Pioneer factor Foxa2 enables ligand-dependent activation of type II nuclear receptors FXR and LXRα

Abstract

Objective Type II nuclear hormone receptors, including farnesoid X receptors (FXR), liver X receptors (LXR), and peroxisome proliferator-activated receptors (PPAR), which serve as drug targets for metabolic diseases, are permanently positioned in the nucleus and thought to be bound to DNA regardless of the ligand status. However, recent genome-wide location analysis showed that LXRα and PPARα binding in the liver is largely ligand-dependent. We hypothesized that pioneer factor Foxa2 evicts nucleosomes to enable ligand-dependent binding of type II nuclear receptors and performed genome-wide studies to test this hypothesis. Methods ATAC-Seq was used to profile chromatin accessibility; ChIP-Seq was performed to assess transcription factors (Foxa2, FXR, LXRα, and PPARα) binding; and RNA-Seq analysis determined differentially expressed genes in wildtype and Foxa2 mutants treated with a ligand (GW4064 for FXR, GW3965, and T09 for LXRα). Results We reveal that chromatin accessibility, FXR binding, LXRα occupancy, and ligand-responsive activation of gene expression by FXR and LXRα require Foxa2. Unexpectedly, Foxa2 occupancy is drastically increased when either receptor, FXR or LXRα, is bound by an agonist. In addition, co-immunoprecipitation experiments demonstrate that Foxa2 interacts with either receptor in a ligand-dependent manner, suggesting that Foxa2 and the receptor, bind DNA as an interdependent complex during ligand activation. Furthermore, PPARα binding is induced in Foxa2 mutants treated with FXR and LXR ligands, leading to the activation of PPARα targets. Conclusions Our model requires pioneering activity for ligand activation that challenges the existing ligand-independent binding mechanism. We also demonstrate that Foxa2 is required to achieve activation of the proper receptor – one that binds the added ligand – by repressing the activity of a competing receptor.

Integrative analysis reveals multiple modes of LXR transcriptional regulation in liver

“…These finding suggests that FOXA1/2 binding could be important for LXR ligand–dependent repressor function. In accordance with our findings, published data suggest that loss of hepatic Foxa2 abolishes the down-regulation of some of these genes ( Cxcl , Etnppl , Got1 , Nnmt , Slc25a15 , Tbc1d8 , and Tymp ) by LXR agonist (GSE149075) ( 33 ).…”

“…Our ATAC-Seq results showed decreased FOXA motif accessibility across the genome in LXRDKO liver, despite increased expression of the Foxa2 gene. The pharmacological repression by LXR agonist was dependent on the presence of Foxa2 for half of the genes showing direct ligand-dependent repression from our dataset ( 33 ). Kain et al.…”

“…Our analysis of global accessibility changes induced by loss of LXR supported this major mode of LXR action, but also revealed additional mechanisms. We integrated datasets for genes differentially expressed in liver in response to GW3965 or T0901317 treatment with LXR ChIP-Seq data and our RNA-Seq data ( 33 ). We found that the expression of a majority of the differentially expressed genes in WT vs. LXRDKO liver was not altered by agonist treatment ( Fig.…”

“…Unexpectedly, we also identified a small set of genes that were repressed by LXR in the basal state and in response to synthetic agonist. These genes had putative LXR-binding sites by ChIP-Seq ( 27 ), showed increased expression in LXRDKO liver, and showed decreased expression with LXR agonist treatment of WT mice ( 33 ). Such a direct ligand-dependent repressor function for LXR has not been demonstrated in liver previously.…”

“…We also profiled the differences in activity of other transcription factors in response to loss of LXRs using the accessibility of their binding motifs. We integrated our results from the LXRDKO model with data from synthetic agonist treatment studies ( 33 ) to define distinct modes of LXR action in the liver, including the ability to act as a ligand-dependent repressor. These findings contribute to a more thorough understanding of how LXRs impact the transcriptional landscape and orchestrate hepatic metabolism.…”