(A) CRED9 knockdown in Hep3B cells with 200 nM gapmer 4 reveals a loss of H3K27ac at the +9 kb CEBPA enhancer as measured by ChIP-seq RT-qPCR. Control H3K27ac-enriched regions within the ACTB and GAPDH promoters do not change upon CRED9 knockdown. Minimal amplification of a “gene desert” product within Chr: 12, devoid of H3K27ac, confirmed low nonspecific pull-down with the H3K27ac-specific antibody. (B) ChIP-seq binding profiles of liver-enriched transcription factors HNF4A, FOXA1, FOXA2, CEBPA, and CEBPB are depicted around the TSS of CRED9. The binding profiles clearly demonstrate that liver-enriched transcription factors bind the +9 kb CEBPA enhancer in liver and a hepatocellular carcinoma cell line. (CAGE-seq data [Fantom5 project, RIKEN]), ChIP-seq data: Degree of shading is indicative of the degree of transcription factor binding from lower (lighter) to higher (darker). Blue and red bars indicate transcription factor consensus sequence (ENCODE). Smaller rectangles represent binding sites of the indicated transcription factors from a separate data source (Unibind). (n = 2 biological replicates; error bars: std; P-values from unpaired t-tests where significance “*” cut-off is [**] P ≤ 0.01, [ns] not significant).
