bPrevious studies have shown that CCAAT/enhancer-binding protein ␣ (C/EBP␣) plays a very important role during adipocyte terminal differentiation and that AP-2␣ (activator protein 2␣) acts as a repressor to delay the expression of C/EBP␣. However, the mechanisms by which AP-2␣ prevents the expression of C/EBP␣ are not fully understood. Here, we present evidence that Suv39h1, a histone H3 lysine 9 (H3K9)-specific trimethyltransferase, and G9a, a euchromatic methyltransferase, both interact with AP-2␣ and enhance AP-2␣-mediated transcriptional repression of C/EBP␣. Interestingly, we discovered that G9a mediates dimethylation of H3K9, thus providing the substrate, which is methylated by Suv39h1, to H3K9me3 on the C/EBP␣ promoter. The expression level of AP-2␣ was consistent with enrichment of H3K9me2 and H3K9me3 on the C/EBP␣ promoter in 3T3-L1 preadipocytes. Knockdown of Suv39h markedly increased C/EBP␣ expression and promoted adipogenesis. Conversely, ectopic expression of Suv39h1 delayed C/EBP␣ expression and impaired the accumulation of triglyceride, while simultaneous knockdown of AP-2␣ or G9a partially rescued this process. These findings indicate that Suv39h1 enhances AP-2␣-mediated transcriptional repression of C/EBP␣ in an epigenetic manner and further inhibits adipocyte differentiation.
bAdipogenesis is a multistep process by which 3T3-L1 preadipocytes differentiate into mature adipocytes through mitotic clonal expansion (MCE) and terminal differentiation. The CCAAT/enhancer-binding protein  (C/EBP) is an important transcription factor that takes part in both of these processes. C/EBP not only transactivates C/EBP␣ and the peroxisome proliferatoractivated receptor ␥ (PPAR␥), which cause 3T3-L1 preadipocytes to enter terminal adipocyte differentiation, but also is required to activate cell cycle genes necessary for MCE. The identification of potential cofactors of C/EBP will help to explain how C/EBP undertakes these specialized roles during the different stages of adipogenesis. In this study, we found that activating transcription factor 5 (ATF5) can bind to the promoter of C/EBP␣ via its direct interaction with C/EBP (which is mediated via the p300-dependent acetylation of ATF5), leading to enhanced C/EBP transactivation of C/EBP␣. We also show that p300 is important for the interaction of ATF5 with C/EBP as well as for the binding activity of this complex on the C/EBP␣ promoter. Consistent with these findings, overexpression of ATF5 and an acetylated ATF5 mimic both promoted 3T3-L1 adipocyte differentiation, whereas short interfering RNA-mediated ATF5 downregulation inhibited this process. Furthermore, we show that the elevated expression of ATF5 is correlated with an obese phenotype in both mice and humans. In summary, we have identified ATF5 as a new cofactor of C/EBP and examined how C/EBP and ATF5 (acetylated by a p300-dependent mechanism) regulate the transcription of C/EBP␣.
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