Figure 3.

The BEND3-BD4 binding site explains direct binding at rDNA and CALR. (A) We subjected the BEND3-BD1 and BEND3-BD4 domains to PBM assays, with only BD4 enriching for a candidate binding site that was consistent between both array designs. We also subjected BEND3-BD4 to HT-SELEX assays, which retrieved a similar apparently monomeric site, as well as a longer consensus that includes a palindrome. (B) Gel shift assay using recombinant GST fusion protein confirmed that BEND3-BD4 binds directly and specifically to a probe bearing a CCCACGC core. (C) UCSC genome browser view at a representative human rDNA repeat. Total RNA-seq highlights rRNA transcripts, and sequence matches to the core BEND3-BD4 motif are marked. rDNA regions previously assayed for BEND3 association are labeled, of which the promoter-proximal H41.9 region was bound most strongly by BEND3 (Khan et al. 2015). (D) Alignment of BD4 sequence matches in BEND3 target regions in rRNA. Note that H41.9 contains two adjacent BD4 sites. (E) H41.9 wild-type and mutant transcriptional reporters. (F) eYFP-BEND3 represses H41.9-wt but not H41.9-mut reporters. Data are mean ± SD; n = 3 experiments; two-tailed t-test applied. (G) The conserved basal promoter of the CALR gene contains a human-specific nucleotide divergence, which coincides with a SNP in multiple schizophrenia and bipolar cases (Aghajanirefah et al. 2016). (H) The human-specific CALR promoter sequence that is affected in psychiatric disease resides within a strong BEND3 ChIP-seq peak.