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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.


Physical and genetic interactions of Ccr4. a Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). Arrow the position of the specific band corresponding to Chp1-myc, star a non-specific band. bLeft: ccr4Δ growth defect is partially suppressed by deletion of mei4. Cells of the indicated genotypes were grown in YE medium and the cell number estimated from the optical density of the culture. Right: ccr4 mutants show synthetic negative interactions with red1 mutants.
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Fig6: Physical and genetic interactions of Ccr4. a Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). Arrow the position of the specific band corresponding to Chp1-myc, star a non-specific band. bLeft: ccr4Δ growth defect is partially suppressed by deletion of mei4. Cells of the indicated genotypes were grown in YE medium and the cell number estimated from the optical density of the culture. Right: ccr4 mutants show synthetic negative interactions with red1 mutants.

Mentions: If Ccr4-Not is involved in heterochromatin formation and/or maintenance, it would be expected to interact with other proteins important for this process. Indeed, Ccr4 coprecipitated with the chromodomain protein Chp1 in an RNA-independent manner. Moreover, the Ccr4-Chp1 interaction did not require Mmi1, in agreement with our results that Ccr4 is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways (Fig. 6a). We then used ChIP-seq to monitor the distribution of Chp1 on heterochromatin in wild-type and ccr4 mutants (Additional file 1: Table S10). In wild-type cells, Chp1-TAP was enriched in a few heterochromatin islands (mcp7, mei4 and SPNCRNA.1506) (Additional file 1: Table S11), subtelomeric regions (Additional file 1: Table S12) and centromeres (Additional file 1: Table S13). Consistent with our previous results, Chp1-TAP levels were reduced in ccr4 mutants on islands and subtelomeres, but not on centromeres (Additional file 1: Tables S11–S13). Thus, Ccr4-Not is required for normal accumulation of Chp1 in both Mmi1-dependent and Mmi1-independent heterochromatin.Fig. 6


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)

Physical and genetic interactions of Ccr4. a Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). Arrow the position of the specific band corresponding to Chp1-myc, star a non-specific band. bLeft: ccr4Δ growth defect is partially suppressed by deletion of mei4. Cells of the indicated genotypes were grown in YE medium and the cell number estimated from the optical density of the culture. Right: ccr4 mutants show synthetic negative interactions with red1 mutants.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4536793&req=5

Fig6: Physical and genetic interactions of Ccr4. a Cell extracts were prepared from the indicated strains and mock-treated (−) or treated with RNase (+). Immunoprecipitations were carried using antibodies against TAP. Samples were probed with antibodies against myc (top) or TAP (bottom). Arrow the position of the specific band corresponding to Chp1-myc, star a non-specific band. bLeft: ccr4Δ growth defect is partially suppressed by deletion of mei4. Cells of the indicated genotypes were grown in YE medium and the cell number estimated from the optical density of the culture. Right: ccr4 mutants show synthetic negative interactions with red1 mutants.
Mentions: If Ccr4-Not is involved in heterochromatin formation and/or maintenance, it would be expected to interact with other proteins important for this process. Indeed, Ccr4 coprecipitated with the chromodomain protein Chp1 in an RNA-independent manner. Moreover, the Ccr4-Chp1 interaction did not require Mmi1, in agreement with our results that Ccr4 is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways (Fig. 6a). We then used ChIP-seq to monitor the distribution of Chp1 on heterochromatin in wild-type and ccr4 mutants (Additional file 1: Table S10). In wild-type cells, Chp1-TAP was enriched in a few heterochromatin islands (mcp7, mei4 and SPNCRNA.1506) (Additional file 1: Table S11), subtelomeric regions (Additional file 1: Table S12) and centromeres (Additional file 1: Table S13). Consistent with our previous results, Chp1-TAP levels were reduced in ccr4 mutants on islands and subtelomeres, but not on centromeres (Additional file 1: Tables S11–S13). Thus, Ccr4-Not is required for normal accumulation of Chp1 in both Mmi1-dependent and Mmi1-independent heterochromatin.Fig. 6

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.