PUFA partitioning into condensates as a new mechanism for gene regulation

SUMMARY: Understanding the mechanisms underlying the cardioprotective and other health benefits of polyunsaturated fatty acids (PUFAs) is critical for providing sound nutritional advice. A major explanation for how PUFAs mediate their effects is by altering gene expression. Through gene expression, specific PUFAs reduce triglyceride levels and inflammation. Yet, we do not clearly understand the basis for the specificity of some, but not other fatty acids in regulating gene expression. This is a major problem for fully understanding the health implications of eating foods with complex mixtures of these different fatty acids. One of the major transcription factor families that mediates the effect of PUFAs are the peroxisome proliferator- activated receptors (PPARs). PUFAs have been found to bind to PPARs and to promoter their activation of target gene transcription. However, there has been disagreement on the effective binding affinity of PUFAs for PPARs and whether it is physiologically relevant. This has led to confusion over the role of PUFAs as PPAR agonists. However, these studies assumed that PPAR is localized to the aqueous nucleoplasm and that they interact with PUFAs in this environment. Instead, at least one PPAR protein, PPARγ, localizes to condensates, which are membraneless organelles enriched in proteins and nucleic acids. We have recently shown that these environments enrich multiple classes of lipids, including PUFAs. Additional analysis of our datasets has also demonstrated that only specific fatty acids enrich in condensates and that this correlates with their number of unsaturated bonds. In this project, we will explore a new explanation for why specific PUFAs regulate PPARγ-mediated transcription initiation more than others. Rather than different abilities to bind the PPARγ ligand binding domain, the extent to which a specific PUFA species regulates PPARγ may be determined by its ability to enrich in PPARγ condensates. Additionally, this novel form of regulation may specifically effect PPARγ-bound genes in condensates, while not affecting other PPARγ-bound genes. In Aim 1, we will determine if PPARγ agonists enrich in PPARγ-containing condensates. First, we will measure the extent to which different PUFAs and other putative PPARγ agonists enrich in PPARγ condensates in vitro. We will then determine whether a subset of PUFAs also enrich in cellular PPARγ-containing condensates using super-resolution, lipid expansion microscopy. Finally, we will determine if transcription activation by PPARγ agonists correlates with their ability to enrich in PPARγ condensates. In Aim 2, we will test whether the condensate pool of PPARγ proteins are preferentially activated by PUFAs and other PPARγ agonists. We will address this question by measuring co-activator binding to PPARγ after addition of different agonists, both in vitro and in multiple cell-base assays. Together, these two aims will establish whether specific PUFAs preferentially activate the condensate pool of PPARγ proteins.