Genome-wide profiles of hZSCAN4 binding sites. (A) Specificity of mouse anti-human ZSCAN4 antibodies. Whole-cell lysates of hES cells transfected with (+) or without (−) synthetic RNA encoding hZSCAN4-3xFLAG-HA were probed with anti-hZSCAN4 antibodies raised in mice. Expression of ZSCAN4-3xFLAG-HA was probed by an anti-HA antibody as a positive control. (B) Rabbit anti-hZSCAN4 antibodies were examined, as in A. (C) hES cells were transfected with (+) or without (−) synthetic mRNA encoding for DUX4. Immunostaining analysis was performed using hZSCAN4 antibodies at 20 h post-transfection. DNA was counterstained with DAPI. Bar, 20 µm. (D) Experimental scheme for hZSCAN4 ChIP-seq. ChIP-seq experiments were conducted using mouse antibody (mAb) and rabbit antibody (rAb) against hZSCAN4 in hES cells overexpressed with DUX4 (hES + DUX) and non-treated hES cells. (E) The number of hZSCAN4 ChIP peaks in hES and hES +DUX identified by mAb and rAb ChIP-seq. (F) Heatmaps showing hZSCAN4 ChIP-seq signals at the hZSCAN4 binding sites identified by mAb and rAb in hES and hES+DUX cells. (G) Sequence motifs enriched in hZSCAN4 binding regions identified by mAb and rAb ChIP-seq. (H) Genomic distribution of hZSCAN4 binding sites (N = 18,770).