Supplementary Figure 7: Analysis and comparison of Prdm15Δ/Δ and Prdm14–/– ESC cistromes.
From: PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK–ERK signaling
(a) Immunofluorescence staining for PRDM15 (red) in wild-type and PRDM15-overexpressing ESCs. (b) Pie chart showing the distribution of PRDM15-specific ChIP–seq peaks across the ESC genome under 2i conditions. (c) Venn diagram depicting the overlap between PRDM15-bound gene promoters in SL versus 2i. (d) Venn diagram showing the overlap between PRDM15 peaks in Prdm15fl/fl and Prdm15Δ/Δ in SL (left and middle) and 2i (right). (e) Venn diagram depicting the overlap between PRDM15 versus PRDM14 peaks (top) and PRDM15 versus MTGR1 peaks (bottom) in SL (left) and 2i (right). (f) PRDM15 binding motif in 2i identified by de novo motif discovery analysis. (g) The in silico prediction tool (http://zf.princeton.edu/) (bottom) is compared to the PRDM15 consensus motif derived from the ChIP–seq analysis (top). (h) Sequence comparison of probe 1 (containing the PRDM15-binding motif) and probe 2 (carrying random mutations of the same motif) used in EMSA. (i) EMSA performed using nuclear lysate from Prdm15fl/fl versus Prdm15Δ/Δ ESCs with probes described in h; an asterisk indicates unknown protein–DNA binding. (j) EMSA using wild-type probe 1 (left) or mutated probe 2 (right) with increasing amounts of nuclear lysate from Prdm15fl/fl ESCs. (k) Competition assay performed with non-labeled (cold) Probe#1 in excess. (l) EMSA performed using nuclear lysates from Prdm15fl/fl vs. Prdm15Δ/Δ ESCs with AP1-binding motif (AP1 was used as a reference/control transcription factor). (m) Supershift assay performed with PRDM15 antibody and matching IgG control. PRDM15–DNA–antibody complex is indicated with an arrow. In i–m, representative images of uncropped gels are shown (n = 3).
