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Histone H3 tail clipping regulates gene expression.

Santos-Rosa H, Kirmizis A, Nelson C, Bartke T, Saksouk N, Cote J, Kouzarides T - Nat. Struct. Mol. Biol. (2008)

Bottom Line: Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters.A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells.These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.

ABSTRACT
Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

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Histone tails are removed prior core depletion from promoters.(A) Histone H3 tails are removed specifically from promoters. Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 antibody to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(B) Histone H3 tails are removed on induction of the sporulation.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated promoters. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.(C) Histone H3 tails are removed on the transition to stationary phase.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in glucose to OD600nm: 0.6 (green bars), OD600nm: 5 (read bars), OD600nm: 11 (blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated genes. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.
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Figure 4: Histone tails are removed prior core depletion from promoters.(A) Histone H3 tails are removed specifically from promoters. Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 antibody to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(B) Histone H3 tails are removed on induction of the sporulation.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated promoters. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.(C) Histone H3 tails are removed on the transition to stationary phase.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in glucose to OD600nm: 0.6 (green bars), OD600nm: 5 (read bars), OD600nm: 11 (blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated genes. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.

Mentions: We next sought to establish an assay for the detection of H3 clipping on chromatin. To do this we used strains expressing a N-terminal myc tagged H3 allele (5). This strategy allows us to monitor the H3 N-terminus using a myc antibody and H3 C-terminus using a C-teminal H3 antibody. We studied genes induced during sporulation and stationary phase, since the endopeptidase activity is enhanced under these conditions. Figure 4A (green bars) shows that during exponential growth similar amounts of H3 N- and C-termini are detected at the promoter of the early meiotic gene IME1. However, 2 hours after induction of sporulation we observed a loss of the N-terminus relative to the H3 core (Figure 4A, red bars). After 12 hours of induction, histone H3 appears to be displaced from the IME1 promoter since a loss of both the N- and C-terminal H3 signals is observed (Figure 4A, blue bars). Tail-loss and histone H3 displacement were not observed within the open reading frame (ORF) of IME1 (Figure 4A). Analysis of a stationary induced gene ACH1 showed a very similar pattern of tail-loss followed by H3 displacement in the promoter, suggesting that tail-loss might be a promoter specific event.


Histone H3 tail clipping regulates gene expression.

Santos-Rosa H, Kirmizis A, Nelson C, Bartke T, Saksouk N, Cote J, Kouzarides T - Nat. Struct. Mol. Biol. (2008)

Histone tails are removed prior core depletion from promoters.(A) Histone H3 tails are removed specifically from promoters. Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 antibody to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(B) Histone H3 tails are removed on induction of the sporulation.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated promoters. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.(C) Histone H3 tails are removed on the transition to stationary phase.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in glucose to OD600nm: 0.6 (green bars), OD600nm: 5 (read bars), OD600nm: 11 (blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated genes. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.
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Figure 4: Histone tails are removed prior core depletion from promoters.(A) Histone H3 tails are removed specifically from promoters. Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 antibody to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(B) Histone H3 tails are removed on induction of the sporulation.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium (read and blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated promoters. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.(C) Histone H3 tails are removed on the transition to stationary phase.Left panel: Chromatin immunoprecipitation experiments were performed in yeast cells cultured in glucose to OD600nm: 0.6 (green bars), OD600nm: 5 (read bars), OD600nm: 11 (blue bars) using anti-myc antibody to detect the N-terminus of histone H3 (N) and anti C-terminal H3 to detect the H3 core (C). The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated genes. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V. Right panel: RT-PCR analysis was performed on the same cultures described (left panel), using primers specific to the indicated genes. The expression level of each gene was normalized to the RNA levels of RTG2.
Mentions: We next sought to establish an assay for the detection of H3 clipping on chromatin. To do this we used strains expressing a N-terminal myc tagged H3 allele (5). This strategy allows us to monitor the H3 N-terminus using a myc antibody and H3 C-terminus using a C-teminal H3 antibody. We studied genes induced during sporulation and stationary phase, since the endopeptidase activity is enhanced under these conditions. Figure 4A (green bars) shows that during exponential growth similar amounts of H3 N- and C-termini are detected at the promoter of the early meiotic gene IME1. However, 2 hours after induction of sporulation we observed a loss of the N-terminus relative to the H3 core (Figure 4A, red bars). After 12 hours of induction, histone H3 appears to be displaced from the IME1 promoter since a loss of both the N- and C-terminal H3 signals is observed (Figure 4A, blue bars). Tail-loss and histone H3 displacement were not observed within the open reading frame (ORF) of IME1 (Figure 4A). Analysis of a stationary induced gene ACH1 showed a very similar pattern of tail-loss followed by H3 displacement in the promoter, suggesting that tail-loss might be a promoter specific event.

Bottom Line: Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters.A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells.These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.

ABSTRACT
Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

Show MeSH