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
A histone H3 endopeptidase activity in S. cerevisiae.(A) A H3 endopeptidase activity is present in the yeast nuclei.Nuclear extracts from early exponential, sporulation or stationary phase cultures were assayed for endopeptidase activity on recombinant H3. The reactions were stained with Ponceau (15 μl reaction, to visualize the products) and analyzed by western blot with anti C-terminal H3 antibody (5 μl reaction, to avoid signal saturation). The clipped H3 product is highlighted.(B) The H3 endopeptidase activity is enriched upon nutrient starvation.Extracts from early exponential, sporulation or stationary phase cultures, purified on sepharose beads, were assayed for endopeptidase activity on calf H3. The reactions were analyzed by western blot with anti C-terminal H3 antibody. The clipped H3 product is highlighted.(C) The Q19L20A21 is the recognition sequence for the H3 endoppetidase.Extract from yeast cell on stationary phase was pull down on Sepharose beads and assayed against recombinant wild type H3 and recombinant Q19L20->AA mutant H3.The reactions were analysed by western blot with anti C-terminal H3 antibody.(D) Activity against different histones.Stationary phase pull down on Sepharose beads was assayed against identical amounts of calf histones. The reactions were analysed by western blot with antibodies specific for each histone. The clipped H3 product is highlighted.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3350865&req=5

Figure 1: A histone H3 endopeptidase activity in S. cerevisiae.(A) A H3 endopeptidase activity is present in the yeast nuclei.Nuclear extracts from early exponential, sporulation or stationary phase cultures were assayed for endopeptidase activity on recombinant H3. The reactions were stained with Ponceau (15 μl reaction, to visualize the products) and analyzed by western blot with anti C-terminal H3 antibody (5 μl reaction, to avoid signal saturation). The clipped H3 product is highlighted.(B) The H3 endopeptidase activity is enriched upon nutrient starvation.Extracts from early exponential, sporulation or stationary phase cultures, purified on sepharose beads, were assayed for endopeptidase activity on calf H3. The reactions were analyzed by western blot with anti C-terminal H3 antibody. The clipped H3 product is highlighted.(C) The Q19L20A21 is the recognition sequence for the H3 endoppetidase.Extract from yeast cell on stationary phase was pull down on Sepharose beads and assayed against recombinant wild type H3 and recombinant Q19L20->AA mutant H3.The reactions were analysed by western blot with anti C-terminal H3 antibody.(D) Activity against different histones.Stationary phase pull down on Sepharose beads was assayed against identical amounts of calf histones. The reactions were analysed by western blot with antibodies specific for each histone. The clipped H3 product is highlighted.

Mentions: While assaying yeast protein complexes for their capacity to demethylate histone H3, we noticed an endopeptidase activity in our nuclei preparations which cleaves the exogenously provided substrate (calf H3). The activity was low in the nuclei of cells growing exponentially in rich medium but increased in cells grown into stationary phase or shifted to sporulation medium (Figure 1A). In an attempt to biochemically purify this activity (see Materials and Methods) we found that it was retained on sepharose-based matrices (Figure 1B). To identify the site of cleavage on calf H3, we analyzed the reactions by mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) on the substrate (calf H3) identified a broad peak representing H3 carrying combinations of post-translational modifications (Supplementary Fig. 1, dark blue circle). When reactions were performed in the presence of the endopeptidase activity from stationary cells, two additional peaks were detected (Supplementary Fig.1, red and light blue circles). Electrospray sequencing of the smaller peak revealed products corresponding to the first 21 amino acids of calf H3 carrying various modifications (Supplementary Fig. S2). The N-terminal tail of H3 is sufficient for endopeptidase recognition as peptides spanning amino acids 1 to 30 are also cleaved after alanine 21 (Supplementary Fig. S3). To establish whether flanking residues represent a recognition site for the enzyme, full-length H3 was mutated at position 19 and 20 from QL to AA, expressed in E. coli and used as a substrate for the endopeptidase activity. Figure 1C shows that the wild type full length H3 is cleaved whereas the QL to AA mutant is resistant to the endopeptidase activity.


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)

A histone H3 endopeptidase activity in S. cerevisiae.(A) A H3 endopeptidase activity is present in the yeast nuclei.Nuclear extracts from early exponential, sporulation or stationary phase cultures were assayed for endopeptidase activity on recombinant H3. The reactions were stained with Ponceau (15 μl reaction, to visualize the products) and analyzed by western blot with anti C-terminal H3 antibody (5 μl reaction, to avoid signal saturation). The clipped H3 product is highlighted.(B) The H3 endopeptidase activity is enriched upon nutrient starvation.Extracts from early exponential, sporulation or stationary phase cultures, purified on sepharose beads, were assayed for endopeptidase activity on calf H3. The reactions were analyzed by western blot with anti C-terminal H3 antibody. The clipped H3 product is highlighted.(C) The Q19L20A21 is the recognition sequence for the H3 endoppetidase.Extract from yeast cell on stationary phase was pull down on Sepharose beads and assayed against recombinant wild type H3 and recombinant Q19L20->AA mutant H3.The reactions were analysed by western blot with anti C-terminal H3 antibody.(D) Activity against different histones.Stationary phase pull down on Sepharose beads was assayed against identical amounts of calf histones. The reactions were analysed by western blot with antibodies specific for each histone. The clipped H3 product is highlighted.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: A histone H3 endopeptidase activity in S. cerevisiae.(A) A H3 endopeptidase activity is present in the yeast nuclei.Nuclear extracts from early exponential, sporulation or stationary phase cultures were assayed for endopeptidase activity on recombinant H3. The reactions were stained with Ponceau (15 μl reaction, to visualize the products) and analyzed by western blot with anti C-terminal H3 antibody (5 μl reaction, to avoid signal saturation). The clipped H3 product is highlighted.(B) The H3 endopeptidase activity is enriched upon nutrient starvation.Extracts from early exponential, sporulation or stationary phase cultures, purified on sepharose beads, were assayed for endopeptidase activity on calf H3. The reactions were analyzed by western blot with anti C-terminal H3 antibody. The clipped H3 product is highlighted.(C) The Q19L20A21 is the recognition sequence for the H3 endoppetidase.Extract from yeast cell on stationary phase was pull down on Sepharose beads and assayed against recombinant wild type H3 and recombinant Q19L20->AA mutant H3.The reactions were analysed by western blot with anti C-terminal H3 antibody.(D) Activity against different histones.Stationary phase pull down on Sepharose beads was assayed against identical amounts of calf histones. The reactions were analysed by western blot with antibodies specific for each histone. The clipped H3 product is highlighted.
Mentions: While assaying yeast protein complexes for their capacity to demethylate histone H3, we noticed an endopeptidase activity in our nuclei preparations which cleaves the exogenously provided substrate (calf H3). The activity was low in the nuclei of cells growing exponentially in rich medium but increased in cells grown into stationary phase or shifted to sporulation medium (Figure 1A). In an attempt to biochemically purify this activity (see Materials and Methods) we found that it was retained on sepharose-based matrices (Figure 1B). To identify the site of cleavage on calf H3, we analyzed the reactions by mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) on the substrate (calf H3) identified a broad peak representing H3 carrying combinations of post-translational modifications (Supplementary Fig. 1, dark blue circle). When reactions were performed in the presence of the endopeptidase activity from stationary cells, two additional peaks were detected (Supplementary Fig.1, red and light blue circles). Electrospray sequencing of the smaller peak revealed products corresponding to the first 21 amino acids of calf H3 carrying various modifications (Supplementary Fig. S2). The N-terminal tail of H3 is sufficient for endopeptidase recognition as peptides spanning amino acids 1 to 30 are also cleaved after alanine 21 (Supplementary Fig. S3). To establish whether flanking residues represent a recognition site for the enzyme, full-length H3 was mutated at position 19 and 20 from QL to AA, expressed in E. coli and used as a substrate for the endopeptidase activity. Figure 1C shows that the wild type full length H3 is cleaved whereas the QL to AA mutant is resistant to the endopeptidase activity.

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