BAZ2A safeguards genome architecture of ground-state pluripotent stem cel (2020)
Figure 7

Figure 7

BAZ2A regulates H3K27me3 occupancy in ground-state ESCs

A. Western blot showing total levels of H3K27me3 in ESC+2i transfected with siRNA-control and siRNA-Baz2a. Histone H3 is shown as a protein loading control.

B. Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC+2i and ESC+serum. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Mean of normalized H3K27me3 read counts in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a over all TSSs (+/- 1kb) were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted relative to the mean of normalized H3K27me3 occupancies. The red line represents the mean of H3K27me3 fold-changes.

C. Representative images showing the H3K27me3 occupancy.

D. Average density plot of ChIPseq read counts of H3K27me3 at -/+ 5 kb from the TSS of refseq genes in ESCs treated with siRNA-control or siRNA-Baz2a Q1: first quartile corresponding to sequences with high H3K27me3 content. Q2-4: quartiles 2-4 corresponding to sequences with low H3K27me3 content.

E. ChIP-qPCR of H3K27me3 in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a. Data were measured by qPCR and normalized to input and to Tshz1 value in control cells. Control represents an intergenic sequence that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).

F. Boxplot showing changes in H3K27me3 in ESC+2i upon BAZ2A at BAZ2A-regulated and random genes. Mean of normalized H3K27me3 read counts in ESC+2i treated with siRNA-control or siRNA-Baz2a over TSSs (+/- 1kb) of upregulated, downregulated and random genes were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01 and *** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.

G. Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC+2i or ESC+serum transfected with siRNA-control or siRNA-Baz2a. Data were ranked by H3K27me3 content in the corresponding siRNA-control ESCs. Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels.

H. H3K27me3 ChIP-qPCR in ESC+2i (top panel) or ESC+serum (bottom panel) transfected with siRNA-control or siRNA-Baz2a. Values show H3K27me3 occupancy at four sequences (#1-#4) with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic sequence that does not contain H3K27me3. Data were measured by qPCR and normalized to input and to sequence #1 value in control cells. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01).

I. Hi-C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster (10-kb resolution in upper panels, 5 kb resolution in lower panels). Blue arrows (A, B, and C) indicate gain or increase of contacts in ESC+siRNA-Baz2a compared to control cells (siRNA-control). The orange arrow (D) indicates loss or decrease in contacts. Quantifications are shown in Table EV5.