Perturbation of BRD4 and p300 activity suppresses super enhancer‑driven KLF6 expression in renal carcinoma

Super enhancers (SEs) are involved in regulating cell identity and lineage‑specific gene expression, and drive cancer‑associated gene expression. The transcription factor Kruppel‑like factor 6 (KLF6) promotes the growth and progression of clear cell renal cell carcinoma (ccRCC), with its high expression driven by one of the strongest SEs in ccRCC. However, the mechanisms that establish and maintain KLF6 SE activity, particularly the roles of epigenetic regulators bromodomain-containing 4 (BRD4) and p300, remain poorly understood. This study investigated the roles of BRD4 and p300 in modulating KLF6 SE activity. The effects of JQ1‑mediated BRD4 and A‑485‑mediated p300 inhibition were assessed using cell viability and colony formation assays. Reverse transcription‑quantitative (q)PCR and ChIP‑qPCR were employed to evaluate the impact of BRD4 and p300 inhibition, as well as CRISPR‑mediated deacetylation of individual constituent enhancers, on KLF6 expression and SE activity. Chemical inhibition of BRD4 and p300 significantly reduced ccRCC cell viability and colony formation, and decreased KLF6 expression and levels of acetylation at lysine 27 of histone H3 (H3K27ac) at KLF6 enhancer regions SE_1, SE_2 and SE_3, suggesting decreased chromatin accessibility. On the other hand, deacetylation of SE_1 using dead Cas9 fused to histone deacetylase 3, led to KLF6 downregulation, which was associated with decreased H3K27ac signals at this region. The present results demonstrated that BRD4 and p300 are key for maintaining KLF6 SE activity and driving high KLF6 expression in ccRCC. However, deacetylation of  individual enhancer regions using CRISPR was insufficient to fully suppress KLF6 transcription, emphasizing the robustness of the KLF6 SE and its modular role in sustaining high KLF6 expression. Overall, the present study deepens the understanding of growth‑promoting KLF6 transcriptional networks in ccRCC and offers insights to support the development of diagnostic or therapeutic strategies.