Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

Abstract

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes.

Nuclear receptors and chromatin: an inducible couple

“…ChIP assays demonstrated the recruitment of Set7/9 and the presence of the associated H3K4 monomethylation activation mark at the loci of two of the known FXR hepatic targets, the small heterodimer partner NR0B2 (SHP) and BSEP. This significantly enhanced the transcription of these two FXR target genes (Balasubramaniyan et al 2012). Similar data for AR (Ko et al 2011) and ER (Subramanian et al 2008) indicate that this mechanism could also possibly stabilize FXR and promote its interaction with its heterodimerization partner RXR and the FXRE (Balasubramaniyan et al 2012).…”

“…This significantly enhanced the transcription of these two FXR target genes (Balasubramaniyan et al 2012). Similar data for AR (Ko et al 2011) and ER (Subramanian et al 2008) indicate that this mechanism could also possibly stabilize FXR and promote its interaction with its heterodimerization partner RXR and the FXRE (Balasubramaniyan et al 2012). Furthermore, recruitment of chromatin-remodelling proteins and NR-dependent gene transcription is a very dynamic process and different co-regulators and chromatin-remodelling proteins can occupy different REs of the same NR.…”

“…The LBD is also a target for post-translational modifications, which govern a variety of cellular functions, including NR activity, DNA-binding affinity, ligand sensitivity, receptor stability and subcellular distribution. Therefore, NR post-translational modifications can ultimately result in either increased or decreased target gene expression (Balasubramaniyan et al 2012) or in transrepression of other pathways (such as sumoylation of PPARg (Pascual et al 2005) and FXR, leading to NF-kB transrepression (Vavassori et al 2009)). …”

Identification of Functionally Relevant Lysine Residues That Modulate Human Farnesoid X Receptor Activation

“…Moreover, Li et al (2007) and Kemper et al (2009) reported that SIRT1 [sirtuin (silent mating type information regulation 2 homolog) 1; a highly conserved NAD 1 -dependent protein deacetylase] is a positive regulator of liver X receptor and FXR. The data from our laboratory also demonstrated that direct methylation of FXR by lysine methyltransferase Set7/9 regulates the expression of FXR target genes (Balasubramaniyan et al, 2012). Posttranslational modifications at specific lysine residues of NRs dx.doi.org/10.1124/mol.113.084772.…”

“…The full-length coding regions of hFXRa (NR1H4) were amplified by polymerase chain reaction (PCR) and cloned into KpnI-BamHI sites of pcDNA3.1(1) vector (Invitrogen, San Diego, CA) or into PstI-KpnI sites of pEnhanced GFP (green fluorescent protein)-N2 vector (Clontech, Mountain View, CA), respectively, to produce expression construct (pcDNA-hFXR) and GFP fusion FXRa (hFXR-GFP). The full-length cDNAs of retinoid X receptor (RXR) a and Set7/9 and SHP promoter were generated as described previously (Balasubramaniyan et al, 2012). The luciferase reporter plasmid of human bile salt export pump (hBSEP) was created by inserting its 59 untranslated region upstream (from bases 21250 to 165) at NheI-XhoI sites of the pGL4.17 [luc2/Neo] vector (Promega, Madison, WI).…”