Comparative analysis of metazoan chromatin organization

Abstract

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Extended Data Fig. 1. Chromatin features at TSSs and gene bodies and co-occurrence of histone modifications

a, Comparative analysis of promoter architecture at Transcription Start Sites (TSSs). From the top, H3K4me3 (human GM12878, fly L3, and worm L3), DNase I hypersensitivity sites (DHS), GC content, and nascent transcript (GRO-seq in human IMR90 and fly S2 cells). Human promoters, and to a lesser extent worm promoters (as defined using recently published capRNA-seq data), exhibit a bimodal enrichment for H3K4me3 and other active marks around TSSs. In contrast, fly promoters clearly exhibit a unimodal distribution of active marks, downstream of TSSs. Since genes that have a neighboring gene within 1 kb of a TSS or TES (Transcription End Site) were removed from this analysis, any bimodal histone modification pattern cannot be attributed to nearby genes. This difference is also not explained by chromatin accessibility determined by DNase I hypersensitivity (DHS), or by fluctuations in GC content around the TSSs, although the GC profiles are highly variable across species. b, Average gene body profiles of histone modifications on protein-coding genes in human GM12878, fly L3, and worm L3. c, Genome-wide correlations between histone modifications show intra- and inter-species similarities and differences. Upper left half: pairwise correlations between marks in each genome, averaged across all three species. Lower right half: pairwise correlations, averaged over cell types and developmental stages, within each species (pie chart), with inter-species variance (grey-scale background) and intra-species variance (grey-scale small rectangles) of correlation coefficients for human (h), fly (f), and worm (w). Modifications enriched within or near actively transcribed genes are consistently correlated with each other in all three organisms. In contrast, we found a major difference in the co-occurrence pattern of two key repressive chromatin marks (black cell in bottom left): H3K27me3 (related to Polycomb (Pc)-mediated silencing) and H3K9me3 (related to heterochromatin). These two marks are strongly correlated at both developmental stages analyzed in worm, whereas their correlation is low in human (r = −0.24 ~ −0.06) and fly (r = −0.03 ~ −0.1).

Extended Data Fig. 2. Histone modifications in heterochromatin

a, Genomic coverage of H3K9me3 in multiple cell types and developmental stages. Embryonic cell lines/stages are marked with an asterisk and a black bar. b, Evidence that overlapping H3K9me3 and H3K27me3 ChIP signals in worm are not due to antibody cross-reactivity. ChIP-chip experiments were performed from early embryo (EE) extracts with three different H3K9me3 antibodies (from Abcam, Upstate, and H. Kimura) and three different H3K27me3 antibodies (from Active Motif, Upstate, and H. Kimura). The H3K9me3 antibodies show similar enrichment profiles (upper panel) and high genome-wide correlation coefficients (lower left). The same is true for H3K27me3 antibodies. There is significant overlap between the H3K9me3 and H3K27me3 ChIP signal, especially on chromosome arms, resulting in relatively high genome-wide correlation coefficients (Extended Data Fig. 1c). The Abcam and Upstate H3K9me3 antibodies showed low level cross-reactivity with H3K27me3 on dot blots, and the Abcam H3K9me3 ChIP signal overlapped with H3K27me3 on chromosome centers. The Kimura monoclonal antibodies against H3K9me3 and H3K27me3 showed the least overlap and smallest genome-wide correlation. In ELISA assays using histone H3 peptides containing different modifications, each Kimura H3K9me3 or H3K27me3 antibody recognized the modified tail against which it was raised and did not cross-react with the other modified tail^,, providing support for their specificity. Specificity of the Kimura antibodies was further analyzed by immunostaining germlines from wild type, met-2 set-25 mutants (which lack H3K9 HMT activity), and mes-2 mutants (which lack H3K27 HMT activity) in the lower right panel. Staining with anti-HK9me3 was robust in wild type and in mes-2, but undetectable in met-2 set-25. Staining with anti-HK27me3 was robust in wild type and in met-2 set-25, but undetectable in mes-2. Finally, we note that the laboratories that analyzed H3K9me3 and H3K27me3 in other systems used Abcam H3K9me3 (for human and fly) and Upstate H3K27me3 (for human), and in these cases observed non-overlapping distributions. Chandra et al. also reported non-overlapping distributions of H3K9me3 and H3K27me3 in human fibroblast cells using the Kimura antibodies. The overlapping distributions that we observe in worms using any of those antibodies suggest that H3K9me3 and H3K27me3 occupy overlapping regions in worms. Those overlapping regions may exist in individual cells or in different cell sub-populations in embryo and L3 preparations. c, Average gene body profiles of H3K9me3 and H3K27me3 on expressed and silent genes in euchromatin and heterochromatin in human K562 cells, fly L3, and worm L3.

Extended Data Fig. 3. Organization of silent domains

a, The correlation of H3K27me3 and H3K9me3 enrichment for human K562 (left most), fly L3 (second left), and worm EE chromosome arms (second right) and centers (right most) with a 10 kb bin (upper) and a 1 kb bin (lower). The density was calculated as a frequency of bins that fall in the area in the scatter plot (darker grey at a higher frequency). r indicates Pearson correlation coefficients between binned H3K27me3 fold enrichment (log₂) and H3K9me3 fold enrichment (log₂). Worm chromosome arms have a distinctly high correlation between H3K27me3 and H3K9me3. The lower correlation in worm chromosome centers is due to the overall absence of H3K9me3 in these regions. b, Schematic diagrams of the distributions of silent domains along the chromosomes in human (H1-hESC), fly (S2), and worm (EE). In human and fly, the majority of the H3K9me3-enriched domains are located in the pericentric regions (as well as telomeres), while the H3K27me3-enriched domains are distributed along the chromosome arms. H3K27me3-enriched domains are negatively correlated with H3K36me3-enriched domains, although in human, there is some overlap of H3K27me3 and H3K36me3 in bivalent domains. CENP-A resides at the centromere. In contrast, in worm the majority of H3K9me3-enriched domains are located in the arms, while H3K27me3-enriched domains are distributed throughout the arms and centers of the chromosomes and are anti-correlated with H3K36me3-enriched domains. In arms and centers, domains that are permissive for CENP-A incorporation generally reside within H3K27me3-enriched domains.

Fig. 1. Dataset overview

a, Histone modifications, chromosomal proteins, and other profiles mapped in at least two species (see Supplementary Fig. 1 for full dataset and Supplementary Table 1 for detailed descriptions). Different protein names for orthologs are separated by slash. (see Supplementary Table 2). b, Number of all datasets generated by this and previous consortia publications^– (new: 815; old: 638). Each dataset corresponds to a replicate-merged normalized profile of a histone, histone variant, histone modification, non-histone chromosomal protein, nucleosome, or salt-fractionated nucleosome. c, Number of unique histone marks or non-histone chromosomal proteins profiled.

Fig. 2. Shared and organism-specific chromatin states

16 chromatin states derived by joint segmentation using hiHMM (hierarchical HMM; see Supplementary Methods) based on enrichment patterns of 8 histone marks. The genomic coverage of each state in each cell type or developmental stage is also shown (see Supplementary Figs. 26–32 for detailed analysis of the states). States are named for putative functional characteristics.

Fig. 3. Genome-wide organization of heterochromatin

a, Enrichment profiles of H3K9me1/me2/me3 and H3K27me3 and identification of heterochromatin domains based on H3K9me3 (illustrated for human H1-hESC, fly L3, and worm L3). For fly chr2, 2L, 2LHet, 2RHet and 2R are concatenated (dashed lines); C: centromere, Het: heterochromatin. b, Genome-wide correlation among H3K9me1/me2/me3, H3K27me3, and H3K36me3 in human K562 cells, fly L3, and worm L3; no H3K9me2 profile is available for human. c, Comparison of Hi-C-based and chromatin-based topological domains in fly LE. Heatmaps of similarity matrices for histone modification and Hi-C interaction frequencies are juxtaposed (see Supplementary Fig. 40).