4A). Similar trends were observed from the samples treated with GW4064 for a short time only (1 hour) (Supporting Fig. 12), which is consistent with a direct effect of FXR on gene expression, rather than delayed indirect responses. For the genes unique to obese mice, mRNA levels were increased significantly for 5 of 13 genes and decreased significantly in one (Fig. 4B). These results suggest that a large fraction of potential FXR target genes examined are likely repressed by agonist-activated FXR. Direct gene repression by agonist-activated FXR was expected to be rare, because

Ibrutinib manufacturer nearly all of the direct FXR target genes have been reported to be activated.2, 3, 19 We therefore further examined epigenetic

histone markers of gene activation and repression, as well as occupancy by RNAPII.21, 24, 25 Genes with increased expression, as well as representative genes that were repressed or not Small molecule library concentration significantly affected (Fig. 4A), were examined. Two-step re-ChIP and qRT-PCR analyses were performed, and the known FXR target gene, Shp, was first analyzed as a control. Co-occupancy of RNAPII with FXR and acetylated histone H3K9/K14 at the Shp promoter was increased, whereas co-occupancy of RXRα with FXR was not changed after GW4064 treatment (Fig. 5A). As expected, mRNA levels for Shp were increased after a 1-hour treatment with GW4064 (Fig. 5B). For selected potential FXR genomic targets, occupancy of FXR was increased in nearly all of the genes examined after GW4064 treatment (Fig. 5C). For the three genes with increased mRNA levels after GW4064 treatment (Fig. 4A), aldehyde dehydrogenase 1 and 2 (Aldh1/2), 3-oxoacid coenzyme A transferase 2B (Oxct2b), and cell division cycle-associated 4 (Cdca4), co-occupancy of RXRα and RNAPII with FXR was increased and acetylated histone H3K9/K14 levels were increased, medchemexpress as expected, for activated genes (Fig. 5D-F). For the remaining genes, RNAPII

occupancy and acetylated histone H3 levels were decreased or unchanged, which would be consistent with either no induction or suppression of these genes, as observed from the mRNA levels. Finally, to directly determine whether binding of agonist-activated FXR leads to the repression of genes examined, ChIP assays were performed to measure the levels of known epigenetic histone marks for gene repression, such as H3K9 di- and H3K27 tri-methylation.24, 25 After treatment with GW4064, occupancies of FXR and RNAPII were increased and tri-methylated H3K9 and H3K27 levels were decreased at the control Shp gene promoter (Fig. 6A). For the potential FXR target genes with increased expression, such as Ald1/2, Oxc2b, and Cdca4, levels of repressed histone marks (H3K27 and H3K9) were unchanged or decreased after GW4064 treatment (Fig. 6B).