A critical step toward understanding the cell-type-specific functions of PPARγ is elucidating its recruitment across the genome in various cell types. In this study, PPARγ binding was compared in mouse macrophages and adipocytes, both of which play important roles in regulating metabolism (54
). PPARγ cistromes in adipocytes and macrophages are largely distinct, although a small subset of the binding locations is shared between cell types. The ability of PPARγ to occupy cell-type-unique locations is associated with chromatin remodeling and active chromatin marks such as H3K9Ace, while access to binding sites of a different cell type may be prevented through chromatin silencing. In macrophages, PPARγ colocalizes with C/EBPβ as well as the hematopoietic factor PU.1, which may explain the ability of PPARγ to regulate not only a subset of the metabolic genes it binds in adipocytes but also a unique set of immune genes.
PPARγ binding in macrophages occurs at DR1 elements through heterodimerization with RXR, as previously described for adipocytes (31
). It is interesting that, although macrophages express only PPARγ1 and their overall level of PPARγ protein is much lower than that of adipocytes, the ChIP enrichment levels at regions common to both cell types are similar. A notable difference, however, is that there are fewer regions occupied by PPARγ in macrophages than in any of the three adipocyte data sets discussed here. Thus, the higher protein levels in adipocytes and/or the expression of the γ2 isoform may allow PPARγ to bind and activate a larger number of genes in adipocytes, where it is required for differentiation, mature function, and survival (69
). In contrast, macrophages do not require PPARγ for differentiation, phagocytic activity, or proinflammatory cytokine production (50
) but employ PPARγ in more specialized functions, such as maintenance of the alternative macrophage phenotype (5
), cholesterol uptake and efflux in atherosclerotic plaques (49
), antigen cross-presentation to T lymphocytes (34
), and dendritic cell immunogenicity (51
One of the major differences at PPARγ-binding regions in macrophages compared to adipocytes is the nearby presence of the hematopoietic transcription factor PU.1. It belongs to the Ets family of transcription factors and is most highly expressed in myeloid cells (13
), from which mature macrophages are derived. PU.1 is absolutely required for the development of monocytes (19
) and can reprogram lymphocytes and fibroblasts into monocytes when expressed exogenously together with C/EBP (18
). In fact, the genome-wide binding studies performed here demonstrate that PU.1 colocalizes extensively with C/EBPβ across the genome. Importantly, the motif search performed on the PPARγ-binding regions did not reveal enrichment for NF-κB sites, although motifs for other transcription factors associated with immunity and inflammation were identified, including AP-1 and STAT, suggesting that PPARγ may also cooperate with those factors in addition to PU.1 and C/EBP. Thus, through colocalization with potent regulators of immune function in macrophages, PPARγ may be able to access and regulate a number of immune genes in addition to a subset of the metabolic genes it binds in adipocytes (40
Further support for the notion that PPARγ can regulate both immune and metabolic genes in macrophages is gained from the distribution of PPARγ-binding regions relative to differentially expressed genes in PPARγ-deficient macrophages (29
). A higher percentage of the downregulated genes have PPARγ-binding regions than the upregulated genes (39% compared to 15%), consistent with findings in adipocytes following PPARγ knockdown (62
). This suggests that in both cell types direct PPARγ binding has activating rather than repressive effects on target gene transcription.
To begin to address the mechanisms driving cell-type-specific binding of PPARγ in macrophages and adipocytes, several chromatin features were examined in the vicinity of PPARγ sites. In the absence of PPARγ occupancy, DNA accessibility is low at PPARγ-binding sites, suggesting that the DNA may be sequestered in nucleosomes (60
). In contrast, occupied PPARγ sites are characterized by a high degree of DNA accessibility, which indicates that the chromatin organization has been altered and histone octamers have been released or repositioned (26
). This type of chromatin remodeling may occur prior to or following PPARγ binding. Although the former possibility cannot be excluded, PPARγ was able to induce chromatin remodeling at adipocyte regions when retrovirally expressed in undifferentiated preadipocytes. PPARγ can interact with SWI/SNF ATP-dependent remodeling complexes (8
), and, therefore, the increased accessibility may be a consequence of PPARγ-dependent recruitment of chromatin remodelers. However, it remains unclear which, if any, remodeling complexes are responsible for this activity and how PPARγ recruits them to target sites. Nevertheless, a similar induction of DNA accessibility has been described for other factors, including FOXA1 (16
), glucocorticoid receptor (33
), and others (4
In addition to altering DNA accessibility, PPARγ was also found to increase H3K9Ace at sites to which it bound in retrovirally transduced preadipocytes. Acetylation of histone tails, including H3K9 acetylation, is mediated by histone acetyltransferases such as p300/CBP and Gcn5 (35
), some of which have been shown to interact with and coactivate PPARγ (11
). Notably, histone acetylation occurs at enhancers and promoters (27
) and is predominantly associated with transcriptional activation (35
). It should be noted that in addition to coactivators, PPARγ can also associate with corepressors such as NCoR and SMRT, which in turn recruit histone HDAC3 (25
). Therefore, it is also possible that the high level of acetylation observed at occupied PPARγ binding sites in macrophages and adipocytes reflects decreased recruitment of corepressors.
Although PPARγ was able to induce both DNA accessibility and H3K9Ace in PPARγ-transduced preadipocytes, it did so selectively: only at a subset of adipocyte regions tested and not at sites that are unique to macrophages. This finding was correlated with the presence of repressive H3K9Me2 chromatin marks at macrophage-unique PPARγ-binding regions in preadipocytes. The presence of these modifications, along with the concurrent hypoacetylation and lack of DNA accessibility, strongly suggests chromatin silencing. H3K9Me2/3 and H3K27Me2/3 are associated with transcriptional repression across the human genome (3
). They can occur in constitutive heterochromatin, i.e., gene-poor repetitive domains at centromeres and telomeres, as well as in facultative heterochromatin, which contains genes that can undergo silencing or activation depending on the cellular context (7
). Given the developmental divergence between macrophages and adipocytes, it is possible that the observed silencing effects may be established early during the differentiation programs of these cell types and may span large domains, including subsets of PPARγ-binding regions and the genes they regulate. Indeed, the fibroblast-like preadipocytes are committed to adipocyte differentiation and, consistent with this, our data implicate H3K9Me2 as a mark that restricts PPARγ binding to sites bound in the mature adipocyte.
The findings presented here demonstrate that cell-type-specific PPARγ binding can facilitate the formation of cell-type-specific enhancers that target unique sets of genes related to the specification of a cell type. The ability of PPARγ to access binding sites is limited and may be defined by repressive mechanisms like chromatin silencing and active mechanisms such as cell-type-specific colocalization with factors like PU.1 and C/EBPβ. Moreover, our data support the existence of an epigenomic hierarchy in which PPARγ binding to cell-specific sites not marked by repressive marks, including H3K9Me2, opens chromatin and leads to local activation marks, including H3K9 acetylation. A more comprehensive understanding of the mechanisms by which PPARγ regulates transcription in cell-type-specific ways may be helpful in designing pharmacological agents that can target individual cell types or distinct pathways in order to optimize therapeutic results and minimize side effects.