Fig 2: Binding of FXR and LXRa is induced by acute activation. (A) The accepted paradigm regarding ligand activation of type II nuclear receptors (NR) is a two-step process: 1) the receptor as a heterodimer with retinoid X receptor (RXR) is bound to the DNA in complex with a co-repressor in the absence of the ligand; 2) binding of the ligand induces a conformational change, co-repressor/co-activator exchange, and initiation of transcription. (B) Experimental design showing the mice (both WT and Foxa2 mutants) were treated with an agonist (GW4064 for FXR, GW3965, specific for LXR, or T0901317, which binds LXR and few other related receptors) using oral gavage; Livers were harvested after 4 h of treatment and used for ATAC-Seq, ChIP-Seq (Foxa2, FXR, LXRa), and RNA-Seq experiments. (C) ChIP-seq track view in Integrative Genomics Viewer (IGV) of common (left) and ligand-dependent (right) sites for FXR (top panel, Nr0b2 chr4:133,551,376–133,558,686, Nr1i2 chr16:38,266,836–38,296,668) and LXRa (bottom panel, Srebf1 chr11:60,207,979–60,213,098, Dcdc2a chr13:25,054,003–25,056,498). (D) Venn diagrams showing increased FXR and LXRa binding with acute ligand activation (FXR 7,883 regions for vehicle, 22,705 regions for GW4064, PeakSeq, FDR < 5%, q-value < 0.015 vs Input control; LXRa 7,019 regions for vehicle, 20,472 regions for GW3965 and 12,669 regions for T09, PeakSeq, FDR < 10%, q-value < 0.05, vs. Input control). (E) Scanning motif of positional weight matrices in Jaspar and TRANSFAC databases in FXR (left) and LXRa (right) bound regions. PscanChIP identified highly enriched consensus sites for FXR (IR-1 element) in FXR bound regions and LXR (DR-4 element) in LXRa sites for both common and ligand-dependent targets. Consensus for nuclear receptor half-site and forkhead motif was highly enriched in both FXR and LXRa sites. (F) EnrichR analysis of bound regions mapped the sites to closest genes and reported overrepresented pathways, including “triacylglyceride synthesis” and “bile acid synthesis” among common FXR targets and “cholesterol and lipid homeostasis” and “SREBP signaling” among common LXRa targets. Different pathways were enriched among ligand-dependent targets, including “glycogen metabolism”, “NAD metabolism”, and “ketone body metabolism” (GW4064, GW3965, T09).

Fig 3: LXRa protein expression is correlated with Pgp expression and is a molecular marker of poor prognosis in ER-negative breast cancer patients.A Correlation analysis between LXR protein expression and matched mRNA of Pgp in 47 ER-negative breast tumors (relative to HPRT). Line indicates linear regression, correlations calculated using Pearson correlation. Kaplan–Meier survival plots following stratification of the cohort into high and low LXRa (B) and Pgp (C) expression. Log-rank tests used to calculate significance. D Tissue microarray of 148 TNBC tumors stained for indicated proteins determined by immunohistochemistry and correlated with Pgp protein (left) and disease-free survival (right). E scOHC concentrations determined by LC-MS/MS and correlated with Pgp protein (left) and disease-free survival (right). Correlations calculated with Pearson’s test and survival differences with log-rank test and Kaplan–Meier curves.

Fig 4: LXR splicing events in BCa validated by TCGA Splicing Variants database (TSVdb)(A) Heatmap visualization of normalized RSEM (RNA-Seq by Expectation Maximization) value expression of LXRa and LXRß in 1,103 TCGA BCa samples. Note: Transcript uc002prv was only reported in TSVdb and is not recorded in NCBI, ENSEMBL, or UNIPROT databases.(B) Comparison of LXR transcript variant expression, for a1.1, a1.2, a3.1, and ß1.1, in matched tumor and normal tissues grouped by BCa subtype, ER+ (n = 78), and TNBC (n = 18). Statistical analysis by Mann-Whitney two-tailed U tests. P = 0.05 was considered significant. The line shows the median value, the box shows 10th to 90th percentile, and whiskers show minimum-to-maximum value.(C and D) Kaplan-Meier survival curves plotting disease-free survival of patients with TCGA BCa. The TCGA BCa samples (downloaded from TSvdb TCGA splicing database) were grouped by BCa subtype, (C) ER+ (n = 803), and (D) TNBC (n = 101). Survival curves of each subtype were separated into two groups: no event (ER+ = 675, TNBC = 79) and event (ER+ = 128; TNBC = 22) based on their overall survival data reported in cBioportal. p value is based on the log rank test, and data groups were considered significantly different if p < 0.05.

Fig 5: Foxa2 interacts with FXR and LXRa in a ligand-dependent manner. (A) Foxa2/NR interaction model. Foxa2 and liganded nuclear receptor, either FXR or LXRa, form a complex, with pioneer factor binding DNA at the forkhead (Fox) binding site. (B) ChIP-seq track view in IGV of colocalized Foxa2 and FXR binding at Nr1i2 (chr16:38,277,930–38,282,000, left panel) and Abcb11 (chr2:69,340,945–69,344,719, right panel) loci. FXR binding at these loci is absent in Foxa2 mutants. ChIP-seq track view in IGV of colocalized Foxa2 and LXR binding at Agmo (chr12:37,400,211–37,405,190, left panel) and Gm31588 (chr9:92,219,475–92,229,805, right panel) loci. LXR binding at these loci is absent in Foxa2 mutants. (C) Co-immunoprecipitation experiments in livers of mice treated with FXR ligand (left) and LXR agonists (right). Immunoprecipitation with an antibody to FXR and subsequent blotting with an antibody to Foxa2 shows a strong interaction between FXR and Foxa2 with GW4064 treatment, but not in IgG and vehicle controls (left panel). Similarly, immunoprecipitation with an antibody to LXRa and subsequent blotting with an antibody to Foxa2 shows a strong interaction between LXRa and Foxa2, in both GW and T09 treatment, but not in IgG and vehicle controls (right panel). (D) Heatmaps of Foxa2 and FXR ChIP-Seq at ATAC-Seq regions induced by GW4064 (60,416 regions, PeakSeq FDR 5%, q-value < 0.05; left panel) and Foxa2 and LXRa ChIP-Seq signal at ATAC-Seq regions induced by LXR ligands GW3965 and T09 (59,054 regions GW3965, 59,783 regions T09, FDR 5%, q-value < 0.05, middle, right panel). (E) Ligand-responsive binding of FXR and LXRa is absent in Foxa2 mutants and is Foxa2-dependent. Heatmaps of FXR and Foxa2 ChIP-Seq signal at sites of FXR binding induced by GW4064 in wildtype controls (left panel) and absent in Foxa2 mutants. Heatmaps of LXRa and Foxa2 ChIP-Seq signal at sites of LXRa binding induced by GW3965 (middle panel) and T09 (right panel) in wildtype controls and absent in Foxa2 mutants.