Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae

Abstract

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.

Fig. 1.

Histone H3-specific acetyltransferases were required for H3K4me3. (A and C) Whole cell extracts from the indicated strains were subjected to immunoblot analysis for H3K4me3, H3K4me2, H3K4me1, H3K4me0, H3K14ac, or H3. (B and D) Immunoblots shown in A and C were repeated with three independent cultures and the results quantified after normalization for histone H3. Shown is H3K4me3 (red) or H3K14ac (blue) normalized for histone H3. Error bars indicate mean ± the SE. (E) Corrected cumulative count plot (SeqMonk) of H3K4me3 levels relative to the TSS of 4,637 yeast genes in the indicated strains.

Fig. 2.

Deletion of JHD2 restored H3K4me3 levels in cells lacking histone H3 acetylation. (A) Whole cell extracts from the indicated strains were subjected to immunoblot analysis for H3K4me3 and H3. A yellow color represented equal red and green intensities. (B) The immunoblot in A was repeated with three independent cultures and the results quantified. Shown is H3K4me3 (red) or H3K14ac (blue) normalized for histone H3 and shown relative to wild-type (WT) levels. Error bars indicate mean ± SE. (C) Corrected cumulative count plot of H3K4me3 levels relative to the TSS of 4,637 yeast genes in the indicated strains.

Fig. 3.

H3K14ac negatively regulates demethylation by Jhd2 in vivo and in vitro. (A) Whole cell extracts were subjected to immunoblot analysis for H3 (red) and H3K4me3 (green) and the images merged. Yellow indicated equal red and green intensities. The strains expressed either WT or mutant (mut) versions of H3 as shown. (B) ChIP-on-chip analysis of genome-wide levels of H3K14ac and H3K4me3 relative to the TSS. All genes with known TSSs were divided into five transcript length classes: very short (XS) ≤ 750 bp, short (S) 750–1,500 bp, medium (M) 1,500–2,250, long (L) 2,250–3,000, and very long (XL) 3,000–3,750. The five groups comprised 542, 1,859, 1,266, 631, and 291 genes, respectively. All genes in each group were partitioned into 150 bp bins, and the average H3K4me3 enrichment values were calculated and plotted. (C) Jhd2, purified from yeast, was subjected to histone demethylase assays using the indicated peptides. Removal of H3K4me3 was analyzed by immunoblot with H3K4me3-specific antibodies.

Fig. 4.

H3K14ac regulates H3K4 demethylation during gene repression. (A and B) Models for coordinated control of histone acetylation and methylation on the H3 tail. (A) In the presence of an activating stimulus, transcriptional activators (TAs) bind DNA sequences and recruit HAT complexes, which acetylate histone H3. Subsequent recruitment of Set1 by RNAP II results in generation of all three methylation states of H3K4. H3K14ac prevents Jhd2 demethylase activity on H3K4me3, stabilizing this mark. (B) After loss of an activating stimulus or in the presence of a repressive signal, a transcriptional repressor (TR) binds to its DNA sequence and recruits histone deacetylase (HDAC) complexes. HDACs deacetylate the H3 tail, allowing Jhd2 to demethylate H3K4me3. (C) Schematic representation of the GAL1 gene. The arrow indicates the TSS and the line below shows the PCR product detected in D. (D) Yeast strains, expressing WT or mutant versions of histone H3 as indicated, were grown in galactose before transfer to dextrose-only media for the indicated times. ChIP–qPCR was used to measure H3K4me3 levels. Error bars indicate mean ± SE from three independent cultures.