Fig. 4: An in vitro system that recapitulates Kipf-independent Rhi binding.
a, A schematic showing the experimental workflow for the CD-binding assay. b, UCSC genome browser tracks of a 72 kb region on chromosome 4 displaying the binding profiles of the indicated histone modifications and CD constructs measured by CUT&RUN (cpm across pooled replicates). The dashed boxes indicate representative areas with either both H3K9me3 and H3K27me3, only H3K27me3 or only H3K9me3, respectively. The mappability of 100 nt reads is indicated below the tracks. c, A Venn diagram showing overlap between CD2xCBX1 binding, H3K9me3, H3K27me3 and CD2xRhi binding across 125,499 genomic 1 kb bins. Signal was considered to be present if the bin overlapped a MACS2 broad peak (q < 0.05, broad cutoff <0.1) present in at least two biological replicates. d, The same as c, but showing CD2xRhi binding, H2AK118ub, H3K27me3 and CD2xPc binding. e, The same as c, but showing CDPc-CBX1 and CD2xRhi binding, H3K27me3 and H3K9me3. f, A heat map and hierarchical clustering (Euclidean distance) of CUT&RUN signal detected for the indicated CD constructs and histone modifications (log10(FPKM)). The signal is shown as the mean across biological replicates. Individual replicates shown in Extended Data Fig. 6d. g, The same as f, but across a subset of the genome classified as heterochromatin (10,180 out of 125,499 1 kb bins). Individual replicates shown in Extended Data Fig. 6e. h, The same as c, but showing H3K9me3, CD2xRhi binding and H3K27me3. ORs for CD2xRhi binding to H3K9me3, H3K27me3, both or neither were calculated using Fisher’s exact test. See Extended Data Fig. 6f for all major overlaps. i, An overview of differentially bound regions (called using DiffBind, two-sided Wald test, Padj <0.05) following kipf or E(z) knockdown. Consensus peaks (n = 11,053) were called using DiffBind with MACS2 broad peaks from the indicated samples. j, Heat maps showing the log2-fold change of E(z) knockdown compared to control knockdown for the indicated constructs and histone modifications across Pc-CBX1 differentially bound regions.
