Interaction between GATA and the C/EBP family of transcription factors is critical in GATA-mediated suppression of adipocyte differentiation.

We have previously demonstrated that GATA-2 and GATA-3 are expressed in adipocyte precursors and control the preadipocyte-to-adipocyte transition. Constitutive expression of both GATA-2 and GATA-3 suppressed adipocyte differentiation, partially through direct binding to the peroxisome proliferator-activated receptor (PPAR) promoter and suppression of its basal activity. In the present study, we demonstrate that both GATA-2 and GATA-3 form protein complexes with CCAAT/enhancer binding protein (C/EBP) and C/EBP, members of a family of transcription factors that are integral to adipogenesis. We mapped this interaction to the basic leucine zipper domain of C/EBP and a region adjacent to the carboxyl zinc finger of GATA-2. The interaction between GATA and C/EBP factors is critical for the ability of GATA to suppress adipocyte differentiation. Thus, these results show that in addition to its previously recognized function in suppressing PPAR transcriptional activity, interaction of GATA factors with C/EBP is necessary for their ability to negatively regulate adipogenesis. Adipocyte differentiation is a process controlled by multiple regulators, principally the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and the peroxisome proliferator-activated receptor (PPAR), a nuclear hormone receptor (12, 15). Upon hormonal stimulation, the expression of C/EBP and C/EBP temporally increases (3), followed by expression of PPAR and C/EBP (3, 16). A cooperative interaction between PPAR and C/EBP drives the expression of genes that are necessary for the generation and maintenance of the adipogenic phenotype, such as genes producing morphological changes, lipid accumulation, and insulin sensitivity (22). The C/EBP family of transcription factors contains a highly conserved basic leucine zipper domain (bZip) that mediates homo- or heterodimerization with other isoforms in this protein family. Gain- or loss-of-function studies of preadipocyte cell lines and loss-of-function experiments in vivo all indicate that C/EBP is a key regulator of adipogenesis (6, 20, 23, 25). Furthermore, two other members of this family, C/EBP and C/EBP, are also highly expressed in adipose tissue and have been demonstrated to be vital components of the signaling cascade which initiates adipocyte differentiation (13). Clearly, the coordinated activity of these three members of the C/EBP family makes key contributions to adipogenesis. We have previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed predominantly in white and not brown adipose tissue in vivo and that their expression is restricted to preadipocytes and down-regulated upon adipocyte differentiation. Constitutive expression of both GATA-2 and GATA-3 suppressed adipocyte differentiation and trapped cells at the preadipocyte stage, as found when morphology and gene expression were assessed. This effect was mediated, at least in part, through direct binding of PPAR and inhibition of its basal promoter activity. However, coexpression of PPAR and GATA can only partially rescue the suppression of adipogenesis by GATA (14), which suggests the existence of additional mechanisms by which GATA regulates adipocyte differentiation. In this study, we demonstrated that both GATA-2 and GATA-3 can form protein complexes with C/EBP or C/EBP and that this interaction is critical for the suppression of adipocyte differentiation by GATA. We mapped the interaction domain on GATA-2 and discovered that mutations within this region abolish protein interaction between GATA-2 and C/EBP and subsequently diminish the ability of GATA-2 to suppress adipocyte differentiation.