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Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

Cotobal C, Rodríguez-López M, Duncan C, Hasan A, Yamashita A, Yamamoto M, Bähler J, Mata J - Epigenetics Chromatin (2015)

Bottom Line: Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant.Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.

Results: Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

No MeSH data available.


Effect of ccr4 catalytic mutations, and of not2 and mot2/not4 inactivation, on heterochromatin integrity of islands and subtelomeric regions. Enrichment in H3 or H3K9-me2 in heterochromatin islands determined using ChIP-seq. The y axes show the normalized enrichment in the indicated immunoprecipitate and the x axes correspond to the position along the chromosome. Data are shown for wild-type cells and for the indicated mutants. amei4 locus in wild-type, ccr4-D558A and ccr4-H665A. b Right subtelomere of chromosome 2 in wild-type, ccr4-D558A and ccr4-H665A. cmei4 locus in wild-type, not2Δ and mot2/not4Δ. d Right subtelomere of chromosome 2 in wild-type, not2Δ and mot2/not4Δ.
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Fig5: Effect of ccr4 catalytic mutations, and of not2 and mot2/not4 inactivation, on heterochromatin integrity of islands and subtelomeric regions. Enrichment in H3 or H3K9-me2 in heterochromatin islands determined using ChIP-seq. The y axes show the normalized enrichment in the indicated immunoprecipitate and the x axes correspond to the position along the chromosome. Data are shown for wild-type cells and for the indicated mutants. amei4 locus in wild-type, ccr4-D558A and ccr4-H665A. b Right subtelomere of chromosome 2 in wild-type, ccr4-D558A and ccr4-H665A. cmei4 locus in wild-type, not2Δ and mot2/not4Δ. d Right subtelomere of chromosome 2 in wild-type, not2Δ and mot2/not4Δ.

Mentions: We next investigated whether the deadenylase activity of Ccr4-Not is important for its role in heterochromatin formation. We generated two independent point mutants in key residues of the catalytic site of Ccr4 (D558A and H665A) [50]. Similar to cells carrying a deletion in ccr4, both mutants grew slowly and displayed low efficiency of mating. In addition, their microarray profiles overlapped with that of ccr4Δ (Additional file 4: Figure S3). ChIP-seq for histone H3K9-me2 revealed a pattern almost identical to those of ccr4Δ and caf1Δ, including a decrease in several heterochromatin islands (mei4, mcp7 and SPNCRNA.1506) and a prominent reduction in subtelomeric regions (Additional file 1: Tables S4–S9; Fig. 5a, b). These results suggest that the deadenylase catalytic activity of the complex is essential for its regulation of heterochromatin.Fig. 5


Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

Cotobal C, Rodríguez-López M, Duncan C, Hasan A, Yamashita A, Yamamoto M, Bähler J, Mata J - Epigenetics Chromatin (2015)

Effect of ccr4 catalytic mutations, and of not2 and mot2/not4 inactivation, on heterochromatin integrity of islands and subtelomeric regions. Enrichment in H3 or H3K9-me2 in heterochromatin islands determined using ChIP-seq. The y axes show the normalized enrichment in the indicated immunoprecipitate and the x axes correspond to the position along the chromosome. Data are shown for wild-type cells and for the indicated mutants. amei4 locus in wild-type, ccr4-D558A and ccr4-H665A. b Right subtelomere of chromosome 2 in wild-type, ccr4-D558A and ccr4-H665A. cmei4 locus in wild-type, not2Δ and mot2/not4Δ. d Right subtelomere of chromosome 2 in wild-type, not2Δ and mot2/not4Δ.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Effect of ccr4 catalytic mutations, and of not2 and mot2/not4 inactivation, on heterochromatin integrity of islands and subtelomeric regions. Enrichment in H3 or H3K9-me2 in heterochromatin islands determined using ChIP-seq. The y axes show the normalized enrichment in the indicated immunoprecipitate and the x axes correspond to the position along the chromosome. Data are shown for wild-type cells and for the indicated mutants. amei4 locus in wild-type, ccr4-D558A and ccr4-H665A. b Right subtelomere of chromosome 2 in wild-type, ccr4-D558A and ccr4-H665A. cmei4 locus in wild-type, not2Δ and mot2/not4Δ. d Right subtelomere of chromosome 2 in wild-type, not2Δ and mot2/not4Δ.
Mentions: We next investigated whether the deadenylase activity of Ccr4-Not is important for its role in heterochromatin formation. We generated two independent point mutants in key residues of the catalytic site of Ccr4 (D558A and H665A) [50]. Similar to cells carrying a deletion in ccr4, both mutants grew slowly and displayed low efficiency of mating. In addition, their microarray profiles overlapped with that of ccr4Δ (Additional file 4: Figure S3). ChIP-seq for histone H3K9-me2 revealed a pattern almost identical to those of ccr4Δ and caf1Δ, including a decrease in several heterochromatin islands (mei4, mcp7 and SPNCRNA.1506) and a prominent reduction in subtelomeric regions (Additional file 1: Tables S4–S9; Fig. 5a, b). These results suggest that the deadenylase catalytic activity of the complex is essential for its regulation of heterochromatin.Fig. 5

Bottom Line: Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant.Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT

Background: Heterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.

Results: Ccr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.

Conclusions: Taken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.

No MeSH data available.