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Background <p>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).</p> Methods <p>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 <i>KLF6</i> on adipogenesis and adipogenic gene levels were evaluated. Chromatin immunoprecipitation assays were performed to identify transcriptional regulators binding to the promoter regions of <i>KLF6</i> and the adipogenesis-inhibitory Delta-like non-canonical Notch ligand 1 (<i>DLK1</i>) gene during adipogenesis.</p> Results <p>In silico screening identified <i>KLF6</i> 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 <i>KLF6</i> promoter. Treatment with the SE inhibitor JQ1 resulted in a dose-dependent decrease in <i>KLF6</i> mRNA expression and reduced ORO staining. Knockdown of eRNA expressed from the SE_00159 domain decreased <i>KLF6</i> levels during adipogenesis. Consistently, <i>KLF6</i> knockdown during hADSC adipogenesis downregulated the adipogenic genes <i>PPARG</i> and <i>CEBPA</i>, while upregulating <i>DLK1</i>. Additionally, KLF6 together with histone deacetylase (HDAC)3 bound to the <i>DLK1</i> promoter and concomitantly caused dissociation of histone acetyltransferase p300 during adipogenesis.</p> Conclusions <p>SE activation upregulates <i>KLF6</i> through PPARγ/p300- and eRNA-mediated transcriptional induction during hADSC adipogenesis. KLF6, in turn, represses <i>DLK1</i> 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 <i>KLF6</i> and <i>DLK1</i> as a potential therapeutic axis in human obesity.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月19日

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  • Mai-Phuong Nguyen,
  • Kaoru Yamagata,
  • Anh Phuong Nguyen,
  • Tong Zhang,
  • Hidenori Sakai,
  • Uyen Thi Ngo,
  • Yu Shan,
  • Takashi Otsuka,
  • Ngo Thi Dieu Huong,
  • Takafumi Aritomi,
  • Meng Yuan,
  • Shigeaki Kato,
  • Yoshiya Tanaka,
  • Shingo Nakayamada

摘要

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.