No abstract

The proximal promoter of the C/EBP␤ gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBP␤ promoter-reporter genes with 5-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBP␤ promoter in mouse embryonic fibroblasts (MEFs) from CREB(؊/؊) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBP␤ gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBP␤ promoter-reporter genes, induced expression of endogenous C/EBP␤, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBP␤, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(؊/؊) MEFs exhibit greatly reduced expression of C/EBP␤ and differentiation. The low level of expression of C/EBP␤ and differentiation in CREB(؊/؊) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.

Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocorticoid injection. Upon activation of the glucocorticoid receptor (GR), proximal regions of activated and repressed genes are remodelled, and these remodelling events correlate with RNA polymerase II occupancy of regulated genes. GR is exclusively associated with accessible chromatin and 62% percent of GR-binding sites are occupied by C/EBPβ. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPβ for GR recruitment. Disruption of C/EBPβ binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin remodelling specifically at sites co-occupied by GR and C/EBPβ. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPβ regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell-specific priming proteins and chromatin remodellers.