Altogether, potent BCL6 repression of promoters in B cells is linked to ternary BCL6-SMRT-BCOR corepressor complex formation within a specific chromatin context featuring loss of activating and gain of repressive marks and suppression of RNA Pol II elongation but not Pol II recruitment (Figure?S7D). Most <a href="https://en.wikipedia.org/wiki/Ceftiofur
">Ceftiofur</a> BCL6-SMRT binding (85%) occurred outside of promoters, suggesting that the BCL6 mechanism may differ at these sites and is perhaps linked to enhancer regions (Figure?4A). Enhancers are characterized by the presence of H3K4me1 and absence of H3K4me3 (Heintzman et?al., 2007?and?Heintzman et?al., 2009). We therefore performed H3K4me1 ChIP-seq to map enhancer regions in DLBCL cells. The vast majority of BCL6-SMRT distal/intronic peaks were H3K4me3NEG/H3K4me1POS (n?= 2,162), suggesting that these complexes are within transcriptional enhancers (Figure?4A). We first focused on distal BCL6-SMRT enhancer binding sites (n?= 818, >5 kb away from TSSs). BCL6 and SMRT peak summits were precisely colocalized at enhancers and generally restricted to a narrow region of less than 400?bp framed by two adjacent nucleosomes as indicated by H3K4me1 read density (Figure?4B). These BCL6-SMRT enhancers were significantly conserved as compared to adjacent control regions, which is suggestive of their functional relevance (Figure?S8A). We next examined whether BCL6-SMRT binding to enhancers?has a cis-regulatory function. Since most BCL6-SMRT enhancers were located within 80 kb from the nearest transcript ( Figure?S8B), <a href="http://www.selleckchem.com/products/3-methyladenine.html
</a> we identified the most proximal gene for every BCL6-SMRT distal enhancer (n?= 553). <a href="http://www.selleckchem.com/products/Sunitinib-Malate-
(Sutent).html">Sunitinib purchase</a> Using GSEA, we found that the group of genes with BCL6-SMRT-bound enhancers were significantly enriched in genes derepressed after BCL6 knockdown (FDR?= 0.005 at 24?hr and FDR?= 0.03 at 48?hr; Figures 4C and S8C). In contrast, genes associated with distal enhancers bound by BCL6 without SMRT (n?= 654) were not enriched among BCL6 siRNA-derepressed genes (FDR?= 0.38 at 24?hr and FDR?= 0.68 at 48?hr; Figures 4C and S8C). Similarly, BCL6-SMRT enhancer linked genes (but not BCL6 only) were significantly upregulated after BCL6 knockdown (BCL6-SMRT: p?< 0.0001 at 24?hr and p?= 0.032 at 48?hr; BCL6 only: p?= 0.07 at 24?hr and p?= 0.49 at 48?hr; Mann-Whitney U) compared to control genes ( Figures 4D and S8D). To further investigate whether BCL6 can repress through enhancer binding we performed reporter assays using constructs containing a BCL6-SMRT enhancer identified by our ChIP-seq, located 13 kb upstream of the CDKN1A promoter and containing a BCL6 consensus binding motif ( Figures 4E and S8E). Addition of CDKN1A distal enhancer induced 3-fold repression of CDKN1A promoter when transfected in DLBCL cells, and this repressor activity was markedly attenuated by BCL6 knockdown (p?< 0.0001, Mann-Whitney U; Figure?4F).