Previously, we demonstrated that GATA transcription factors suppress adipocyte differentiation, partially through inhibition of PPARγ expression by direct suppression of the basal PPARγ2 promoter activity. In the present study, we examined additional pathways that GATA utilizes to suppress adipogenesis and demonstrated that both GATA-2 and GATA-3 are capable of forming protein complexes with either C/EBPα or C/EBPβ. Through deletion analysis, we mapped the interaction domain to the bZip region of C/EBPα and to aa residues 381 to 385, which follow the carboxyl zinc finger of GATA-2. This domain is critical for C/EBP interaction but not for nuclear localization or DNA binding. Finally, we demonstrated that the interaction between GATA and C/EBP is critical for GATA suppression of adipocyte differentiation, since isolated disruption of the C/EBP-GATA interaction impairs the ability of GATA to suppress adipocyte differentiation.
Both GATA-2 and GATA-3 can form protein complexes with either C/EBPα or C/EBPβ. Our previous data indicated that the expression of both GATA-2 and GATA-3 are significantly down-regulated at the onset of adipogenesis, while expression of C/EBPα increases during differentiation. However, expression of C/EBPβ and C/EBPδ remains relatively low in preadipocytes and surges during the early stages of differentiation upon hormonal stimulation (3
). The temporal coexpression of GATA with these members of the C/EBP protein family in adipocytes therefore suggests that suppression of the C/EBPβ or C/EBPδ expression function may also be the target of the antagonism between GATA and the C/EBP family of transcription factors. The ability of GATA to interact with C/EBPα may result from interactions at homologous regions of the C/EBP family or indicate that constitutive expression of GATA during adipogenesis may impair differentiation by inappropriately interacting with C/EBPα. Since GATA down-regulation partially overlaps with C/EBPα induction, it remains possible that under normal conditions GATA factors can interact with C/EBPα, perhaps in order to sequester its ability to activate the adipogenic program until derepression is appropriate. It is also possible that interaction of GATA with C/EBPα could be triggered at other times upon exposure to specific antiadipogenic stimuli. The ability of GATA to interact with multiple C/EBP isoforms with various temporal expression patterns suggests that their proadipogenic contribution may be counterregulated by GATA in a stage-specific fashion.
Our work collectively demonstrates that GATA suppresses adipocyte differentiation through at least two pathways: inhibition of PPARγ expression by binding to its promoter, and interference with C/EBP function through protein-protein interaction. When various deletion mutants were investigated for their ability to suppress the PPARγ gene promoter, we found that loss of DNA binding (in mutant A) rendered GATA incapable of suppressing PPARγ promoter activation, whereas a functionally intact mutant (mutant B) exerted the same effect as wild-type GATA-2. More detailed analysis indicated that a GATA protein which retained its ability to bind DNA but could not interact with C/EBP (mutant C) likewise failed to suppress adipocyte differentiation as well as C/EBPα-activated PPARγ promoter activity, indicating that protein interaction with C/EBP was independent of DNA binding and critical for GATA suppression of differentiation and PPARγ gene transcription in the presence of C/EBP, which can both interact with C/EBP and bind its cognate DNA sequence, has little or no effect on PPARγ promoter activity, although when constitutively expressed in NIH 3T3 cells, it was able to hamper lipid accumulation and adipogenic gene expression (data not shown). Taken together, these findings highlight the significance of GATA interaction with C/EBP, which allows GATA to exert its full effect on adipogenesis. The dual mechanism by which GATA acts underscores the necessity for tight control of the initiation of adipogenesis in preadipocytes, so that perturbation of either pathway may result in only partial loss of GATA suppression of adipocyte differentiation. It is not yet known whether temporal release of these suppressive effects is critical for proper adipogenesis to proceed.
GATA transcription factors interact with many proteins, such as FOG (18
), PU.1 (11
), Rb (21
), Trap220 (5
), and CBP (1
). Interestingly, many of these GATA-interacting factors have been implicated in the process of adipogenesis. For example, Rb and Trap220 were shown to be necessary for adipocyte differentiation (4
), and CBP heterozygous mice are lipodystrophic (24
). These proteins may serve as integral partners for the action of GATA during adipogenesis to impart the specificity of its action in various cellular, temporal, or spatial contexts. The involvement of other cofactors interacting with GATA in adipogenesis remains to be investigated and may elucidate additional pathways by which GATA factors regulate adipogenesis. This study introduces a new mechanism by which GATA interferes with adipocyte differentiation. In addition to its classical role as a DNA-binding transcription factor, we have shown here that GATA interaction with C/EBP proteins is also a critical component of this regulation.