Relationship between between REST binding affinities, RE1 motifs, and clearance of REST from binding sites. (A) REST protein levels during siRNA knockdown. (Top panel) Nuclear protein extracts were prepared from K562 cells and subject to Western blotting using an anti-REST antibody, to confirm REST protein knockdown at 48, 72, and 96 h post-transfection with REST and luciferase (Lucif) siRNAs. (Lower panel) Membrane was reprobed using an anti-CSTF64 antibody as a loading control. (B) Treeview diagram of hierarchical clustering of REST-enriched genomic tiles that were identified by HMM analysis in REST and luciferase siRNA knockdowns in the K562 cell line. (Left) Tree diagram. Each tile is represented by a horizontal bar and is shaded in red scale or green scale to denote the level of enrichment or depletion, respectively. Knockdown experimental states are labeled at the top of each column of tiles. (Right) Recurring RE1 sequence motifs found in tiles of the three principle clusters of the tree (groups 1, 2, and 3). The overall discovered motif shown in Fig. 3 is also shown. (Asterisks) Nucleotides that are highly conserved in the discovered motif, or that were found to be invariant in previous studies. (C) Mean REST ChIP-chip enrichments (y-axis) for the three groups of REST binding sites (groups 1, 2, and 3 as defined in Results) in both luciferase and REST siRNA knockdown conditions (x-axis) at 72 and 96 h post-transfection. The mean fold reductions in enrichment levels at both time points (based on luciferase/REST ChIP-chip enrichments) are also shown at the right of the panel. (D) Induction of gene expression of REST target genes during siRNA knockdown. Total RNA was extracted from cells at 48, 72, and 96 h time points, from either REST or luciferase (luc) siRNA transfected K562 cells. Quantitative RT-PCR was performed using oligonucleotide primers specific to 11 known (*) or putative REST target gene transcripts and six control genes not associated with REST binding sites. Fold changes in mRNA levels (REST siRNA/luciferase siRNA) are shown on the y-axis; genes are shown along the x-axis.
