Supplementary Materialssupp_fig1. an MNase-resistant nucleosome at the 3 end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or unfavorable functions via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers take action in an reverse manner. These findings show that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. We applied a genome-wide remodeller-nucleosome conversation assay4 (MNase digestion to define nucleosomes followed by remodeller ChIP-seq) to ES cells, focusing on the 5 ends of genes (Extended Data Fig. 1 and Supplementary Table 1). We first examined remodeller co-enrichment with other factors such as pol II, selected histone marks, and transcription factors, over broad (500-bp) windows centred on DNase-I hypersensitive sites (DHS) (i.e., promoters and enhancers; N = 138,582) (Fig. 1a). High Pearson correlation scores were observed among the remodellers Brg1, Ep400, Chd1, Chd4, Chd6 and Chd8, suggesting that these factors tend to occupy the same genomic regions in ES cells. When we focused on Crizotinib inhibitor active promoter regions within DHSs, most remodellers were correlated with components of the general transcription machinery, including pol II S5ph and TBP (Fig. 1b and Extended Data Fig. 2). Open in a separate window Physique 1 Correlated occupancies Crizotinib inhibitor across remodeller-bound nucleosomal regionsa, Warmth map representing Pearson correlations between remodellers and other factors within 500 bp of 138,582 DHS midpoints. b, Same as (a) but for 16,300 promoter-like, H3K4me3-, TBP- and Pol II S5ph-positive DHSs. c, Distribution of remodeller-nucleosome interactions (MNase ChIP-seq tags for the indicated remodellers in blue) aligned at 14,623 individual RefSeq TSSs (rows), sorted by H3K4me3 levels. Corresponding RNA expression levels (reddish) are shown. We next examined remodeller distribution in more detail by focussing on annotated TSSs (Fig. 1c and Extended Data Fig. 3). Amazingly, some remodellers like Brg1, Chd4, Chd6 bound comparable nucleosome positions at all active genes, regardless of their H3K4me3 enrichment (which is a mark of transcriptional activity), while others, such as Chd1, Chd2, Chd9 and Ep400, were tightly linked to H3K4me3/transcription levels. Chd8 experienced an intermediate pattern. Chd1 and Chd2, which are both related to (yeast) Chd1, showed strikingly different distributions. Whereas Chd1 is present near the 5 ends of genes, the Chd2-nucleosome enrichment pattern encompassed the entire transcription unit and shared high correlation with H3K36me3. (Fig. 1a, c and Col4a5 Extended Data Fig. 2). This is consistent with how yeast Chd1 works5,6, and thus mammalian Chd2 and yeast Chd1 may be functionally comparative. We next investigated more closely the relationship between Crizotinib inhibitor individual remodeller-bound nucleosomes and all nucleosomes defined by MNase-resistant mononucleosome-sized DNA fragments7. Plots of individual genes were aligned by their NFR midpoint and sorted by NFR width into thin and wide groups (Fig. 2a). We validated the experimental approach and its improved resolution by comparison to an existing sonication-based (rather than MNase) ChIP-seq approach8 (Extended Data Fig. 4). Importantly, this sonication-based method, which reports on both nucleosomal and non-nucleosomal interactions, exhibited that Chd4 was not bound within NFRs in a non-nucleosomal manner. Open in a separate windows Physique 2 Patterns of remodeller-nucleosome interactions and chromatin features around promoter NFRsa, Distribution of remodeller-nucleosome interactions, as in Fig. 1c, except aligned by NFR midpoint and sorted by NFR length. Standard MNase-defined nucleosomes (grey) and TSS (green) are shown. Narrow and wide NFRs are delineated by the dashed collection. b, Same as in (a) for other genomic features. c, Averaged distribution of remodeller-nucleosome interactions from (a, b) at thin and wide NFRs, aligned to the dyad of ?1 (left.