO, Japan Science and Technology Corporation, Japan. 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. PMID: 15372076 [PubMed - indexed for MEDLINE] A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe.Sigova A, Rhind N, Zamore PD. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways. Copyright 2004 Cold Spring Harbor Laboratory Press PMID: 15371329 [PubMed - indexed for MEDLINE] Calcineurin homologous protein isoform 2 (CHP2), Na+/H+ exchangers-binding protein, is expressed in intestinal epithelium.Inoue H, Nakamura Y, Nagita M, Takai T, Masuda M, Nakamura N, Kanazawa H. Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan. hirokii@bio.sci.osaka-u.ac.jp The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s). PMID: 12576672 [PubMed - indexed for MEDLINE] Calcineurin homologous protein isoform 2 (CHP2), Na+/H+ exchangers-binding protein, is expressed in intestinal epithelium.Inoue H, Nakamura Y, Nagita M, Takai T, Masuda M, Nakamura N, Kanazawa H. Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan. hirokii@bio.sci.osaka-u.ac.jp The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s). PMID: 12576672 [PubMed - indexed for MEDLINE]Calcineurin homologous protein isoform 2 (CHP2), Na+/H+ exchangers-binding protein, is expressed in intestinal epithelium.Inoue H, Nakamura Y, Nagita M, Takai T, Masuda M, Nakamura N, Kanazawa H. Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan. hirokii@bio.sci.osaka-u.ac.jp The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s). PMID: 12576672 [PubMed - indexed for MEDLINEcis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site.Partridge JF, Scott KS, Bannister AJ, Kouzarides T, Allshire RC. MRC Human Genetics Unit, Crewe Road, EH4 2XU, Edinburgh, United Kingdom. BACKGROUND: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established. RESULTS: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9. CONCLUSIONS: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin. PMID: 12361567 [PubMed - indexed for MEDLINE] ]
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