NuclearReceptors2018 Poster Presentations (1) (7 abstracts)
Single-molecule analysis of peroxisome proliferator-activated receptor γ2 and α reveal subtype specific differences in chromatin binding dynamics
Rikke AM Jensen 1, , Ville Paakinaho 1 , Diego M Presman 1 , Erin E Swinstead 1 , R Louis Schiltz 1 , David A Ball 1 , Tatiana S Karpova 1 , Susanne Mandrup 2 & Gordon L Hager 1,
1Laboratory of Receptor Biology and Gene Expression, NCI, NIH, Bethesda, Maryland, USA; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. *Chief of the Laboratory of Receptor Biology and Gene Expression, NCI, NIH, Bethesda, Maryland, USA.
The peroxisome proliferator-activated receptors (PPARs) display a high degree of conservation in the DNA- and ligand-binding domains. Despite these sequence similarities, the PPARs show distinct functions, even when co-expressed. Many experimental approaches have been employed to determine the molecular mechanisms that underlie their subtype-specific characteristics. However, these approaches have largely relied on cell population based studies, such as ChIP-seq. Here we have used single-molecule tracking (SMT) to investigate the unexplored intranuclear dynamics of two PPAR subtypes. Using HILO illumination, HaloTags, and the bright and stable fluorophore JF549, we have examined the behavior of PPARγ2 and PPARα in vivo at the single-molecule level. We detect slow and fast stops which we hypothesize to be functional- and non-functional binding events, respectively. Consistent with this model, we find that most long-lived binding events are lost upon mutation of the PPAR heterodimerization- and DNA-binding domains. The residence time and bound fraction (BF, slow stops) of both PPARs are unaffected by agonist or antagonist treatment. Interestingly however, both the BF and the residence time are greater for PPARγ2 than for PPARα, indicating that PPARγ2 and PPARα display subtype-specific dynamic binding behavior at the single-molecule level. This subtype specificity is found to be dependent on the N-terminal domain. Furthermore, we show that the BF and residence time of PPARγ2 significantly increase in the presence of C/EBPα, which we have previously shown can facilitate PPARγ binding to chromatin. The ability of C/EBPα to facilitate PPARγ binding is dependent on the AF-2 domain, consistent with a model wherein the interplay between multiple TFs relies on the recruitment of coactivators and chromatin remodelers. Overall, we have demonstrated that SMT provides a unique ability to resolve unanswered questions concerning the highly dynamic properties of transcription factors, including properties that are linked to specific subtypes of closely related transcription factors.
Volume 54
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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