Detailed investigations of the molecular events involved in activation of endogenous chromatin-embedded target genes by nuclear receptors have so far been limited to steroid receptors, such as the progesterone, glucocorticoid, estrogen, and androgen receptors (28
), and a few RXR-dependent nuclear receptors such as the thyroid hormone and vitamin D3
). In the absence of agonists these receptors are either transcriptionally inactive due to interaction with heat shock proteins or nuclear exclusion or they are kept in a repressive state due to interaction with transcriptional corepressors. Addition of agonists promotes release from heat shock protein complexes, import to the nucleus, and/or exchange of transcriptional corepressors with coactivators (43
). Thus, exogenous agonists can be used to set clean on/off states of these receptors, and the sequence of events of transcription factor recruitment to target promoters can be closely followed using ChIP. However, since many of the nuclear receptors, including the PPARs, are considered to be metabolic sensors activated by lipid metabolites produced within the cells (14
), the agonist concentration is difficult to control. Furthermore, transcriptional activity may not be strictly agonist dependent. These built-in difficulties have hampered the investigations of the molecular events involved in the activation of genomic target genes by this large group of nuclear receptors.
Here, we demonstrate that the combination of adenoviral delivery and ChIP is ideal for studying the acute molecular effects of PPAR activation on PPAR target sites and promoters. We show that adenoviral transduction of the HA-PPARs in NIH-CAR fibroblasts results in increased PPAR mRNA levels after 2 h and potent PPAR protein expression within 4 h posttransduction. The HA-PPAR proteins reach maximal levels, similar to that of PPARγ protein in 3T3-L1 adipocytes, 4 to 8 h after transduction (Fig. ). Binding of PPARγ, RXR, cofactors, and basal transcription factors to the enhancer of the well-established PPARγ target gene, A-FABP, is maximal at 5 h. At this point, the expression from the A-FABP gene is significantly activated. Recruitment of PPARγ to the locus also leads to a significant increase in histone H3 and H4 acetylation at the promoter as well as at the enhancer (Fig. ). Thus, the adenoviral transgene delivery provides a unique system to tightly control the expression of PPARs and to study the acute and direct effects of PPAR binding to target sites of endogenous genes.
As PPARs are activated by fatty acids and derivatives thereof, the “basal” transcriptional activity (i.e., activity in the absence of exogenous agonists) of PPARs is often ascribed to endogenous ligands or ligands present in the serum. In addition, the ligand-independent activation function of the receptors may contribute to this activity; however, the relative contribution of endogenous ligands and ligand-independent function to “basal” transcriptional activity is unknown. Using the adenoviral delivery system, we show here that PPARγ2 displays significant ligand-dependent as well as -independent transcriptional activity. PPARγ2 leads to profound activation of the A-FABP gene and other target genes like LPL and perilipin in the absence of exogenous ligands. This activation is only slightly decreased by the addition of a specific PPARγ antagonist, GW9662 (Fig. and results not shown). Thus, the considerable transcriptional potential of PPARγ2 in NIH-CAR fibroblasts in the absence of exogenous agonists is predominantly a consequence of the high ligand-independent activity of PPARγ2.
The ligand-independent transcriptional activity of PPARγ is likely to be mediated at least in part by ligand-independent activation function 1 (AF1) in the N-terminal part of the receptor. However, the AF2 of the ligand binding domain (LBD) may also contribute to the ligand-independent transactivation. Helix 12 has been reported to be in an “agonist position” in crystal structures of apo-PPARγLBD (46
), and recent investigations indicated that this is due to the stabilization of helix 12 in the agonist position by specific charged amino acids in the LBD (42
). In keeping with this, PPARγLBD has recently been shown to interact with CBP/p300 (33
) as well as p160 CoA (42
) in the absence of ligands. Presence of a synthetic ligand further induces stabilization of helix 12, thereby leading to an increased interaction with coactivators (42
). Future investigations in our adenoviral system with truncated and mutated PPARs will help clarify whether the ligand-independent transactivation of endogenous genes is due primarily to the AF1 or the AF2 function and whether these effects are promoter specific. Recent data from the Lazar laboratory indicate that there is indeed promoter specificity in the dependency of ligand for cofactor recruitment (21
Intriguingly, our data show that PPARγ occupancy of the A-FABP PPRE is considerably increased in the presence of the thiazolidinedione-type agonist, rosiglitazone. The increased occupancy is paralleled by increased recruitment of basal transcription factors and of coactivators such as CBP. This ligand-dependent increase in occupancy may in part be explained by a stabilization of the PPAR/RXR/DNA interaction, as suggested by electrophoretic mobility shift assays of PPARα/RXR (15
). In addition, ligand-induced stabilization of receptor-cofactor interactions may lead to a longer residence time of the receptor at the locus. Interestingly, however, the relative importance of agonist for PPARγ2/RXR binding to target sites is highly dependent on the target gene, and for some genes like ADRP, agonists did not appear to affect PPARγ2/RXR binding although it potentiated expression of the gene (Fig. ).
In general, the effect of the agonist Wy14.643 on PPARα binding to target sites and activation of target genes is minor (Fig. ). However, the lack of efficient synthetic PPARα antagonists for cell culture experiments precludes investigations similar to those shown in Fig. for PPARγ to determine whether the activity in the absence of exogenous agonist is due to agonist-independent transactivation or to the presence of endogenous agonists.
It is clear from numerous in vivo and ex vivo experiments that the different PPAR subtypes serve different physiological functions. However, it is unclear to what extent these different actions are due to intrinsic biochemical properties or to differences in the cellular context in which the PPARs are expressed. Only a few studies have sought to mutually compare PPAR subtype-specific activation of endogenous genes in a particular cell type (7
). Although these comparisons have been very useful, they were performed with stable ectopic expression, which makes it difficult to distinguish between direct and indirect target genes. Furthermore, constitutive expression of the receptors hampers investigations of the molecular mechanism involved in target gene activation. The combination of adenoviral delivery and ChIP provides a unique possibility to investigate the molecular mechanisms underlying PPAR subtype specificity by comparing the acute actions of the different PPAR subtypes on endogenous chromatin-embedded target genes. We show that, when adenovirally expressed at equal levels, HA-tagged PPARs have highly different potentials to activate endogenous target genes in the NIH-CAR cells. While PPARα had more transcriptional activity than PPARγ in transient transfections with a reporter construct consisting of a mulitmerized ACOx1 PPRE, PPARγ was the most potent activator of the majority of the endogenous genes investigated. However, a small number of genes were most potently activated by PPARα. Surprisingly, however, in NIH-CAR cells as well as in MEFs, PPARβ/δ was either unable to activate or a poor activator of all genes investigated (Fig. and Table ). The reason for this is unclear, as adenovirally expressed PPARβ/δ was active in transient transfections of NIH-CAR cells and able to activate endogenous genes in AML-12 and MIN6 cells. Previous experiments with stable retroviral expression of PPARβ/δ in NIH 3T3 cells have shown that, although PPARβ/δ is by far the least potent PPAR subtype in these cells, constitutive PPARβ/δ expression induces low levels of several PPAR target genes (7
). However, from these long-term experiments it is impossible to conclude whether the induction is due to direct but slow induction of the target genes or due to indirect activation of the target genes via induction of another transcription factor, e.g., PPARγ. The lack of target gene activation by adenovirally expressed PPARβ/δ suggests the existence of a global mechanism (e.g., posttranslational modification or lack of a particular cofactor) that renders PPARβ/δ less transcriptionally active on endogenous genes in the NIH 3T3 fibroblasts.
To address the question how the PPARs retain gene specificity, we used ChIP to investigate if PPAR subtype-specific transcriptional activation is a consequence of differential PPAR binding to promoters or to PPAR subtype-specific recruitment of coactivators. Interestingly, ChIP analysis demonstrated that, in general, the transcriptional potential of the PPAR subtypes is correlated with the level of PPAR/RXR heterodimer binding to the promoters of activated genes (Fig. ). There are clear exceptions where a subtype binds without further activating; however, for all target genes investigated, the subtype that binds best is also the subtype that activates most efficiently. This indicates that a major, but clearly not the only, rate-limiting step in establishing a transcriptionally active complex at the target genes investigated is the ability of the receptor subtypes to gain access to the chromatin-embedded binding sites.
Some target genes remained transcriptionally silent in NIH-CAR cells irrespective of the PPAR subtype. As exemplified by the L-FABP gene, we did not observe PPAR/RXR binding to these target sites, and hence these are likely to be embedded in inaccessible chromatin or to require synergy with other cell-type-specific transcription (co)factors to gain access. Other genes (e.g., ME and thiolase B) were transcriptionally active but insensitive to further activation by PPARs. Interestingly, however, PPARγ2/RXR displays significant binding and PPARγ2-dependent cofactor recruitment to the PPREs of the ME gene (Fig. ). This suggests that other (co)factors than PPAR/RXR, CBP, and TRAP220 are limiting the expression of this gene. Further detailed ChIP analyses of factor recruitment to this and other loci will be required to understand why receptor binding is unable to further enhance the transcription of such genes.
The differential binding of PPAR subtypes to different PPREs may in part be explained by the sequence composition of the PPREs. In vitro experiments performed in the absence of ligands have shown that PPREs with a relatively low match to the consensus have a preference for PPARγ heterodimers (27
). Such “weak” PPREs include the two PPREs of the A-FABP enhancer. However, the LPL PPRE has been shown to bind both PPARα and -γ in vitro and is a recognized PPARα as well as PPARγ target gene (39
). In addition, the ACOx1 PPRE(s) match the consensus very well and is generally regarded as a PPARα-type PPRE (64
). For both the LPL and ACO PPREs, we find that they preferably bind PPARγ heterodimers in the context of chromatin in NIH-CAR cells, indicating that factors other than the affinity for the naked DNA sequence play a role. Furthermore, as discussed below, PPAR subtype specificity differs between the cell lines. Thus, the abilities of the PPAR subtypes to gain access to PPREs in vivo cannot be explained only by their differential affinity for a given PPRE in vitro. It is likely that an important determining factor is the synergy with other transcription factors and the joint ability to effectively recruit the right combination of chromatin remodeling and modifying complexes present in the cell.
In addition to the ability of the PPAR/RXR heterodimer to gain access to target sites, the ability of the PPAR/RXR heterodimer to bind cofactors prior to DNA-binding or to recruit cofactors to target genes once bound to DNA is likely to greatly influence the level of expression of the target genes. An exhaustive analysis of this aspect is beyond the scope of this article; however, we have investigated the differential ability of the PPAR subtypes to recruit TRAP220 and CBP to a small number of selected target genes in NIH-CAR cells (Fig. ). Importantly, for most genes the recruitment of these more general cofactors correlates with binding of the PPAR subtypes and therefore does not add to the subtype specificity. An exception may be the ADRP gene to which all PPAR subtypes bind, whereas only PPARα and PPARγ2 are able to significantly recruit TRAP220. Interestingly, this may in part explain why PPARβ/δ is unable to activate the ADRP gene. Further insight into the role of cofactors in PPAR subtype specificity for different genes awaits large-scale ChIP analysis of more specific cofactors.
The poor PPARα and PPARβ/δ transcriptional activity of endogenous genes in NIH 3T3 fibroblasts prompted us to investigate the ability of PPARα and PPARβ/δ to activate endogenous genes in other cell lines such as AML-12 hepatocytes and MIN6 pancreatic β cells. From these experiments it is evident that the cell type is indeed a very important parameter of PPAR subtype specificity toward several genes (Fig. ). PPARγ remains the subtype able to activate the largest number of genes in the cell types investigated. However, for the target genes investigated PPARα and PPARβ/δ are on average more transcriptionally active in AML-12 and MIN6 cells than in NIH-CAR cells. Irrespective of the cell line, the A-FABP and CD36 genes remain highly PPARγ specific, but other genes that are PPARγ specific in NIH-CAR cells can be induced by PPARα and/or PPARβ/δ in AML-12 and MIN6 cells. Similarly, HMG-CoA S2 and HD, which are PPARα selective in NIH-CAR cells, are induced by other PPAR subtypes in AML-12 and MIN6 cells. These results show that PPAR subtype specificity is a highly complex phenomenon likely to be dependent on the cell-type-specific setting of the individual target sites, i.e., a combination of other transcription factors, cofactors, and chromatin modifications in a cell. Importantly, however, as observed for the NIH-CAR cells, there was a good correlation between the ability of the PPAR subtypes to bind to target sites and their ability to activate the corresponding gene (Fig. ).
In conclusion, we demonstrate that the combination of adenoviral delivery and ChIP is an ideal method for investigating the basic molecular events involved in the activation of genomic target genes by PPARs in a broad range of cell lines. Using this approach, we show that PPARγ2 possesses considerable agonist-dependent as well as -independent transactivation potential and that agonist increases the occupancy of PPARγ2/RXR at most but not all PPREs. The ability of a particular PPAR subtype to gain access to a PPRE is highly dependent on the subtype as well as the cell type. However, in general (although there are clear exceptions) there is a good correlation between the extent of PPAR binding and the magnitude of the target gene induction. Future challenges will be to investigate why the PPAR subtypes differ in their ability to gain access to endogenous chromatin-embedded PPREs and how the cell type regulates this ability. In addition, large-scale ChIP analyses will be required to define the role of different cofactors in PPAR subtype-specific transactivation.