Unraveling the Hierarchy of cis and trans Factors That Determine the DNA Binding by Peroxisome Proliferator-Activated Receptor γ

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor essential for adipocyte development and the maintenance of the alternatively polarized macrophage phenotype. Biochemical studies have established that as an obligate heterodimer with retinoid X receptor (RXR), PPARγ binds directly repeated nuclear receptor half sites spaced by one nucleotide (direct repeat 1 [DR1]). However, it has not been analyzed systematically and genome-wide how cis factors such as the sequences of DR1s and adjacent sequences and trans factors such as cobinding lineage-determining transcription factors (LDTFs) contribute to the direct binding of PPARγ in different cellular contexts. We developed a novel motif optimization approach using sequence composition and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) densities from macrophages and adipocytes to complement de novo motif enrichment analysis and to define and classify high-affinity binding sites. We found that approximately half of the PPARγ cistrome represents direct DNA binding; both half sites can be extended upstream, and these are typically not of equal strength within a DR1. Strategically positioned LDTFs have greater impact on PPARγ binding than the quality of DR1, and the presence of the extension of DR1 provides a remarkable synergy with LDTFs. This approach of considering not only nucleotide frequencies but also their contribution to protein binding in a cellular context is applicable to other transcription factors.