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H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells.

Karmodiya K, Krebs AR, Oulad-Abdelghani M, Kimura H, Tora L - BMC Genomics (2012)

Bottom Line: Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac.This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation.In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U 964, Université de Strasbourg, BP 10142-67404 ILLKIRCH Cedex, CU de Strasbourg, France.

ABSTRACT

Background: Transcription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression.

Results: Here we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genome-wide H3K9ac and H3K14ac show very high correlation between each other as well as with other histone marks (such as H3K4me3) suggesting a coordinated regulation of active histone marks. Moreover, the levels of H3K9ac and H3K14ac directly correlate with the CpG content of the promoters attesting the importance of sequences underlying the specifically modified nucleosomes. Our data provide evidence that H3K9ac and H3K14ac are also present over the previously described bivalent promoters, along with H3K4me3 and H3K27me3. Furthermore, like H3K27ac, H3K9ac and H3K14ac can also differentiate active enhancers from inactive ones. Although, H3K9ac and H3K14ac, a hallmark of gene activation exhibit remarkable correlation over active and bivalent promoters as well as distal regulatory elements, a subset of inactive promoters is selectively enriched for H3K14ac.

Conclusions: Our study suggests that chromatin modifications, such as H3K9ac and H3K14ac, are part of the active promoter state, are present over bivalent promoters and active enhancers and that the extent of H3K9 and H3K14 acetylation could be driven by cis regulatory elements such as CpG content at promoters. Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac. This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation. In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.

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Genomic distribution of H3K9ac and H3K14ac in mES cells. (A) Distribution of H3K9ac and H3K14ac peaks over the promoters (2000 bp upstream of TSS,), coding exons, introns and distal intergenic regions. Many of the H3K9 and H3K14 acetylation peaks are at distal intergenic regions. (B) Dot plot representation of genome-wide co-localization analysis of the H3K9ac and H3K14ac modifications over the 15595 combined promoter list of H3K9 and H3K14ac suggests a strong correlation between these two modifications at promoters. (C) Average input normalized profile of 15595 combined promoter list of H3K9 and H3K14ac around the transcription starts sites shows bimodal distribution. (D) Average input normalized whole gene profiles for H3K9ac and H3K14ac modifications over 15595 combined promoter list of H3K9 and H3K14ac. (E) Sequential ChIP–qPCR quantification for co-occupancy of H3K9ac (primary ChIP) and H3K14ac (secondary ChIP) at randomly selected H3K9 and H3K14 acetylated loci suggest that these loci are co-marked with H3K9 as well H3K14 acetylation. Enrichment after first ChIP using H3K9ac followed by re-ChIP with no antibody was used as a control. Primer sequences used in ChIP-qPCR is provided in Additional file 4: Table S1. Error bars represent the standard deviation for three technical replicates.
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Figure 1: Genomic distribution of H3K9ac and H3K14ac in mES cells. (A) Distribution of H3K9ac and H3K14ac peaks over the promoters (2000 bp upstream of TSS,), coding exons, introns and distal intergenic regions. Many of the H3K9 and H3K14 acetylation peaks are at distal intergenic regions. (B) Dot plot representation of genome-wide co-localization analysis of the H3K9ac and H3K14ac modifications over the 15595 combined promoter list of H3K9 and H3K14ac suggests a strong correlation between these two modifications at promoters. (C) Average input normalized profile of 15595 combined promoter list of H3K9 and H3K14ac around the transcription starts sites shows bimodal distribution. (D) Average input normalized whole gene profiles for H3K9ac and H3K14ac modifications over 15595 combined promoter list of H3K9 and H3K14ac. (E) Sequential ChIP–qPCR quantification for co-occupancy of H3K9ac (primary ChIP) and H3K14ac (secondary ChIP) at randomly selected H3K9 and H3K14 acetylated loci suggest that these loci are co-marked with H3K9 as well H3K14 acetylation. Enrichment after first ChIP using H3K9ac followed by re-ChIP with no antibody was used as a control. Primer sequences used in ChIP-qPCR is provided in Additional file 4: Table S1. Error bars represent the standard deviation for three technical replicates.

Mentions: In order to test whether H3K9ac and H3K14ac modifications have differential preference over various chromatin regions, we compared their presence over promoters (2000 bp upstream of transcription start sites (TSSs)), coding exons, introns and distal intergeneic regions (Figure1A), which represent 2.5%, 1.6%, 38.7% and 57.2% of the total genome, respectively [22]. First, peaks of H3K9ac and H3K14ac local enrichment were determined after sequence alignment and normalization to input DNA using MACS [23]. Our analyses show that H3K9ac and H3K14ac peaks are distributed over all four genomic regions and the frequency of distribution over promoters is 13.2% and 12.4%, respectively (Figure1A). However, approximately 85% of both the H3K9ac and H3K14ac marks are observed in distal intergenic and intronic regions with significant enrichments, comparable to promoters ( Additional file 5: Figure S4), suggesting that these two modifications may have a role at distal intergeneic and intronic regions. Further to compare H3K9 and H3K14ac marks, a combined list of binding sites at transcription start sites (TSSs) was established containing 15595 TSSs. The comparison of H3K9ac and H3K14ac over these TSSs shows a Pearson correlation coefficient of 0.73 (Figure1B), suggesting that the studied two H3 acetylation marks are present simultaneously on promoters (Figure1C). Moreover, H3K9 and H3K14 acetylations have a characteristic bimodal distribution around the TSSs, with one peak upstream of the TSS, another single peak (stronger in the case of H3K14ac) downstream of the TSS, and depletion of the signal right on the TSS (Figure1C). To examine the distribution of these two histone marks over the gene body, composite profile of both marks spanning the entire gene body and extending 5 kb upstream from TSSs and 5 kb downstream of the 3’ end of the genes for combined list of binding sites over TSSs (15595) was generated (Figure1D). The H3K9ac and H3K14ac distribution profiles around TSSs suggest that both marks are predominantly located in regions surrounding the TSSs of genes. Further to confirm that the co-occupancy of H3K9 and H3K14 observed is not because of cellular heterogeneity, we performed sequential ChIP for H3K9ac followed by H3K14ac (Figure1E). Sequential ChIP demonstrates that genomic loci are acetylated simultaneously both at H3K9 as well H3K14. Thus, our analyses suggest that on the genome H3K9ac and H3K14ac are mostly present at distal intergenic and intronic regions, specifically enriched at promoters when localized in the vicinity of genes and that the two acetylation marks co-occur at promoters as well as other locations.


H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells.

Karmodiya K, Krebs AR, Oulad-Abdelghani M, Kimura H, Tora L - BMC Genomics (2012)

Genomic distribution of H3K9ac and H3K14ac in mES cells. (A) Distribution of H3K9ac and H3K14ac peaks over the promoters (2000 bp upstream of TSS,), coding exons, introns and distal intergenic regions. Many of the H3K9 and H3K14 acetylation peaks are at distal intergenic regions. (B) Dot plot representation of genome-wide co-localization analysis of the H3K9ac and H3K14ac modifications over the 15595 combined promoter list of H3K9 and H3K14ac suggests a strong correlation between these two modifications at promoters. (C) Average input normalized profile of 15595 combined promoter list of H3K9 and H3K14ac around the transcription starts sites shows bimodal distribution. (D) Average input normalized whole gene profiles for H3K9ac and H3K14ac modifications over 15595 combined promoter list of H3K9 and H3K14ac. (E) Sequential ChIP–qPCR quantification for co-occupancy of H3K9ac (primary ChIP) and H3K14ac (secondary ChIP) at randomly selected H3K9 and H3K14 acetylated loci suggest that these loci are co-marked with H3K9 as well H3K14 acetylation. Enrichment after first ChIP using H3K9ac followed by re-ChIP with no antibody was used as a control. Primer sequences used in ChIP-qPCR is provided in Additional file 4: Table S1. Error bars represent the standard deviation for three technical replicates.
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Figure 1: Genomic distribution of H3K9ac and H3K14ac in mES cells. (A) Distribution of H3K9ac and H3K14ac peaks over the promoters (2000 bp upstream of TSS,), coding exons, introns and distal intergenic regions. Many of the H3K9 and H3K14 acetylation peaks are at distal intergenic regions. (B) Dot plot representation of genome-wide co-localization analysis of the H3K9ac and H3K14ac modifications over the 15595 combined promoter list of H3K9 and H3K14ac suggests a strong correlation between these two modifications at promoters. (C) Average input normalized profile of 15595 combined promoter list of H3K9 and H3K14ac around the transcription starts sites shows bimodal distribution. (D) Average input normalized whole gene profiles for H3K9ac and H3K14ac modifications over 15595 combined promoter list of H3K9 and H3K14ac. (E) Sequential ChIP–qPCR quantification for co-occupancy of H3K9ac (primary ChIP) and H3K14ac (secondary ChIP) at randomly selected H3K9 and H3K14 acetylated loci suggest that these loci are co-marked with H3K9 as well H3K14 acetylation. Enrichment after first ChIP using H3K9ac followed by re-ChIP with no antibody was used as a control. Primer sequences used in ChIP-qPCR is provided in Additional file 4: Table S1. Error bars represent the standard deviation for three technical replicates.
Mentions: In order to test whether H3K9ac and H3K14ac modifications have differential preference over various chromatin regions, we compared their presence over promoters (2000 bp upstream of transcription start sites (TSSs)), coding exons, introns and distal intergeneic regions (Figure1A), which represent 2.5%, 1.6%, 38.7% and 57.2% of the total genome, respectively [22]. First, peaks of H3K9ac and H3K14ac local enrichment were determined after sequence alignment and normalization to input DNA using MACS [23]. Our analyses show that H3K9ac and H3K14ac peaks are distributed over all four genomic regions and the frequency of distribution over promoters is 13.2% and 12.4%, respectively (Figure1A). However, approximately 85% of both the H3K9ac and H3K14ac marks are observed in distal intergenic and intronic regions with significant enrichments, comparable to promoters ( Additional file 5: Figure S4), suggesting that these two modifications may have a role at distal intergeneic and intronic regions. Further to compare H3K9 and H3K14ac marks, a combined list of binding sites at transcription start sites (TSSs) was established containing 15595 TSSs. The comparison of H3K9ac and H3K14ac over these TSSs shows a Pearson correlation coefficient of 0.73 (Figure1B), suggesting that the studied two H3 acetylation marks are present simultaneously on promoters (Figure1C). Moreover, H3K9 and H3K14 acetylations have a characteristic bimodal distribution around the TSSs, with one peak upstream of the TSS, another single peak (stronger in the case of H3K14ac) downstream of the TSS, and depletion of the signal right on the TSS (Figure1C). To examine the distribution of these two histone marks over the gene body, composite profile of both marks spanning the entire gene body and extending 5 kb upstream from TSSs and 5 kb downstream of the 3’ end of the genes for combined list of binding sites over TSSs (15595) was generated (Figure1D). The H3K9ac and H3K14ac distribution profiles around TSSs suggest that both marks are predominantly located in regions surrounding the TSSs of genes. Further to confirm that the co-occupancy of H3K9 and H3K14 observed is not because of cellular heterogeneity, we performed sequential ChIP for H3K9ac followed by H3K14ac (Figure1E). Sequential ChIP demonstrates that genomic loci are acetylated simultaneously both at H3K9 as well H3K14. Thus, our analyses suggest that on the genome H3K9ac and H3K14ac are mostly present at distal intergenic and intronic regions, specifically enriched at promoters when localized in the vicinity of genes and that the two acetylation marks co-occur at promoters as well as other locations.

Bottom Line: Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac.This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation.In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U 964, Université de Strasbourg, BP 10142-67404 ILLKIRCH Cedex, CU de Strasbourg, France.

ABSTRACT

Background: Transcription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression.

Results: Here we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genome-wide H3K9ac and H3K14ac show very high correlation between each other as well as with other histone marks (such as H3K4me3) suggesting a coordinated regulation of active histone marks. Moreover, the levels of H3K9ac and H3K14ac directly correlate with the CpG content of the promoters attesting the importance of sequences underlying the specifically modified nucleosomes. Our data provide evidence that H3K9ac and H3K14ac are also present over the previously described bivalent promoters, along with H3K4me3 and H3K27me3. Furthermore, like H3K27ac, H3K9ac and H3K14ac can also differentiate active enhancers from inactive ones. Although, H3K9ac and H3K14ac, a hallmark of gene activation exhibit remarkable correlation over active and bivalent promoters as well as distal regulatory elements, a subset of inactive promoters is selectively enriched for H3K14ac.

Conclusions: Our study suggests that chromatin modifications, such as H3K9ac and H3K14ac, are part of the active promoter state, are present over bivalent promoters and active enhancers and that the extent of H3K9 and H3K14 acetylation could be driven by cis regulatory elements such as CpG content at promoters. Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac. This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation. In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.

Show MeSH