Pivotal contribution of super-enhancer-driven KLF6 expression to the adipogenesis of human adipose-derived stem cells.

Abstract

Background: Super-enhancers (SEs), characterized by dense clusters of enhancer elements enriched with transcriptional activator binding sites, are involved in cell differentiation. However, little is known about SE-mediated regulation of adipogenic genes. The aim of this study was to elucidate the functional role of the KLF6-proximal SE during the adipogenesis of human adipose-derived stem cells (hADSCs).

Methods: Adipogenic induction medium (AIM) was used for differentiation of hADSCs into adipocytes, which were evaluated for adipogenic gene expression and adipogenesis using quantitative PCR and Oil Red O (ORO) staining, respectively. The effects of SE inhibitors, locked nucleic acid-mediated enhancer RNA (eRNA) knockdown, and small interfering RNA-mediated knockdown of KLF6 on adipogenesis and adipogenic gene levels were evaluated. Chromatin immunoprecipitation assays were performed to identify transcriptional regulators binding to the promoter regions of KLF6 and the adipogenesis-inhibitory Delta-like non-canonical Notch ligand 1 (DLK1) gene during adipogenesis.

Results: In silico screening identified KLF6 as an obesity-susceptibility gene associated with single-nucleotide polymorphisms and located within the domain of SE_00159, which was activated in adipocytes. AIM-cultured hADSCs exhibited time-dependent increases in KLF6 mRNA and protein expression. During adipogenesis, the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) bound to the KLF6 promoter. Treatment with the SE inhibitor JQ1 resulted in a dose-dependent decrease in KLF6 mRNA expression and reduced ORO staining. Knockdown of eRNA expressed from the SE_00159 domain decreased KLF6 levels during adipogenesis. Consistently, KLF6 knockdown during hADSC adipogenesis downregulated the adipogenic genes PPARG and CEBPA, while upregulating DLK1. Additionally, KLF6 together with histone deacetylase (HDAC)3 bound to the DLK1 promoter and concomitantly caused dissociation of histone acetyltransferase p300 during adipogenesis.

Conclusions: SE activation upregulates KLF6 through PPARγ/p300- and eRNA-mediated transcriptional induction during hADSC adipogenesis. KLF6, in turn, represses DLK1 expression through recruitment of HDAC3 to the promoter and p300 dissociation, thereby facilitating adipocyte differentiation. These findings support a working model in which the epigenetic regulation of KLF6 and DLK1 as a potential therapeutic axis in human obesity.