H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress

Abstract

The selectivity of transcriptional responses to extracellular cues is reflected by the deposition of stimulus-specific chromatin marks. Although histone H3 phosphorylation is a target of numerous signaling pathways, its role in transcriptional regulation remains poorly understood. Here, for the first time, we report a genome-wide analysis of H3S28 phosphorylation in a mammalian system in the context of stress signaling. We found that this mark targets as many as 50% of all stress-induced genes, underlining its importance in signal-induced transcription. By combining ChIP-seq, RNA-seq, and mass spectrometry we identified the factors involved in the biological interpretation of this histone modification. We found that MSK1/2-mediated phosphorylation of H3S28 at stress-responsive promoters contributes to the dissociation of HDAC corepressor complexes and thereby to enhanced local histone acetylation and subsequent transcriptional activation of stress-induced genes. Our data reveal a novel function of the H3S28ph mark in the activation of mammalian genes in response to MAP kinase pathway activation.

Figure 1.

Stress-induced H3S28ph targets active promoters. (A) Enrichment of H3S28ph ChIP-seq regions over six gene features compared with the genome background. “Promoter” is defined as the region located within 1 kb upstream of the annotated transcription start site (TSS). “CDS” refers to the coding sequence. “Gene” consists of 5′UTR, coding exons, introns, and 3′UTR. “Intergenic” refers to all regions located outside of genes extended by 1 kb from both gene ends. The significance of the observed enrichment was determined by a one-sided binomial test. (B) Average density plots of normalized H3S28ph ChIP-seq tags centered at transcription start sites (TSS, left panel), transcription end sites (TES, right panel) for a control (ctrl), and stress-induced state (aniso). (Middle panel) The metagene profile of H3S28ph ChIP-seq densities. The genes are grouped according to their expression level: highly and moderately expressed (in red; ctrl: 3429 genes, aniso: 3181 genes), lowly expressed (in blue; ctrl: 7271 genes, aniso: 7369 genes), and not expressed (in green; ctrl: 10,908 genes, aniso: 11,059 genes). The average profile derived from all genes is depicted in black. The classification of the genes is based on mRNA-seq data (see Supplemental Methods and Supplemental Fig. 4B).

Figure 2.

Stress signaling via p38/MAPK mainly targets the regulators of signal transduction characterized by the high CG content of their promoters. (A) Changes in mRNA abundance of 21,608 RefSeq genes upon stress stimulation determined by full-length mRNA-seq. (B) Histogram showing the distribution of promoter classes according to CpG content. Weak and strong CpG promoters are enriched in the group of up-regulated genes (P-value = 1.052 × 10⁻¹⁴, χ² test). (C) Transcription-factor motifs enriched in promoter regions (located between 400 bp upstream of and 100 bp downstream from the TSS) of up-regulated genes.

Figure 3.

Stress-induced deposition of H3S28ph is dependent on MSK1/2 activity. (A) Genome browser representations of H3S28ph, H3K9ac, H3K4me3, RNAPIIS5ph, and RNAPIIS2ph normalized tag density profiles of representative genes (Dusp1 and Mafk) up-regulated after 1 h of treatment with anisomycin. The profiles derived from untreated cells are depicted in blue and profiles of cells under stress-induced conditions are shown in red. (B,D) ChIP-qPCR analysis of stress-induced H3S28ph levels upon MSK1/2 inhibition with H89 at Dusp1 and Mafk genes (left panels); and RT-qPCR analysis of mRNA expression of Dusp1 and Mafk genes (right panels) in control (C) and anisomycin-treated cells (A) in the absence or presence of H89. Error bars represent SDs (n = 3). (*) P < 0.05. (C,E) ChIP-qPCR analysis of stress-induced H3S28ph in knockdown control (NT) and Rpska5 knockdown (MSK) cells at Dusp1 and Mafk genes (left panels); and RT-qPCR analysis of mRNA expression of Dusp1 and Mafk genes (right panels) upon anisomycin treatment in knockdown control (NT) and Rpska5 knockdown (MSK) cells. Error bars represent SDs (n = 3). (*) P < 0.05. Knockdown efficiency is shown in Supplemental Figure 10B–D.

Figure 4.

Classification of stress-induced genes. (A) Normalized RNAPIIS5ph ChIP-seq tag density profiles of untreated (blue line) and stress-induced (red line) serum-deprived mouse Swiss 3T3 fibroblasts. The genes are divided into four groups according to their pausing indices and the presence of scRNA. (B) Violin plots showing the distribution of pausing indices among groups of genes associated with different RNAPII profiles. (C) Histogram showing the distribution of H3S28ph-marked genes among different regulatory groups. None of the groups is significantly enriched in H3S28ph marked genes (P-value = 0.07, Fisher’s exact test).

Figure 5.

A subset of H3S28ph target genes is primed for later activation. (A) Western blot analysis of H3S28ph levels in serum deprived mouse Swiss 3T3 fibroblasts treated with anisomycin for 1, 3, and 6 h. H3 C-terminus antibody was used as a loading control. (B) Barplot showing the overlap between genes associated with H3S28ph after 1 h of anisomycin treatment and genes up-regulated after 1, 3, and 6 h after stress induction. P-values were calculated using a hypergeometric test. (C) ChIP-qPCR analysis of stress-induced H3S28ph at Optn and Ube2v2 genes in control (C) and anisomycin-treated cells (A) in the absence or presence of H89. Error bars represent SDs (n = 3). (*) P < 0.05. (D) RT-qPCR analysis of Optn and Ube2v2 gene expression after 1, 3, and 6 h of anisomycin treatment in the absence and presence of H89. Error bars represent SDs (n = 3). (*) P < 0.05.

Figure 6.

H3S28ph-marked genes show higher increase in histone acetylation levels upon 1 h of anisomycin stimulation. (A) Boxplots showing log₂ fold change in normalized read counts (RPKM) for H3K9ac and H3K4me3 ChIP-seq after 1 h of treatment with anisomycin at 1hA, 3hA, and 6hA targets. Fold change was calculated as the ratio of RPKM in anisomycin condition to RPKM in the control state at the regions from −1 kb to +3 kb surrounding the TSS. P-values were determined by the Mann-Whitney U test. (B,C) ChIP-qPCR analysis of H3K9ac, H3K27ac, and H4ac levels at Dusp1 and Ube2v2 genes in control (C) and anisomycin-treated cells (A) in the absence and presence of H89. Error bars represent SDs (n = 3). (*) P < 0.05.

Figure 7.

H3S28ph mediates the dissociation of HDAC-containing complexes from target promoters. (A) Western blot analysis of histone pull-down assays with nuclear extracts from HeLa cells treated with anisomycin for 1 h and synthetic peptides corresponding to aa 3–20 and 19–36 of histone H3, either unmodified or carrying the phosphorylation mark at S10 or S28. The association of SIN3A, HDAC1, HDAC2, MTA1, and 14-3-3 zeta (encoded by Ywhaz) with differentially modified peptides was analyzed. (B–D) ChIP-qPCR analysis of changes in SIN3A, HDAC2, and HDAC1 occupancy at Dusp1 and Ube2v2 genes in control (C) and anisomycin-treated cells (A) in the absence and presence of H89. Error bars represent SDs (n = 3). (*) P < 0.05. (E,F) RT-qPCR analysis of Dusp1, Mafk, Ube2v2, and Optn gene expression upon anisomycin treatment (A) in control (NT) and Hdac2 (HD2) knockdown cells. Error bars represent SDs (n = 3). (*) P < 0.05. (G) A model demonstrating the impact of H3S28 phosphorylation on the local histone acetylation levels at stress-induced genes. Local histone acetylation results from the dynamic interplay between recruited HAT and HDAC activities. Upon stress stimulation, MSK1/2 phosphorylates S28 at histone H3 at stress target promoters. This leads to dissociation of HDAC-containing complexes, thereby inducing an increase in local histone acetylation levels.