Limits...
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.

Show MeSH
The histone H3 endopeptidase is a serine protease with preference for non-activating marks.(A) Histone H3 tails carrying non activatory marks are preferentially clipped.Histone H3 modified at different residues (see Materials and Methods) was used as substrate for the endopeptidase activity pulled down from stationary phase culture. The reactions were analyzed by western blot with anti C-terminal H3 antibody.(B) K4 me3 H3 are not evicted from promoters upon gene induction.Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium for 12 hours (blue bars) using anti K4 me3 H3 antibody and anti C-terminal H3 antibody. The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the IME2 gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(C) H3Δ1-21 occurs in vivo in S. cerevisiae.Yeast chromatin was prepared from early stationary phase culture and analyzed by Coomasie (left panel) or western blot (right panel). The full length and clipped H3 (H3Δ1-21) are highlighted.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3350865&req=5

Figure 2: The histone H3 endopeptidase is a serine protease with preference for non-activating marks.(A) Histone H3 tails carrying non activatory marks are preferentially clipped.Histone H3 modified at different residues (see Materials and Methods) was used as substrate for the endopeptidase activity pulled down from stationary phase culture. The reactions were analyzed by western blot with anti C-terminal H3 antibody.(B) K4 me3 H3 are not evicted from promoters upon gene induction.Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium for 12 hours (blue bars) using anti K4 me3 H3 antibody and anti C-terminal H3 antibody. The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the IME2 gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(C) H3Δ1-21 occurs in vivo in S. cerevisiae.Yeast chromatin was prepared from early stationary phase culture and analyzed by Coomasie (left panel) or western blot (right panel). The full length and clipped H3 (H3Δ1-21) are highlighted.

Mentions: The electrospray analysis on cleaved calf thymus H3 showed clipped tail peptides carrying various modifications (Supplementary Fig. 4-6). Interestingly, peptides containing methylated K4 were absent among the most prominent N-terminal cleavage products. In contrast, K9 di- and tri methylated products could be detected. To test directly whether post-translational modifications alter the activity of the H3 clipping enzyme, we assayed this activity on full length modified H3. The H3 substrate was generated by ligating recombinant H3 spanning residues 32 to 135 to synthetic peptides (residues 1-31) containing trimethylated K4 (an activatory mark) or asymmetrical dimethyl R2 (a repressive mark, see Material and Methods). This procedure enables us to use a fully modified population of histones carrying a single post-translational mark. As shown in Figure 2A, the endopeptidase activity purified from a stationary culture was able to cut the unmodified H3 as well as R2 dimethylated H3. However the endopeptidase activity was reduced when H3 trimethylated at K4 was the substrate, suggesting that methylation of K4 is inhibitory to the H3 clipping activity. Chromatin Inmunoprecipitation analysis of inducible genes support this view. Figure 2B shows that IME2 induction is accompanied by a strong reduction in nucleosome occupancy at the promoter of the gene (left panel, H3 green and blue bars). This reduction is not observed on K4me3 H3 at the same location. In fact, the actual ratio of K4me3 with respect to the H3 content increases significantly from the repressed to the active transcription state of the gene (right panel). This behaviour was observed on other genes when induced (HOP1, SPR3, data not shown) and suggests that the K4 trimethylated H3 nucleomes are not removed from the promoters.


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)

The histone H3 endopeptidase is a serine protease with preference for non-activating marks.(A) Histone H3 tails carrying non activatory marks are preferentially clipped.Histone H3 modified at different residues (see Materials and Methods) was used as substrate for the endopeptidase activity pulled down from stationary phase culture. The reactions were analyzed by western blot with anti C-terminal H3 antibody.(B) K4 me3 H3 are not evicted from promoters upon gene induction.Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium for 12 hours (blue bars) using anti K4 me3 H3 antibody and anti C-terminal H3 antibody. The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the IME2 gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(C) H3Δ1-21 occurs in vivo in S. cerevisiae.Yeast chromatin was prepared from early stationary phase culture and analyzed by Coomasie (left panel) or western blot (right panel). The full length and clipped H3 (H3Δ1-21) are highlighted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3350865&req=5

Figure 2: The histone H3 endopeptidase is a serine protease with preference for non-activating marks.(A) Histone H3 tails carrying non activatory marks are preferentially clipped.Histone H3 modified at different residues (see Materials and Methods) was used as substrate for the endopeptidase activity pulled down from stationary phase culture. The reactions were analyzed by western blot with anti C-terminal H3 antibody.(B) K4 me3 H3 are not evicted from promoters upon gene induction.Chromatin immunoprecipitation experiments were performed in yeast cells cultured in either glucose (green bars) or sporulation medium for 12 hours (blue bars) using anti K4 me3 H3 antibody and anti C-terminal H3 antibody. The precipitated DNA was analyzed by quantitative PCR using primers specific to the indicated positions within the IME2 gene. The diagrams represent relative fluorescent units normalized to an intergenic region on chromosome V (see Material and Methods).(C) H3Δ1-21 occurs in vivo in S. cerevisiae.Yeast chromatin was prepared from early stationary phase culture and analyzed by Coomasie (left panel) or western blot (right panel). The full length and clipped H3 (H3Δ1-21) are highlighted.
Mentions: The electrospray analysis on cleaved calf thymus H3 showed clipped tail peptides carrying various modifications (Supplementary Fig. 4-6). Interestingly, peptides containing methylated K4 were absent among the most prominent N-terminal cleavage products. In contrast, K9 di- and tri methylated products could be detected. To test directly whether post-translational modifications alter the activity of the H3 clipping enzyme, we assayed this activity on full length modified H3. The H3 substrate was generated by ligating recombinant H3 spanning residues 32 to 135 to synthetic peptides (residues 1-31) containing trimethylated K4 (an activatory mark) or asymmetrical dimethyl R2 (a repressive mark, see Material and Methods). This procedure enables us to use a fully modified population of histones carrying a single post-translational mark. As shown in Figure 2A, the endopeptidase activity purified from a stationary culture was able to cut the unmodified H3 as well as R2 dimethylated H3. However the endopeptidase activity was reduced when H3 trimethylated at K4 was the substrate, suggesting that methylation of K4 is inhibitory to the H3 clipping activity. Chromatin Inmunoprecipitation analysis of inducible genes support this view. Figure 2B shows that IME2 induction is accompanied by a strong reduction in nucleosome occupancy at the promoter of the gene (left panel, H3 green and blue bars). This reduction is not observed on K4me3 H3 at the same location. In fact, the actual ratio of K4me3 with respect to the H3 content increases significantly from the repressed to the active transcription state of the gene (right panel). This behaviour was observed on other genes when induced (HOP1, SPR3, data not shown) and suggests that the K4 trimethylated H3 nucleomes are not removed from the promoters.

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