Figure 1. PRC2 establishes H3K27me3 in ESCs independent of REST repression.
(A) A limited number of REST-occupied sites are associated with domains of H3K27me3 enrichment in ESCs, even if defined more broadly (+/− 1 kb). (B) H3K27me3 levels are stable in Rest−/− ESCs in the majority of regions targeted by PRC2. The scatter-plot shows the relative enrichment of H3K27me3 ChIP-Seq signal in wild type (WT, x-axis) and Rest−/− ESCs (y-axis) at regions targeted by PRC2 in WT ESCs. (C) As in (B), but at identified REST-binding sites. (D) Chromatin immunoprecipitation analysis showing H3K27me3-enrichment changes at RE1 sites near PRC2-targeted regions in WT and Rest−/− ESCs (* indicates p < 0.05), normalized for H3 density.
Figure 1—figure Supplement 1. REST is required for recruitment of co-factors to RE1 sites in ESCs.
ChIP assays were performed in WT and
Rest−/− ESCs using
antibody against Sin3a, Cdyl, CoREST1, CoREST2, Hdac2, G9a, and Kdm1a to
compare recruitment of endogenous co-factors at the RE1 sites near
Igsf21, Kcnk9,
Erich1, Npas4, and
Rad51. ChIP assays done in parallel with normal
rabbit IgG are included as a negative control.
Figure 1—figure Supplement 2. Detection of REST binding to PRC2 members is biochemically possible, but a true interaction is unlikely.
(A) Immunoprecipitation was performed with streptavidin
beads from biotin-tagged REST ESCs or BirA control ESCs and the
demarcated proteins were assayed by Western blot. (B)
Previously published ChIP-seq data sets were interrogated over H3K27me3
peaks and REST peaks normalized to the signal found at the H3K27me3
peaks.
Figure 1—figure Supplement 3. Characteristics of REST-bound loci.
(A) Regions with multiple RE1 motifs show a strong
association with REST. REST ChIP-Seq data displayed as sequence tags
aligned to the mouse genome assembly (mm9) viewed in the UCSC genome
browser. Green and red hash marks represent sequences matching the
forward and reverse strands, respectively. Consensus RE1 sequence motifs
are indicated by black hash marks. All views are shown at an equal size
relative to the scale bar. (B) FIMO definition of the RE1
motif was derived from ChIP-seq peaks. (C) Relative
enrichment of REST measured by ChIP-Seq was confirmed by ChIP-qPCR. ChIP
assays were performed using antibody raised against a peptide
corresponding to a fragment of mouse REST in WT (red bars) and
Rest−/− (blue bars) in
ESCs at regions associated with RE1 motifs and regions lacking an RE1
site (Oct4 and MageA8).
(D) REST targets associated with trimethylation of histone
H3K4 or H3K27 enrichment are preferentially localized near gene
promoters. Graph shows the percentage of REST-bound regions that overlap
with domains of the bivalent histone modification pattern consisting of
H3K4me3 and H3K27me3 enrichment (red bars), H3K4me3 enrichment alone
(green bars), or without overlap to either modification (tan bars).
Figure 1—figure Supplement 4. H3K27me3 levels from WT and Rest−/− ESCs are as similar as H3K27me3 levels from different published reports.
(A) Normalized enrichment values for H3K27me3 at defined
H3K27me3 peaks were derived from several previously published data sets
and plotted against one another. Numbers indicate Pearson
coefficient.
Figure 1—figure Supplement 5. Ezh2-enrichment at REST-bound loci.
(A) Ezh2 occupancy was increased at RE1 sites within
H3K27me3 domains that gained H3K27me3 in the absence of REST. ChIP assays
were performed with anti-Ezh2 to compare enrichment at RE1 sites between
WT (light orange) and
Rest−/− (dark orange)
ESCs.