PPARγ is involved in adipocyte differentiation, insulin sensitivity and diabetes, atherosclerosis, and the control of cell proliferation in some cancer cells (reviewed in references 5
). Consequently, its function has been the subject of intense investigation. Relatively little, however, is known about the mechanisms controlling its expression. Here we utilized two different cellular models for adipocyte differentiation to temporally describe the molecular interactions that occur at the promoter of the inducible PPARγ2 gene during adipocyte differentiation, with particular emphasis on the requirement for SWI/SNF chromatin-remodeling enzymes. Through use of a differentiation system driven by introduction of the adipogenic regulator, C/EBPα, we demonstrate a requirement for SWI/SNF enzymes in the activation of the PPARγ regulator as well as in the activation of adipogenic marker genes expressed later during differentiation. Moreover, these experiments revealed that this requirement for SWI/SNF enzymes was relatively late in the cascade of events leading to PPARγ2 activation. Activator binding, Pol II and associated GTF interactions at the promoter, and histone H3 and H4 acetylation occurred prior to and independently of SWI/SNF function. Instead, the data revealed a role for SWI/SNF enzymes in the function of the PIC components at the promoter at the time of transcriptional activation.
Because of the inherent differences between forcing differentiation of fibroblasts into the adipocyte lineage and genuine preadipocyte differentiation, we analyzed differentiation of 3T3-L1 preadipocytes and confirmed both the general order of events that occur during PPARγ2 activation and a role for SWI/SNF enzymes in facilitating PIC function. We expect that the differences exhibited by the two systems reflect the ability of the C/EBPα activator to recruit GTFs and SWI/SNF enzymes (32
) prematurely at the initiation of the forced differentiation program. Despite the differences, it is important to note that the data from both systems are consistent with a need for SWI/SNF enzymes to promote the function of the PIC.
The order of events occurring in differentiating 3T3-L1 cells is diagrammed schematically in Fig. . Acetylation of H4 occurs before the onset of differentiation, followed by concurrent changes in promoter accessibility, binding of the C/EBPβ and -δ activators, and assembly of Pol II and most of the GTFs on day 1 of differentiation. Subsequently, on day 2, SWI/SNF enzymes and TFIIH associate with the promoter, indicating that the SWI/SNF enzymes likely facilitate completion of the preinitiation complex, thereby permitting PPARγ2 transcription to commence. On day 3, there is both an increase in the levels of H3 acetylation and a transition from binding of C/EBPβ and -δ to binding of C/EBPα. Which event, if either, is causal remains to be determined. Following day 4, the SWI/SNF enzymes disappear from the promoter and the rate of PPARγ transcription drops, indicating that the presence of SWI/SNF is required for continued transcription.
Schematic model of the temporal changes in factor interactions at the PPARγ2 promoter during 3T3-L1 preadipocyte differentiation. Nucleosome positions are presented for illustrative purposes only.
The data from differentiating 3T3-L1 preadipocytes indicate that both BRG1 and BRM are present on the PPARγ2 promoter, suggesting that both complexes are contributing to function. Alternatively, the two complexes could be redundant in function, and the presence of either SWI/SNF enzyme might be sufficient. Our studies using cell lines that inducibly express dominant-negative BRG1 or BRM also suggest that both ATPases are required for PPARγ2 activation and adipocyte differentiation. However, because the BRG1 and BRM SWI/SNF complexes share multiple subunits, it is possible that expression of one mutant ATPase deleteriously affects both complexes by sequestering subunits from the other, endogenous ATPase. Thus, we cannot rigorously state at present whether BRG1, BRM, or both are required for PPARγ2 activation and adipocyte differentiation.
One of the interesting results from our studies is the demonstration that the C/EBP binding sites undergo a transition during the time course of differentiation from binding C/EBPβ and -δ to binding C/EBPα. Although the kinetics of expression for these factors has long supported this idea, this is the first documentation that such a transition occurs at a promoter expressed during adipogenesis. These results differ from previously published work that showed that C/EBPα and -δ, but not C/EBPβ, could bind to the C/EBP sites in the PPARγ2 promoter in a gel shift study and could activate a transiently transfected PPARγ2 reporter plasmid (16
). The differences between the studies may be attributed to the likelihood that the chromatin structure at the genomic locus differs from that on a transfected template and undergoes changes during the differentiation process that affect factor interactions.
Previous reports documenting the potential of C/EBPα and -β to physically interact with BRM in cells transfected with both the C/EBP isoform and BRM suggested that these factors may recruit SWI/SNF enzymes (19
). Our temporal analysis of factor interactions on the PPARγ2 promoter during C/EBPα-driven differentiation strongly suggests that targeting of SWI/SNF by C/EBPα can occur. However, C/EBPα is prematurely present on the promoter in this differentiation system; the temporal differences in the appearance of the different C/EBP factors on the PPARγ2 promoter in the differentiating 3T3-L1 adipocytes suggest that in a more natural differentiation context, C/EBPβ and -δ may target SWI/SNF enzymes, which then later recruit C/EBPα.
Changes in nuclease accessibility and the binding of C/EBP factors, Pol II, and many of the GTFs on day 1 prior to the appearance of SWI/SNF enzymes on the PPARγ2 promoter indicate that other factors must control the initial accessibility of these promoter sequences. Changes in H4 acetylation did not correlate with initial factor binding to the promoter. Changes in other histone modifications at the PPARγ2 promoter have not been tested but potentially could mediate factor accessibility. Alternatively, a different ATP-dependent remodeling enzyme(s) could alter chromatin structure and promote activator binding prior to SWI/SNF function. This hypothesis is supported by in vitro studies showing that ISWI containing chromatin-remodeling enzymes facilitated stable interaction of RARα:RXR on chromatin templates prior to SWI/SNF enzyme-mediated stimulation of transcription (15
). Finally, transcriptional regulators present on the promoter prior to differentiation, such as GATA-2 and -3 (43
) and KLF2 (4
), might influence chromatin structure in a manner that promotes the transition to an actively transcribing gene.
Our analysis of GTF interactions on the PPARγ2 promoter also revealed that serine 5-phosphorylated Pol II is present at the promoter before TFIIH. TFIIH contains a kinase activity that is capable of phosphorylating the Pol II CTD; however, the temporal order of factor appearance at the PPARγ2 promoter suggests that either a different kinase is responsible for the CTD phosphorylation or TFIIH mediates CTD phosphorylation independently of promoter binding. Additionally, the presence of Pol II phosphorylated at serine 5 of the CTD raises the possibility that the polymerase may be transcriptionally engaged at day 1 and that the inclusion of SWI/SNF enzymes and TFIIH on day 2 promotes release of the polymerase and/or elongation. Multiple other genes are regulated at the level of transcriptional elongation, including hsp70
, where elongation can be stimulated by SWI/SNF enzymes in vitro and in vivo (6
The concurrent entry of SWI/SNF enzymes and TFIIH onto the PPARγ2 promoter in differentiating 3T3-L1 cells suggests that SWI/SNF facilitates the interaction of TFIIH with the rest of the preinitiation complex. Such a role for SWI/SNF enzymes has not previously been documented. However, the data presented here agree with and extend findings from temporal analyses of other mammalian promoters that show the following: (i) some form of histone hyperacetylation precedes association of SWI/SNF with the promoter, and (ii) SWI/SNF enzymes work late in the activation of many genes, typically after some, if not most, of the components driving transcription have associated with the promoter (1
). Thus, the data we present on PPARγ2 activation during adipocyte differentiation support a general model where SWI/SNF enzymes function subsequent to activator binding by completing or stabilizing preinitiation complex formation and/or by promoting promoter clearance and elongation.