H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells

Abstract

Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these alternatively splice exons. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells.

Figure 1.

KDM5B regulates H3K4me3 in ES cells. (A) Venn diagram showing overlap of KDM5B in control (shLuc) and KDM5B-depleted (shKdm5b) ES cells (See ‘Materials and Methods’ section for SICER-analysis; FDR < 0.001). (B) Scatter plot and (C) average profile of KDM5B densities in shLuc and shKdm5b ES cells. (D) Heat maps of KDM5B and H3K4me3 densities at H3K4me3 regions in ES cells. (E) Depletion of KDM5B leads to decreased H3K4me3 levels at promoters and increased levels in gene body regions (See ‘Materials and Methods’ section for SICER-analysis; fold-change > 1.5, FDR < 0.001). (F) Boxplots of KDM5B densities in shLuc and shKdm5b ES cells at H3K4me3 regions. (G) Annotation of H3K4me3 regions using HOMER (34) software. (H) Western blot of KDM5B and H3K4me3 in shLuc, shKdm5b, shKdm5b+ wild-type KDM5B (WT), shKdm5b+ mutant KDM5B (H499A; mut). Note the increased levels of H3K4me3 in shKdm5b and shKdm5b+mut ES cells relative to control (shLuc) and shKdm5b+WT ES cells. The three arrowheads in the KDM5B western blot represent the size of endogenous KDM5B (middle arrow), FLAG-tagged KDM5B (top arrow) and a non-specific band (bottom arrow).

Figure 2.

KDM5B regulates RNAPII occupancy at active genes in ES cells. (A) Venn diagrams showing overlap of RNAPII and H3K4me3 (left) and KDM5B and RNAPII/H3K4me3 at all SICER-defined islands. (B) Venn diagrams showing co-occupancy of KDM5B and RNAPII at all islands (top) or at promoter regions (bottom). (C) Change in the global distribution of RNAPII SICER-defined islands in shKdm5b ES cells relative to shLuc ES cells. (D) Scatter plots of RNAPII densities in shLuc and shKdm5b ES cells. x-axis: log2 fold-change density (shKdm5b/Input); y-axis: log2 fold-change density (shLuc/Input). (E) Boxplots of RNAPII densities in shLuc and shKdm5b ES cells. (F) Boxplots of KDM5B densities in shLuc and shKdm5b ES cells at RNAPII regions. (G) ChIP-Seq tag density of RNAPII binding normalized by input (log2 fold-change versuss Input) for all refseq genes sorted into quartiles based on their expression level in control (shLuc) ES cells. (H) ChIP-Seq tag density of RNAPII at TSS regions in shLuc and shKdm5b ES cells. (I) Schematic describing the calculation used to determine the traveling index (TI) at RNAPII marked genes in ES cells. The promoter bin is defined as a 1 kb window around the TSS of genes marked by RNAPII, while the transcribed region (gene body) is defined as the region extending to the TES. The TI is calculated from the ratio of the density of RNAPII in the promoter bin to the density of RNAPII in the gene body bin. (J) Empirical cumulative distribution for the TI of RNAPII across all genes for shLuc (black) and shKdm5b (red) ES cells. Y-axis shows the percentage of genes that exhibit a TI less than the value specified by the x-axis. A line shifted to the left means a systematic decrease in the TI. P-value for all <1E-5 (Kolmogorov–Smirnov test). Note the decreased TI for genes marked by RNAPII in shKdm5b ES cells. (K) Scatter plot of the ratio of relative tag densities of RNAPII and H3K4me3 in shKdm5b versus shLuc ES cells. x-axis: log2 fold-change (shKdm5b-RNAPII/shLuc-RNAPII); y-axis: log2 fold-change (shKdm5b-H3K4me3/shLuc-H3K4me3).

Figure 3.

KDM5B regulates RNAPII initiation at active genes in ES cells. (A) Venn diagram showing overlap of RNAPII-Ser5P and KDM5B at promoter regions in ES cells. (B) Change in the global distribution of RNAPII-Ser5P in shKdm5b ES cells. (C) Boxplots of RNAPII-Ser5P densities in shLuc and shKdm5b ES cells. (D) Boxplots of KDM5B densities in shLuc and shKdm5b ES cells at RNAPII Ser5P regions. (E) Average profile of RNAPII-Ser5P binding normalized by input (log2 fold-change versus Input) of all refseq genes (TSS-TES) sorted into quartiles based on their expression level in ES cells. (F) Heat map of change in RNAPII-Ser5P in shKdm5b versus shLuc ES cells (yellow, decrease). (G) Average profile of RNAPII-Ser5P at TSS regions in shLuc and shKdm5b ES cells. (H) UCSC browser view of KDM5B, H3K4me3, RNAPII and RNAPII-Ser5P in shLuc and shKdm5b ES cells.

Figure 4.

KDM5B regulates RNAPII elongation in ES cells. (A) Venn diagram showing overlap of RNAPII-Ser2P and KDM5B in gene body regions in ES cells. (B) Change in the global distribution of RNAPII-Ser2P in shKdm5b versus shLuc ES cells as defined by SICER analysis (see methods, fold-change > 1.5, FDR < 0.001). (C) Annotation of differentially enriched RNAPII-Ser2P regions shLuc and shKdm5b ES using HOMER software (34). (D) Histogram showing the distance of differentially enriched (increased or decreased) RNAPII Ser2P islands from TSS regions. Note that RNAPII Ser2P islands with decreased levels are located further from TSS regions relative to islands with increased levels. (E) Boxplots and (F) scatter plots of RNAPII-Ser2P densities in shLuc and shKdm5b ES cells. (G) Average profile of RNAPII-Ser2P binding normalized by input (log2 fold-change versus Input) at all refseq genes (TSS-TES) sorted into quartiles based on their expression level in control (shLuc) ES cells. The red and orange arrows denote the peak of RNAPII Ser2P in shLuc ES cells. (H) Average profile of RNAPII-Ser2P density at TES regions in shLuc and shKdm5b ES cells. RNAPII-Ser2P is known to be enriched at TES regions of genes. (I) Heat map of change in RNAPII-Ser2P in shKdm5b versus shLuc ES cells (yellow, decrease). (J) KEGG gene (left heatmap) and ChIP-X genes (right heatmap) evaluated using Network2Canvas demonstrates that metabolic and lysine degradation genes have decreased RNAPII-Ser2P in shKdm5b ES cells, and are bound by KDM5B. Each node (square) represents a gene list (shLuc versus shKdm5b bound genes bound by RNAPII-Ser2P associated with a gene-set library (KEGG or ChIP-X). The brightness (white) of each node is determined by its P-value.

Figure 5.

KDM5B regulates RNAPII at genes with broad H3K4me3 domains. (A) Distribution of KDM5B, H3K4me3 and RNAPII SICER-defined ChIP-Seq peaks in ES cells. (B) Venn diagram showing the top 5% broadest H3K4me3 peaks in ES cells. (C) Heatmap of H3K4me3 ChIP-Seq tag density at TSS to pA regions of genes with the broadest (top 5%) H3K4me3 domains. (D) ChIP-Seq tag density of H3K4me3 around TSS regions of genes with the broadest (top 5%) H3K4me3 domains in shLuc and shKdm5b ES cells. (E) Correlation between changes in gene body histone methylation and expression level. Fold-change normalized tag density ratios (shKdm5b-ESC-H3K4me3/shLuc-ESC-H3K4me3) of H3K4me3 at genes with the broadest (top 5%) H3K4me3 domains, which were sorted into four groups based on their absolute expression level in control (shLuc) ES cells (red line, highest 25% expressed; green line, lowest 25% expressed). (F) Boxplots of RNAPII, RNAPII-Ser5P and RNAPII-Ser2P ChIP-Seq tag densities. log2 fold-change (shLuc-RNAPII/shLuc-Input); log2 fold-change (shKdm5b-RNAPII/shKdm5b-Input) at the broadest (top 5%) H3K4me3 domains in ES cells. (G) Heatmaps of RNAPII, RNAPII-Ser5P and RNAPII-Ser2P ChIP-Seq tag densities at the broadest (top 5%) H3K4me3 domains in ES cells. (H) Boxplots of densities of RNAPII, RNAPII-Ser5P and RNAPII-Ser2P ChIP-Seq tag densities at promoter and gene body regions of genes with the broadest (top 5%) H3K4me3 domains in ES cells.

Figure 6.

KDM5B is involved in regulating alternative splicing in ES cells. Average profiles of (A) KDM5B (left panel), (B) H3K4me3, (C) RNAPII-Ser5P and (D) RNAPII-Ser2P densities at exons (Norm. tag density versuss Input). Only exons located in gene body regions (>2 kb from TSS) were used for this analysis. The right panel in (A) shows KDM5B binding at random genomic sequences (Norm. tag density versus Input). An increase of H3K4me3 in gene body regions in KDM5B-depleted ES cells is consistent with previous findings. (E–G) Average profile of H3K4me3 densities at (E) upregulated or (F) downregulated alternatively spliced exon (cassette exons) regions and their flanking constitutively spliced exons in ES cells. Schematic of alternatively spliced exon and the flanking exons is shown below the graph. Densities were calculated at cassette exons from the two alternatively spliced exon groups (upregulated and downregulated) and levels of H3K4me3 are displayed relative to splice acceptor (acc) and donor (don) sites across cassette exons. (G) Average profile of KDM5B densities at upregulated or downregulated alternatively spliced exon regions and constitutively spliced flanking exons in ES cells. P-values for relative enrichment of H3K4me3 or KDM5B at differentially expressed alternatively spliced exons are shown.