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. 2010 Dec;17(12):1495-9.
doi: 10.1038/nsmb.1924. Epub 2010 Nov 7.

Reciprocal intronic and exonic histone modification regions in humans

Jason T Huff  1 , Alex M Plocik, Christine Guthrie, Keith R Yamamoto
Affiliations

Reciprocal intronic and exonic histone modification regions in humans

Jason T Huff et al. Nat Struct Mol Biol. 2010 Dec.

Abstract

While much attention has been focused on chromatin at promoters and exons, human genes are mostly composed of intronic sequences. Analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we identified histone modifications specifically associated with 5' intronic sequences, distinguishable from promoter marks and bulk nucleosomes. These intronic marks were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3), typically transitioning near internal exons. Several marks transitioned near bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we examined whether splicing affects histone marking. Even with considerable changes in regulated alternative splicing, histone marks were stable. Notably, these findings are consistent with exon definition influencing histone marks. In summary, we show that the location of many intragenic marks in humans can be distilled into a simple organizing principle: association with 5' intronic or 3' exonic regions.

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Figures

Figure 1
Figure 1
Groups of histone marks revealed by PCA. PCA of all ChIP-seq and MNase-seq data 10 kb up- and downstream of RefSeq TSSs is presented in a 3D stereoscopic image. Gray points represent the PCA loadings for each of the 41 histone marks and nucleosome occupancy (Nucs). Principal components (Comp.) 1, 2, and 3 are projected to the lower right, lower left, and top, respectively, with the orientation of the axes shown as gray bars radiating from the origin. Individual points representative of each component are colored: H3K4me3 (orange) for Comp. 1, Nucs (purple) for Comp. 2, and H3K79me2 (green) for Comp. 3. For reference, marks enriched at promoters are colored light orange and H3K36me3 is colored blue. Supplementary Figure 1 shows the fully labeled and separated PCA loadings and the transformed PCA scores plotted on the genome with respect to transcription start sites.
Figure 2
Figure 2
Intragenic histone modification regions reflect gene architecture. (a) H3K79me2, and H3K36me3 ChIP-seq reads plotted on two genes (UGCGL1 and SMURF1) of similar length, but different gene architectures. A black line in each density plot shows where a uniform distribution of sequence reads would be. The proposed 5′ intronic, and 3′ exonic histone modification zones are diagrammed below each gene. (b) Heatmaps show the density for H3K4me3, nucleosomes (Nucs), H3K79me2, and H3K36me3 ChIP- and MNase-seq, plotted with respect to transcription start sites (bent arrows) with genes sorted by distances to the beginning of the first internal exons (i.e. first 3′ splice sites). For clarity only the 4286 annotated RefSeq genes that are in the top 50% of expression and with the 50% longest distances to the first internal exon are shown. Full plots are shown in Supplementary Figure 2. Each gene is displayed as a row and columns are 1-kb bins from 10 kb upstream to 50 kb downstream of transcription start sites (bottom). The value of each bin is shaded by the number of ChIP- or MNase-seq reads per kb, scaled to cover data between 0 reads (white) and the 99th percentile of each sequencing experiment (black), indicated at the top right of each heatmap. At far right the aligned transcription start sites (broken line) and the positions of first internal exons (gray line) are shown as a visual reference.
Figure 3
Figure 3
Histone modification profiles at the alternatively spliced exon of YPEL5 are similar between caffeine-treated SW620 cells expressing different YPEL5 mRNA isoforms. (a) Increasing caffeine concentrations result in higher exon inclusion of YPEL5 exon 2. RT-PCR of SW620 cells treated for 7 hours with 0, 6, or 18 mM caffeine. Alternatively (red) and constitutively (black) spliced exons are numbered and diagrammed to the right of the corresponding RT-PCR product. (b) Normalized H3K79me2 and H3K36me3 profiles in SW620 cells treated with 0 (orange), 6 (green), and 18 (blue) mM caffeine for 7 hours. Points indicate log2(ratios) of H3K79me2/H3 (top) and H3K36me3/H3 (bottom) ChIP-qPCR measurements, relative to the lowest value, for each of two SW620 biological replicates. Vertical gray lines provide a visual reference for the 3′ splice site of each internal exon. Arrowheads indicate the positions of qPCR amplicons along the gene (bottom). The alternatively spliced cassette exon 2 is indicated in red. The gene schematic and markers are drawn to scale according to their genomic coordinates except for the second (green) and third (blue) data points at each amplicon, which are offset to the right to facilitate comparison between conditions.
Figure 4
Figure 4
Histone modification profiles at the alternatively spliced exons of CD45 are similar between B cell lines stably expressing different CD45 mRNA isoforms. In all panels, colored points and lines refer to matched BJAB B cell lines expressing a short hairpin targeting knockdown of either RFP as a control (blue) or hnRNPLL (green) or to matched BL41 B cell lines overexpressing hnRNPLL (orange) or mock-treated as a control (red). (a) BJAB and BL41 B cell lines differ in the relative abundance of CD45 and hnRNPLL mRNA. Points indicate mean log2(ratios) ± s.d. of CD45/RPLP0 and hnRNPLL/RPLP0 qRT-PCR measurements from (n=4) biological replicates (not detectable; ND). (b) Confirmation that passaged BJAB and BL41 B cell lines express different CD45 isoforms. Alternatively (red) and constitutively (black) spliced exons are diagrammed to the right of the corresponding RT-PCR product. (c and d) Normalized H3K79me2 and H3K36me3 profiles in matched B cell lines. Points indicate the mean log2(ratios) ± s.d. of H3K79me2/H3 (top) and H3K36me3/H3 (bottom) ChIP-qPCR measurements, relative to the lowest value, from (c) BJAB (n=5) and (d) BL41 (n=6) biological replicates. Positions of the 3′ splice sites and qPCR amplicons along the gene are displayed as vertical grey lines and arrowheads, respectively. All positions are drawn to scale, except for a gap in the second intron and the second point at each amplicon, which is offset to the right to facilitate comparison.
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