Limits...
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.


Related in: MedlinePlus

Caf1 and Ccr4 are required for heterochromatin integrity in 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. The mei4 gene is deleted in the mmi1Δ strain (the deleted region is indicated by a dashed line). amei4 locus. bSPNCRNA.1506 region. c Right subtelomere of chromosome 1. d Right subtelomere of chromosome 2. e Centromere of chromosome 2.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Caf1 and Ccr4 are required for heterochromatin integrity in 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. The mei4 gene is deleted in the mmi1Δ strain (the deleted region is indicated by a dashed line). amei4 locus. bSPNCRNA.1506 region. c Right subtelomere of chromosome 1. d Right subtelomere of chromosome 2. e Centromere of chromosome 2.

Mentions: Mmi1 is also required for the maintenance of heterochromatin in genomic ‘islands’, some of which correspond to Mmi1 target genes. This function is carried out, at least partially, by targeting Red1 and components of the RNAi complex RITS to specific genes [37, 38]. However, inactivation of components of the RITS complex or heterochromatin causes no or only moderate changes in the levels of mmi1 targets [37–39]. To investigate whether Ccr4-Not is involved in this role of Mmi1, we applied chromatin immuno precipitation analysed by sequencing (ChIP-seq) for genome-wide profiling of histone H3K9 di-methylation (H3K9-me2, a marker of heterochromatin) in ccr4Δ, caf1Δ and wild-type cells. As a control for histone occupancy, the levels of histone H3 were measured in parallel and used to normalize the enrichment in H3K9 methylation (Additional file 1: Table S3). We detected reproducible enrichments above background in only 6 of the 21 originally reported heterochromatin islands (Additional file 1: Table S4) [38]. This discrepancy might reflect technical issues, as the previous study used tiling microarrays that provide less resolution and tend to have higher noise levels. The detected islands included four clear targets of Mmi1 (mcp7, ssm4, moa1 and mei4), as well as two ncRNAs (SPNCRNA.1506 and SPNCRNA.394). Five of the islands displayed clear and reproducible decreases in H3K9-me2 in both ccr4 and caf1 mutants, suggesting that the structure of heterochromatin in these loci is compromised (Additional file 1: Table S4; Fig. 4a, b, Additional file 2: Figure S1, Additional file 3: Figure S2). The extent of the reductions was varied, with the island containing mei4 showing the strongest effects (Additional file 1: Table S4; Fig. 4a). The island that includes SPNCRNA.1506 contained two blocks of heterochromatin, one of which appeared to be much more sensitive to mutations in caf1 and ccr4 (Fig. 4b). Mutants in mmi1 showed reduced levels in H3K9-me2 in the four loci corresponding to Mmi1 targets (Additional file 1: Table S4; Fig. 4a, Additional file 3: Figure S2A, B) but not in the two ncRNAs (Additional file 1: Table S4; Fig. 4b and [38]), indicating that Ccr4-Not can regulate heterochromatin independently of Mmi1.Fig. 4


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)

Caf1 and Ccr4 are required for heterochromatin integrity in 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. The mei4 gene is deleted in the mmi1Δ strain (the deleted region is indicated by a dashed line). amei4 locus. bSPNCRNA.1506 region. c Right subtelomere of chromosome 1. d Right subtelomere of chromosome 2. e Centromere of chromosome 2.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Caf1 and Ccr4 are required for heterochromatin integrity in 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. The mei4 gene is deleted in the mmi1Δ strain (the deleted region is indicated by a dashed line). amei4 locus. bSPNCRNA.1506 region. c Right subtelomere of chromosome 1. d Right subtelomere of chromosome 2. e Centromere of chromosome 2.
Mentions: Mmi1 is also required for the maintenance of heterochromatin in genomic ‘islands’, some of which correspond to Mmi1 target genes. This function is carried out, at least partially, by targeting Red1 and components of the RNAi complex RITS to specific genes [37, 38]. However, inactivation of components of the RITS complex or heterochromatin causes no or only moderate changes in the levels of mmi1 targets [37–39]. To investigate whether Ccr4-Not is involved in this role of Mmi1, we applied chromatin immuno precipitation analysed by sequencing (ChIP-seq) for genome-wide profiling of histone H3K9 di-methylation (H3K9-me2, a marker of heterochromatin) in ccr4Δ, caf1Δ and wild-type cells. As a control for histone occupancy, the levels of histone H3 were measured in parallel and used to normalize the enrichment in H3K9 methylation (Additional file 1: Table S3). We detected reproducible enrichments above background in only 6 of the 21 originally reported heterochromatin islands (Additional file 1: Table S4) [38]. This discrepancy might reflect technical issues, as the previous study used tiling microarrays that provide less resolution and tend to have higher noise levels. The detected islands included four clear targets of Mmi1 (mcp7, ssm4, moa1 and mei4), as well as two ncRNAs (SPNCRNA.1506 and SPNCRNA.394). Five of the islands displayed clear and reproducible decreases in H3K9-me2 in both ccr4 and caf1 mutants, suggesting that the structure of heterochromatin in these loci is compromised (Additional file 1: Table S4; Fig. 4a, b, Additional file 2: Figure S1, Additional file 3: Figure S2). The extent of the reductions was varied, with the island containing mei4 showing the strongest effects (Additional file 1: Table S4; Fig. 4a). The island that includes SPNCRNA.1506 contained two blocks of heterochromatin, one of which appeared to be much more sensitive to mutations in caf1 and ccr4 (Fig. 4b). Mutants in mmi1 showed reduced levels in H3K9-me2 in the four loci corresponding to Mmi1 targets (Additional file 1: Table S4; Fig. 4a, Additional file 3: Figure S2A, B) but not in the two ncRNAs (Additional file 1: Table S4; Fig. 4b and [38]), indicating that Ccr4-Not can regulate heterochromatin independently of Mmi1.Fig. 4

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.


Related in: MedlinePlus