Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

The bromodomain and extra-terminal domain (BET) family consists of acetyllysine-binding proteins involved in many cancers, including glioblastoma multiforme. While the BET family activates transcription through binding to hyperacetylated histone H4 with simultaneous acetylation of K5 and K8 (H4K5acK8ac), so far epigenomic analysis of the BET family has mainly focused on H3K27ac, which is not their binding target. Here, we have examined the epigenomic profiles, including H4K5acK8ac and a BET family protein, BRD4, along with transcriptomics using a BET inhibitor, JQ1, in three human glial cell lines. We found that the expression of genes in which H4K5acK8ac is preferred over H3K27ac at promoters was mostly downregulated upon JQ1 in a glioblastoma stem cell (GSC) line. Knockdown of genes with the H4K5acK8ac-preferred promoters diminished the stemness of GSC. By defining super-enhancers (SEs) ranked by H4K5acK8ac, we found that 43% of the H4K5acK8ac-ranked SE were different from H3K27ac-ranked SE in GSC. CRISPR-Cas9-mediated deletion of the H4K5acK8ac-preferred SEs reduced the expression of associated genes including MYCN and NFIC in GSC and diminished its stemness. These results validate a novel strategy to identify genes involved in the regulation of glioblastoma stemness, through histone H4 hyperacetylation. Examination of histone modifications (H4K5acK8ac, H3K27ac, H3K4me3, & H3K4me1) and BRD4 by ChIP-seq in the JQ-treated, DMSO-treated, and untreated human glial cell lines (Glioblastoma stem cell line-GSC316, Glioblastoma cell line-U87, and Microglia cell line-C13NJ). Transcriptomic profiling through CAGE-seq of JQ-treated, DMSO-treated, and untreated of Glioblastoma stem cell line-GSC316, Glioblastoma cell line-U87, and Microglia cell line-C13NJ.