Aim: We previously discovered that 14-3-3-zeta (14-3-3ζ) was essential for adipocyte differentiation. Although 14-3-3ζ scaffold is not a transcription factor (TF) , it undergoes nuclear translocation during adipogenesis. Considering its diverse interactome, we hypothesized that 14-3-3ζ anchors, nucleates and regulates essential transcriptional complexes required for adipogenesis.
Methods: 3T3-L1 pre-adipocytes were modified by CRISPR-Cas9 to express a TAP (Flag-HA) -tagged 14-3-3ζ protein. At 48 first hours of adipogenesis, nuclear TAP-14-3-3-ζ complexes were purified to identify and annotate the 14-3-3ζ interactome by MS and Gene Ontology. Highly enriched proteins functions were assessed for their roles in adipogenesis using pharmacological inhibitors or siRNA. In parallel, chromatin accessibility was assessed by ATAC-Seq from control or 14-3-3ζ-depleted 3T3-L1 cells during early adipogenesis.
Results: We found that the 14-3-3ζ nuclear interactome during the early stages of adipogenesis was significantly enriched with 133 proteins, including CEBP-β, an early adipogenic TF. According to GO analysis, proteins within the 14-3-3ζ interactome were primarily associated (FDR ≤ 0.01) with histone H2B ubiquitination, DNA unwinding for replication, DNA hypermethylation and ribosome assembly. Pharmacological inhibition or depletion by siRNA of these proteins significantly dampened adipocyte differentiation. Principal component analysis of ATAC-seq results revealed that samples from 14-3-3ζ-depleted cells differed from controls in term of whole chromatin accessibility, and DESeq2 identified nearly 3400 (p<0.05) differentially accessible regions of open chromatin following 14-3-3ζ depletion.
Conclusion: These results reveal that via critical protein interactions, 14-3-3ζ has essential roles in chromatin accessibility, DNA replication, and mRNA translation, which are crucial events for the early adipogenesis.
S.A.Rial: None. A.Alkhoury: None. G.Lavoie: None. P.Roux: None. T.M.Durcan: None. A.Martinez-sanchez: None. G.E.Lim: None.
Natural Sciences and Engineering Research Council of Canada (NSERC) ; Canada Research Chairs (CRC) ; Fonds de recherche du Québec-Nature and Technologies (FRQNT)