Modulation of Ezh2 expression, and other H3K27 modifiers, by NF-kB transcription factors in adipose cells

Abstract

The adipose tissue (TA) shows high plasticity when answering different individual metabolic demands. The major cell responsible for these answers is the mature adipocyte, but mesenchymal stem cells (AdSCs) residing in the adipose tissue stromal vascular fraction provide a source of differentiation for new adipocytes. During adipogenesis, epigenetic changes on histone 3 lysine 27 (H3K27) are critical for silencing genes responsible for AdSC commitment with chondrogenesis and osteogenesis, and for activation of adipogenesis key transcription factors, such as Pparg. Ezh2 is responsible for histone 3 lysine 27 trimethylation (H3K27me3) which leads to gene silencing, while Crebbp and Ep300 (Cbp/p300) are responsible for histone 3 lysine 27 acetylation (H3K27ac) which leads to transcription activation. The action of H3K27 demethylases Kdm6a and Kdm6b are necessary between gene silencing and activation. The expression of Ezh2 is regulated by NF-kB transcription factors in cancer, but little is known about Ezh2 transcriptional regulation in TA cells. NF-kB binding can be found in Crebbp, Ep300, Kdm6a, and Kdmb6b regulatory regions. In obese individuals, there is an increase in serum TNF-a and endotoxins that can lead to NF-kB activation in TA. We hypothesize that the expression of Ezh2, Kdm6a, Kdmb6b, Crebbp, and Ep300 in adipocytes and AdSCs are modulated by NF-kB, which shows different levels of activation in obese and non-obese individuals. The alterations in the expression of these histone modifiers can modify the transcription of transcription factors that are key for adipocyte metabolism and differentiation, such as Klf15. Therefore, the goals of this project are: 1) to investigate if the expression of Ezh2, Kdm6a, Kdmb6b, Crebbp, and Ep300 is modulated by NF-kB in murine (3T3-L1) and human (hWA A41) adipocyte and pre-adipocyte with TNF-a, LPS and macrophage conditioned medium, using chromatin immunoprecipitation and siRNA; 2) ) to investigate if the alterations in histone modifiers expression affect the expression of transcription factors such as Klf15; 3) to investigate if the findings in cell lines replicate in obese patients' adipose cells. This study will bring important contributions to the understanding of the effects of inflammation on the expression of histone modifiers and on the epigenetic signature of transcription factors that act during adipogenesis and adipocyte metabolism. (AU)