Fig. 4

Analysis of ZFP57 binding to the LCb478 sequence in EMSA. A Distribution of ZFP57 (above the line) and ZFP57-like (L; below the line) motifs in the H19 ICR’ (top), LCb (middle) and LCb478 (bottom) sequences. CTCF-binding sites and b sequence are indicated by black circles and a gray rectangle, respectively. Position of probes and competitors used for EMSA in panels C–E is shown as thick horizontal lines. B (top) Two out of six nucleotides in the consensus ZFP57 binding site-like sequences (shaded) in the ZFP57-L-1/2 probe were mutated to generate canonical ZFP57 binding sites in the ZFP57-1/2 probe. (bottom) Sequence of competitor oligos each carrying CTCF-binding sites (c1-c4 or c4d [downstream]) within the H19 ICR (yet outside of the 478 sequence). Consensus CTCF and putative ZFP57 binding sites are underlined or shaded, respectively. C GST or GST-ZFP57 (a.a. 137–195) fusion proteins were expressed in and recovered from E. coli. The GST-ZFP57 protein bound robustly to the methylated ZFP57-1/2 probe (lane 6), but not to its unmethylated counterpart (lane 4) or to the ZFP57-L-1/2 probe (lane 2). D Nuclear extracts of HEK293T cells transfected with (+) or without (−) a HA-ZFP57 expression vector were analyzed by ZFP57-L-1/2 or ZFP57-1/2 probes. Combination of cell extracts with forcibly expressed HA-ZFP57 and methylated ZFP57-1/2 probe generated shifted bands (lane 14). The antibody/HA-ZFP57 super-shifted bands are denoted by the open arrowheads (lanes 15 and 16). antibodies a: α HA; b: α ZFP57. E Competitor oligos (50-fold molar excess) were included in the EMSA with HEK293T/HA-ZFP57 nuclear extracts and methylated ZFP57-1/2 probe, to test ZFP57 binding to the LCb/LCb478 sequences