Regulation Of C/EBPs On Fad24During The Early Phase Of Adipogenesis

Fad24(factor for adipocyte differentiation24) is a novel gene transiently expressed during the early stage of adipocyte differentiation. Previous studies demonstrated that knockdown of fad24in3T3-L1cells impairs mitotic clonal expansion (MCE) during the initiation of adipogenesis, and blocks the further terminal differentiation. On the other hand, overexpression of fad24in non-adipogenic NIH/3T3fibroblasts promotes their ability to undergo adipogenesis. Although fad24has been established as a positive regulator of adipocyte differentiation, the underlying mechnisms remain unclear.In the current study, using a combination of methods including cell transfection, dual-luciferase reporter assay, point mutation, chromatin immunoprecipitation (ChIP), real-time PCR and RNA interference, we analyzed the transcriptional regulation of fad24and explored its functionality in the context of transcription cascade controlling adipogenesis. We found that CCAAT enhancer binding proteins (C/EBPP and C/EBPδ) can directly binds to the cis-elements within fad24promoter and regulates its transcriptional activity, which in turn affects MCE in adipogenesis. The major results are as follows:1. A1279bp (-1128/+152) fragment witin the5’upstream region of mouse fad.24was obtained and subjected to bioinformatic analysis for potential C/EBP binding sites. Based on that, a series of deletions (-896/+151,-450/+151,-164/+151) were generated and subcloned into pGL3-basic plasmids. The promoter activity of these fragments was tested in NIH/3T3cells using dual-luciferase reporter assay.2. Co-transfection experiments demonstrated that C/EBP8markedly enhances the promoter activity of fad24, whereas C/EBPβ significantly reduces it.3. Consistent with the results of promoter activity assay, overexpression of C/EBP6in3T3-L1cells remarkably up-regulated the transcription of endogenous fad24, wheras C/EBPβ overexpression had the opposite effect.4. Point mutations in the potential C/EBP binding sites (-605/-591and-354/-340) either significantly compromised or abolished the regulation of C/EBPβ and C/EBP8on fad24promoter.5. ChIP assay confirmed that both C/EBPβ and C/EBPδ can directly bind to fad24promoter at-354/-340. 6. Knockdown of C/EBP8in3T3-L1cells markedly reduced the mRNA level of fad24and cyclinDl, and induced the transcription of p21, which in turn impairs MCE phase in early adipogenesis. Overexpression of fad24in C/EBPδ-knockdown3T3-L1cells could promote proliferation and partially rescue MCE process.