Regulation of CEBPbeta transcription and preadipocyte differentiation by histone deacetylase 1 and GCN5

Abstract

Previous studies in this laboratory demonstrated that the glucocorticoid receptor (GR) ligand binding domain (LBD) can enhance transcription mediated by the CCAAT/enhaner binding protein C/EBPbeta. This result suggests that the receptor can act in a non-classical manner to positively affect transcription without directly binding DNA. To study the physiological impact of the potentiation of C/EBPbeta activity by the GR LBD, we focused on preadipocyte differentiation as both GR and C/EBPbeta influence early events. Using retroviral expression of the GR LBD in 3T3 L1 cells, a 3-fold increase in preadipocyte differentiation was observed when cultures were induced to differentiate in a cocktail containing dexamethasone. The GR LBD was also able to enhance transcription mediated by DNA-bound C/EBPbeta from the C/EBPalpha promoter and its expression increased C/EBPbeta protein levels during preadipocyte differentiation. The effect of the GR LBD was accomplished through the targeted degradation of a sub-population of the transcriptional repressor histone deacetylase 1 (HDAC1), which associates with C/EBPbeta via corepressor protein mSin3A. In the absence of glucocorticoids, HDAC1 maintains the C/EBPalpha promoter in a deacetylated state. The addition of steroid to the differentiation cocktail causes the degradation of the HDAC1 found in the CIEBPbeta-associated complex, a release of mSin3A, increased acetylation of the promoter histone H4 and recruitment of RNA polymerise II leading to maximal transcription from the C/EBPalpha promoter. C/EBPbeta itself is acetylated by the histone acetyltransferases GCN5 and PCAF within lysines 98, 101 and 102. In 3T3 L1 preadipocytes where only GCNS is expressed, GCNS coprecipitates with C/EBPbeta and can be found at the C/EBPalpha promoter by chromatin immunoprecipitation. The acetylation of C/EBPbeta is essential for development of full transcriptional activation potential and for differentiation of preadipocytes. Mutation of these residues leads to loss of transcriptional potentiation by GR and a striking decrease in the number of differentiated cells in both NIH 3T3 and 3T3 L1 cultures. Following glucocorticoid treatment, the mutant C/EBPbeta remains associated with HDAC1 and is inefficient in driving C/EBPalpha transcription. Acetylation may be a required modification to prevent C/EBPbeta from repression by HDAC1.