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

ABSTRACT

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.

KEYWORDS:

• PPARγ
• RXR
• adipocytes
• direct repeat 1
• lineage-determining factors
• macrophages
• nuclear receptors

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SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

ACKNOWLEDGMENTS

We thank Zsolt Czimmerer, Petros Tzerpos, Lajos Szeles, and other members of the Nagy laboratory for discussions and comments on the manuscript.

This work was supported by grants from the National Institutes of Health (R01DK115924 to L.N.) and the Hungarian Scientific Research Fund (OTKA grant no. K124298, K126885, and K116855 to L.N. and OTKA grant no. PD124843 to G.N.). G.N. is supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and by the ÚNKP-19-4-DE-173 New National Excellence Program of the Ministry of Human Capacities. B.D. is supported by an American Heart Association postdoctoral fellowship (17POST33660450). Library preparation and bioinformatics analysis were performed at the Center of Clinical Genomics and Personalized Medicine of the University of Debrecen. Next-generation sequencing was performed at the Centre National de Genotypage Evry by Steven McGinn, Anne Boland, Doris Lechner, and Marie Thérèse Bihoreau and supported by the European Sequencing and Genotyping Infrastructure (funded by the European Commission; FP7/2007-2013) under grant agreement no. 26205 (École Supérieure de Génie Informatique), as part of the ADIPOMACTX transnational access program and also at the Analytical Genomics Core Facility at the Sanford Burnham Prebys Medical Discovery Institute.

We declare that we have no competing interests.