Figure 1.
N-ChIP-seq localizes ISWI and CHD remodelers throughout the genome.
(A) Size distributions of mapped paired-end 2.5′ and 10′ MNase-digested wild-type Isw1, Isw2, and Chd1 ChIP and input fragments. Slight variations in the amount of supernucleosomal (>251 bp) fragments are attributable to minor variation in the degree of MNase digestion for each sample, as evidenced by slight differences in the nucleosomal maxima for each sample. (B) Binding profiles for 10′ MNase-digested Isw1, Isw2, and Chd1 samples across a representative region of the genome. The number of paired-end reads overlapping each genomic position (counts/bp) is indicated on the Y-axis. See Figure S1 for additional remodeler binding profiles.
Figure 2.
Comparison of Isw2 N-ChIP and X-ChIP data.
(A) Aggregate profiles of log2(Isw2 IP/input) at sites bound by catalytically inactive (K227R) Isw2 in ChIP-chip experiments (2128 sites), sites with altered nucleosome positioning in an isw2Δ strain (1399 sites), sites bound by wild-type Isw2 in ChIP-exo experiments (1251 sites), and around random nucleosomes (1399 sites).Also shown are profiles of remodeler binding at the (B) chrIII recombination enhancer and (C) chrIII centromere (marked by vertical lines).
Figure 3.
Isw2 associates with centromeres.
(A) Aggregate plot of Isw1, Isw2, and Chd1 ChIP/input signal at all 16 yeast centromeres. (B) V-plot of Isw2 ChIP data for all 16 yeast centromeres showing enrichment of Isw2 to the CDEIII side of centromeres. (C) Sequence logo of all 16 yeast centromeres spanning 400 bp centered on the centromere midpoint. A+T are represented as red and G+C are represented as blue, demonstrating the high A+T content of centromeres. The binding of Isw2 to centromeres is thus consistent with its preference for association with regions of high A+T content. The sequence logo was generated with WebLogo (http://weblogo.berkeley.edu).
Figure 4.
ISWI and CHD remodelers associate with TFBSs.
(A) V-plots of wild-type Isw1 ChIP and soluble input chromatin at binding sites for the Abf1 TF after 2.5′ and 10′ MNase digestion. Flanking nucleosomes are visualized as well-defined dot clusters on either side of the TFBS. Example fragments contributing to the generation of various V-plot features are shown schematically below each plot. Cyan arrows point to the position of each fragment midpoint within the V-plot. Similar results were seen for wild-type Isw2 and Chd1 at Abf1 sites and for Isw1, Isw2, and Chd1 at Cbf1, Mbp1, and Reb1 binding sites (Figures S3 and S4). (B) Interpretive schematic of V-plot results. DBPs; DNA-binding proteins.
Figure 5.
ISWI and CHD remodeler association with TFBSs is ATP-independent.
(A) Size distributions of mapped paired-end 2.5′ MNase-digested wild-type and catalytically inactive K227R Isw1 ChIP and input fragments. Similar profiles were seen for Isw2 K215R and Chd1 K407R (Figure S4). (B) Profiles of wild-type and K227R Isw1 binding along a representative segment of the genome. (C) V-plots of wild-type and K227R Isw1 ChIP and soluble input chromatin at binding sites for the Reb1 TF, determined by ChIP-exo, after 2.5′ MNase digestion. The overall fragment size in the Isw1 K227R ChIP and input samples is slightly reduced when compared to wild-type, indicative of technical variation in MNase digestion. Similar results were seen for K227R Isw1 and catalytically inactive Isw2 (K215R) and Chd1 (K407R) at Abf1 and other TFBSs (Figures S4 and S6).
Figure 6.
ISWI and CHD remodelers bind NDRs.
Aggregate plots of log2(IP/input) signal ±1 kb of verified ORF 5′ and 3′ ends for (A) Chd1, (B) Isw1, and (C) Isw2 ranked separated into quintiles by average remodeler signal.
Figure 7.
ISWI and CHD remodeler binding is positively associated with histone turnover and transcription rate.
(A) Heat maps of log2(IP/input) signal for Chd1, Isw1, and Isw2 ±1 kb of verified ORF 5′ ranked descending by gene expression. (B) Heat maps of log2(nucleosome turnover) and log2(IP/input) signal for Chd1, Isw1, and Isw2 ±1 kb of verified ORF 5′ ranked descending by average nucleosome turnover across the entire 2-kb window. (C) Aggregate plots of Chd1, Isw1, and Isw2 log2(IP/input) signal ±1 kb of verified ORF 5′ ends, separated by transcription rate in mRNA/hr [47].