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, 7 (3), e1001354

H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

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H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

Benoit Guillemette et al. PLoS Genet.

Abstract

Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me).

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. H3K4 Is Acetylated in S. cerevisiae.
(A) Synthetic peptides derived from the histone H3 N-terminal tail (residues 1 to 10) with modifications at different residues were applied to a nitrocellulose membrane. The membrane was incubated with the H3K4ac antibody, a horseradish peroxidase-conjugated anti-rabbit IgG, and signals visualised by chemiluminescence (left). Ponceau S staining showing that all the peptides bound to nitrocellulose (right). (B) Immunoblots carried out with nuclear extracts from mouse thymocytes. The membrane was cut into strips and incubated with H3K4ac antibody that was pre-incubated with the indicated competitor peptides (1 µg/ml), resulting in a molar ratio of approximately 1000∶1 (peptide:antibody). (C) Dot blots of synthetic peptides (100ng each) containing H3K4ac, H3K9ac, and tetra-acetylated histone H4, (K5, 8, 12, 16)ac, were incubated with H3K4ac and H3K9ac antibodies. Antibody binding was detected as described above. (D) Whole-cell lysates from strains expressing either WT or mutant versions of H3 were analysed by immunoblotting for H3K4ac or a non-modified C-terminal peptide of H3. (E) Coomassie stained gel of histones purified from exponentially growing yeast cells. (F) Doubly charged precursor ion with the expected m/z ratio for tryptic peptide 3-TKacQTAR-8. The experimental and theoretical masses of the non-charged peptides are indicated in the inset along with the mass difference between the empirically determined and the predicted mass of the K4-acetylated peptide. (G) MS/MS spectrum of the doubly charged precursor ion with m/z 373.7113. The peptide sequence is shown from its C-terminus to its N-terminus above the spectrum. (H) Chromatin immunoprecipitation (ChIP) assays were analysed by quantitative PCR to determine the abundance of H3K4ac relative to non-modified H3 ChIP at different loci in WT and H3-K4R cells.
Figure 2
Figure 2. HATs and HDACs That Contribute to H3K4ac In Vivo.
(A–B) Whole-cell lysates prepared from strains with deletions of known HAT encoding genes were analysed by immunoblotting with the indicated antibodies. (C) Quantification of the immunoblots shown in (B). Signals for each band were expressed in Arbitrary Units (AU) and, after background subtraction (AUb), normalised to the H3-C signal measured in each strain. (D–E) Whole-cell lysates obtained from strains with deletions of known HDAC encoding genes were analysed by immunoblotting with the indicated antibodies. (F) Quantification of the immunoblots shown in (E), calculated as described above. (G) Chromatin immunoprecipitation (ChIP) was performed by quantitative PCR to determine the abundance of H3K4ac at different loci in WT, hst1Δ, sir2Δ or hst1Δ sir2Δ strains. The six target regions were divided into three groups: regions targeted by Sir2, gene promoters targeted by Hst1 and other gene regions not targeted by Sir2 or Hst1. The TEL03R primers amplify a portion of ARS319, a unique sequence region approximately 1 Kb from the telomeric repeats on the right arm of chromosome III. Results are expressed as a ratio of ChIP signals obtained from the same extracts with antibodies against H3K4ac and a non-modified H3 C-terminal peptide.
Figure 3
Figure 3. H3K4ac Is Enriched at Promoters of Transcribed Genes.
(A) Examples of the localisation pattern of H3K4ac along a 50 kb segment of chromosome 12, as displayed on the UCSC genome browser (http://genome.ucsc.edu/) . The data (purple) are represented as a log2 ratio of H3K4ac over non-modified H3 C-terminus ChIP signals and represent the average of 3 biological replicates. The names, position and orientation of ORFs (S. cerevisiae assembly Oct. 2003) are shown (blue) at the bottom of each panel. (B–C) Systematic analyses of the genome-wide location of H3K4ac / H3-C ChIP signals at: (B) 5′-ends of ORFs with divergent promoter regions. (C) 3′-ends of ORFs with convergent terminator regions. The data was aligned to the closest 5′- or 3′-end of ORFs, normalised for gene length and sub-divided into 5 groups according to transcriptional activity (mRNA/hr) . For simplicity, the relative position value for each data point was rounded to the nearest hundredth. The y-axes show the abundance of H3K4ac (average of three biological replicates) as a function of relative position along the nearest gene (x-axes).
Figure 4
Figure 4. H3K4ac Is Located Upstream of H3K4me3.
(A) The H3K4ac / H3 (black) ChIP-chip data were aligned with the H3K4me3 (burgundy), -me2 (purple) and -me1 (blue) data from and displayed in the UCSC genome browser (http://genome.ucsc.edu/). Each vertical line represents one probe in the dataset and the intensity of the color is proportional to the enrichment of the modification. For clarity, only the probes with a log2 ratio above 0 are shown for each dataset. (B) Systematic alignement of H3K4ac / H3 (red shades) and H3K4me3 / H3 (blue shades) ChIP-chip data at intergenic regions (IGRs) with divergent promoters (as in Figure 3B). The raw ChIP-chip data were converted into Z-scores in order to be compared on the same scale (see Material and Methods). (C–E) Venn diagrams illustrating the overlapping number of promoters that are enriched for H3K4ac or H3K4me3. The cut-off indicates the H3K4(mod) / H3-C Z-score threshold at which a given promoter was judged to be enriched in either H3K4ac or H3K4me3. For example, with a cut-off at the 90th percentile (C), the red circle contains the number of promoters that have H3K4ac / H3 signal ratios in the top 10% of the genome. (F) A table displaying the data from the Venn diagrams as percentages.
Figure 5
Figure 5. The COMPASS Complex Limits Global Levels of H3K4ac and Confines H3K4ac Localisation to Promoter Regions.
(A) Extracted ion chromatogram showing the retention time of peptide 3-TKacQTAR-8 during reverse phase HPLC and its relative abundance in WT and set1Δ cells. (B–F) Whole-cell lysates from the indicated mutants were analysed by immunoblotting. In (E), a peptide competition assay was performed as described in Figure 1B. (G–H) Examples of the localisation pattern of H3K4ac in either WT (purple) or set1Δ (green) strains at two genomic regions displayed using the UCSC genome browser (genome.ucsc.edu) . The data represents the normalised (Z-score) log2 ratio of H3K4ac over H3-C (see Materials and Methods). (I) Systematic analyses of H3K4ac in WT versus set1Δ cells in all intergenic regions (IGRs) that contain divergent promoters. The raw H3K4ac ChIP-chip data in WT or set1Δ cells were normalised either against their respective H3-C ChIP (black: WT, blue: set1Δ), converted into Z-scores (see Material and Methods) and aligned relative to the 5′-end of the corresponding ORF (normalised as described in Figure 3). The difference between the set1Δ and WT datasets (set1Δ - WT, green) is also shown.
Figure 6
Figure 6. Global mRNA Expression Profiles of set1Δ and H3-K4R Mutant Strains.
(A) Graph of mRNA expression fold-change relative to WT cells in the H3-K4R (blue) and set1Δ (yellow) strains for all the analysed genes. The genes were ranked according to log2 fold-change in the H3-K4R strain and a sliding average (window  =  50, step  =  1) was applied to the data on the y-axis. The bottom panels are heat map illustrations of the log2 fold-change, where yellow indicates an increase in mRNA abundance in the mutant strains relative to WT cells and blue indicates a decrease. (B–C) Venn diagrams showing the overlap between groups of genes up-regulated (B) or down-regulated (C) at least two-fold when either the H3-K4R or the set1Δ strain is compared with WT cells. (D) Graph of the H3K4ac / H3 ratio at the promoter regions of each gene (average for from −200 to +1bp relative to the start codon) derived from the ChIP-chip data as a function of their log2 fold-change in mRNA abundance in the H3-K4R mutant strain versus WT cells (derived from the mRNA expression microarray data). A sliding window average was applied to the data on both axes (window  =  50, step  =  1). (E–H) Localisation of H3K4ac near genes that belong to the ontology group of “amino acid transporters” and are poorly expressed in H3-K4R mutants compared with WT cells: (BAP2/YBR068C, TAT1/YBR069C, BAP3/YDR046C, GNP1/YDR508C, TAT2/YOL020W). The start sites of these genes are indicated by arrows and vertical dashed lines indicate the peaks of H3K4ac in their upstream regions. The display is from the UCSC genome browser (http://genome.ucsc.edu/). (I–M) RT-qPCR analyses of RNA extracted from the indicated strains (WT, H3-K4R, set1Δ, H3-K4R set1Δ). The values were normalised to ACT1. The data represent an average of three biological replicates.

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