β-actin contributes to open chromatin for activation of the adipogenic pioneer factor CEBPA during transcriptional reprograming

Abstract

Adipogenesis is regulated by a cascade of signals that drive transcriptional reprogramming in adipocytes. Here, we report that nuclear actin regulates the chromatin states that establish tissue specific expression during adipogenesis. To study the role of β-actin in adipocyte differentiation we conducted RNA-Sequencing on wild-type (WT) and β-actin knockout (KO) mouse embryonic fibroblasts (MEFs) after reprograming to adipocytes. We found β-actin depletion affects induction of several adipogenic genes during transcriptional reprograming. This impaired regulation of adipogenic genes is linked to reduced expression of the pioneer factor Cebpa and is rescued by reintroducing NLS-tagged β-actin. ATAC-Seq in KO MEFs revealed that actin-dependent reduction in Cebpa expression correlates with decreased chromatin accessibility and loss of chromatin association of the ATPase Brg1. This, in turn, impairs CEBPB's association with its Cebpa promoter-proximal binding site during adipogenesis. We propose a role for the nuclear β-actin pool in maintaining open chromatin for transcriptional reprogramming during adipogenic differentiation.

Mouse Embryonic Fibroblast Adipogenic Potential: A Comprehensive Transcriptome Analysis

“…Therefore, the study of adipogenesis in vitro at molecular and mechanistic levels is essential for development of better therapeutic targets. Many studies that elucidate the processes of adipogenesis have used different cell lines, such as 3T3-L1, 3T3-F442A, MSCs, murine and human adipose stem cells (ASCs), and MEFs [16,[28][29][30][31][32][33][34]. Some of these same models have also been used to explore transcriptomic aspects of adipogenesis [16,19,20,[50][51][52].…”

“…Temporal lipid accumulation was assessed at 0, 3, and 5 days in cells in non-treated, basal medium, and differentiation medium groups. These time points were chosen due to sufficiency of lipid formation in MEFs when exposed to differentiation factors, as observed previously [16]. Where continues incubation of MEFs with differentiation factors can lead continuous cell death [16], which will have biases towards exploring RNA-Seq data especially when targeting DEGs related to adipocyte or fat formation.…”

“…Mechanistically, induction of CEBPB, at an early phase of adipogenesis, promotes induction of PPARG and CEBPA by binding to their proximal promoter regions. Therefore, this results in activating several pro-adipogenic genes [12][13][14][15][16] that support the progression of preadipocytes cells into mature adipocytes [11,17].…”

“…Most knowledge of AT is based on in vitro studies of committed murine cell lines, notably 3T3-L1 and 3T3-F442A [28,29], and human cell lines, particularly mesenchymal stem cells (MSCs) [30,31]. However, some studies have demonstrated the adipogenic potential of mouse embryonic fibroblast (MEF) [16,[32][33][34]. MEFs, isolated from 14-day-old mouse embryos (C57/BL6 52 strain), are classically used as 'feeder layers' for human and mouse embryonic stem cells, for DNA transfection assays, recombinant protein expression, and utilization for epigenome and transcriptome analysis (i.e.…”

“…MEFs, isolated from 14-day-old mouse embryos (C57/BL6 52 strain), are classically used as 'feeder layers' for human and mouse embryonic stem cells, for DNA transfection assays, recombinant protein expression, and utilization for epigenome and transcriptome analysis (i.e. chromatin remodelling) [16,32]. Although MEFs can differentiate into multiple cell types, yet little is known in regard to specific mechanisms underlying their adipogenic potential.…”