(a) MudPIT mass spectrometry analysis of proteins co-immunoprecipitated with H3K36me3-containing mononucleosomes from wildtype yeast. IgG was used as a negative control. ^a Spectral count, total spectra matching peptides detected by tandem mass spectrometry for the indicated protein. ^b Sequence coverage, percentage of protein sequence represented in peptides identified by tandem mass spectrometry. (b) Subunit composition of Isw1 and Chd1 remodelers. Isw1 is the catalytic subunit of two chromatin-remodeling complexes: It associates with Ioc3 to form Isw1a, while Ioc2 and Ioc4 are part of the Isw1b complex. Chd1 is thought to exist largely as a monomer. (c) Schematic representations of the domain organizations for the Isw1 and Chd1 chromatin remodelers.

(a,b) Recombinant, reconstituted H3K36 MLA nucleosomes (5 fmol) were incubated with increasing concentrations (0, 3, 7, 10, 13, 17, 20 and 27 nM) of Ioc4 (a) or Ioc4 with its N-terminal PWWP domain deleted (Ioc4_ΔPWWP) (b) and analyzed using EMSAs. Mononucleosome bands are indicated. (c) Quantitation of Ioc4 and Ioc4_ΔPWWP binding to tri- and unmethylated H3K36 MLA mononucleosomes. Mononucleosome bands were quantitated for each lane. Lanes 2–8 were normalized against input lane 1, while lanes 10–16 were normalized to input lane 9. The percentage of nucleosomes bound by Ioc4 or Ioc4_ΔPWWP was expressed as 100 – % mononucleosomes for each lane. Mean values ± SEM were plotted for at least three independent experiments. P-values were calculated using Student's t-test.

(a,b) ChIP-chip experiments were performed using yeast genomic tiling arrays. The log₂ ratios of IP over input were subjected to average gene analysis (Supplementary Fig. 2a). Whole-genome average data were calculated and plotted as mean ± SEM. SE of mean are shown in grey and represent three independent experiments. The Transcription Start Site (TSS) and TErmination Site (TES) are indicated. Flag-tagged Ioc4 (a) and Ioc3 (b) were immunoprecipitated from wildtype and set2Δ yeast strains.

(a,c,e,g) Total RNA for each strain was isolated and used for Northern blot analysis. The probes used were directed against the 3′ ends of the STE11 and PCA1 genes. ACT1 and rRNA were used as loading controls. The full-length (←) and cryptic transcripts (*) are indicated. (b,d,f,h) For each lane all cryptic transcript bands were quantitated and normalized against the ACT1 loading control. Total levels of cryptic transcription were expressed relative to set2Δ. Data were plotted as mean ± SEM for three independent experiments.

(a) Heatmap of cryptic transcript genes in an isw1Δ chd1Δ mutant. Probe intensities for the 5′ and 3′ ends of all genes were determined. Resulting 3′/5′ ratios with a cutoff value of log₂ > 0.5 were used to define sense cryptic transcripts (n=646). K-means clustering of gene expression profiles was used to identify antisense cryptic transcripts (n=962). Only cryptic transcript genes are shown. (b) Venn diagram showing the overlaps between genes displaying increased histone exchange over ORFs in set2Δ (mean log₂ > 0; n=3,728) and genes with cryptic transcription in set2Δ (n=865) and/or isw1Δ chd1Δ (n=1,437). Cryptic transcript sets contain genes with sense and/or antisense cryptic transcripts, whereby 68 and 171 genes show intragenic initiation in both the sense and antisense direction for set2Δ and isw1Δ chd1Δ, respectively. For the purpose of direct comparison we selected cryptic transcript genes for set2Δ according to the same criteria applied to isw1Δ chd1Δ (a), rather than the previously published data⁸.

(a–b,e–f) ChIP-chip experiments were performed using yeast genomic tiling arrays. Only genes known to display increased exchange over ORFs in a set2Δ mutant (n=3,728) (Supplementary Fig. 4a) were used for average gene analysis and plotted as mean ± SEM (grey) for three independent experiments. The Transcription Start Site (TSS) and TErmination Site (TES) are indicated. (a–b) Histone exchange (Flag-H3/Myc-H3) was determined for wildtype, isw1Δ, chd1Δ, isw1Δ chd1Δ (a) and ioc4Δ (b) strains and presented as difference profiles for mutant (mut) over wildtype. (c–d) Genes were clustered into two groups based on their average histone exchange signals for mutant relative to wildtype profiles over ORFs (mean log₂ < 0, mean log₂ > 0). Venn diagrams show overlaps between genes displaying increased histone exchange over ORFs (mean log₂ > 0) for the mutants indicated. (e–f) Average gene analysis for acetylated K56 (H3K56ac) immunoprecipitated from wildtype, isw1Δ, chd1Δ, isw1Δ chd1Δ (e) and the ISW1^K227R catalytic mutant (f) strains. H3K56ac occupancy was normalized to histone H3 levels. Difference profiles are shown for mutant over wildtype. (g) Venn diagram showing the overlap between genes that exhibit cryptic transcription in an isw1Δ chd1Δ mutant (Fig. 5a,b) and genes that exhibit increased histone exchange over ORFs in an isw1Δ chd1Δ and set2Δ background. (h) Pearson correlation coefficients and P-values were calculated for histone exchange (a) and H3K56ac/H3 (e) profiles for each mutant background using R⁴⁵. (i–l) For whole-genome analysis genes (n=4,894) were clustered into two groups (< 10 mRNA per hour (4,250 genes), ≥ 10 mRNA per hour (644 genes)) based on transcription rates published by Holstege et al.⁴⁶. Average gene analysis for histone exchange (i–j) or H3K56ac/H3 (k–l) difference profiles clustered according to transcription rates are shown. Data were plotted for isw1Δ (i,k) or chd1Δ (j,l) relative to the wildtype.

(a–d) ChIP-chip experiments were performed as described in Fig. 6. Only genes known to display increased exchange over ORFs in a set2Δ mutant (n=3,728) (Supplementary Fig. 4a) were used for average gene analysis and plotted as mean ± SEM (grey) for three independent experiments. The Transcription Start Site (TSS) and TErmination Site (TES) are indicated. (a) Average gene analysis for H3K36me3 immunoprecipitated from wildtype, isw1Δ and chd1Δ yeast strains. (b) Average gene analysis for histone H3 immunoprecipitated from wildtype, isw1Δ and chd1Δ yeast strains. (c–d) Average gene analysis for acetylated H4 (AcH4) immunoprecipitated from wildtype, isw1Δ, chd1Δ, isw1Δ chd1Δ (c) as well as set2Δ, isw1Δ set2Δ and chd1Δ set2Δ (d) yeast strains. AcH4 occupancy was normalized to histone H3 levels. Difference profiles are shown for mutant over wildtype.

Isw1b is recruited to ORFs by interaction of its Ioc4 subunit with H3K36me3, while Chd1 is thought to co-localize with RNAPII due to its interaction with known transcription elongation factors (grey). In the wildtype both remodelers cooperate to preserve chromatin structure by preventing histone exchange over ORFs. Deletion of ISW1, IOC4 or CHD1, or abrogation of Isw1 catalytic activity allows for trans-histone exchange to occur over coding sequences and results in increased levels of histone acetylation over ORFs. This perturbation of ORF chromatin structure exposes normally hidden cryptic promoters and results in the production of internal, cryptic transcripts.