(DCP001) 14-3-3ZETA REGULATES ADIPOGENESIS BY CONTROLLING CHROMATIN ACCESSIBILITY DURING THE EARLY STAGES OF DIFFERENTIATION

Background: Canonical transcription factors that facilitate adipogenesis, such as C/EBPs and PPARγ, act on specific chromatin regions to drive the adipogenic transcriptional program. However, a complete understanding of the endogenous proteins that governs chromatin accessibility during adipogenesis remain unknown. Here, we hypothesized that the scaffold protein, 14-3-3zeta(ζ), which we previously found to be indispensable for adipocyte differentiation, anchors essential protein complexes that control chromatin accessibility during adipogenesis.

METHODS AND RESULTS:
CRISPR-Cas9 editing was used to generate TAP (FLAG-HA)-tagged 14-3-3ζ-expressing 3T3-L1 pre-adipocytes to determine the nuclear 14-3-3ζ interactome by mass spectrometry during adipogenesis. The enzymatic activities of Histone Deacetylases (HDAC) and DNA methyltransferases (DNMT) were measured from nuclear fractions of control or 3T3-L1 cells transfected with 14-3-3ζ siRNA following 0, 24, and 48h of differentiation. ATAC-Seq was used to assess how 14-3-3ζ deletion influenced chromatin accessibility during 3T3-L1 differentiation. To further examine the specific roles of 14-3-3ζ in adipogenesis, Adipoq-Cre:14-3-3ζKO and Pdgfra-Cre:14-3-3ζKO mice were generated to target either mature adipocytes or adipocyte progenitor cells (APCs), respectively. Male and female mice were fed chow or high-fat (HFD) diet for 16 weeks and assessed for body weight (BW) and adiposity.

Results:
At 24h and 48h post-differentiation, the TAP-14-3-3ζ nuclear interactome was primarily enriched with proteins regulating chromatin accessibility (eg. DNMT1/3a, and HDAC1). ATAC-seq analysis revealed that 14-3-3ζ depletion at both timepoints significantly lowered the accessibility of 1,834 chromatin regions (exons, enhancers and promoters) associated with cell differentiation and lipid anabolism. Further supporting decreased chromatin accessibility, 14-3-3ζ depletion decreased DNMT activity and abrogated HDAC activation. Over-laying our ATAC-seq results with our previous transcriptomic analysis (GSE60745) revealed a positive correlation between reduced accessibility of promoter and enhancer regions with decreased expression of metabolic genes (eg. Fabp4).
With our finding that 14-3-3ζ is necessary for the adipogenic program, we explored the impact of deleting 14-3-3ζ in mature adipocytes or APCs. Regardless of the diet, male and female Adipoq-Cre-14-3-3ζKO mice did not exhibit significant differences in BW, total fat, nor epididymal and subcutaneous fat masses, when compared to WT mice. In male Pdgfra-Cre-14-3-3ζKO mice delayed BW gain and adiposity were observed; in contrast, these were significantly potentiated in females. Pdgfra-Cre-14-3-3ζKO mice of both sexes were protected from HFD-induced BW gain and adiposity.

Conclusion: The effects of 14-3-3ζ in adipogenesis are likely specific to APCs, and not mature adipocytes. This is due to the abilities of 14-3-3ζ to control the activities of key effectors of chromatin accessibility during adipocyte differentiation.