Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes - PubMed (original) (raw)

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

Natarajan Balasubramaniyan et al. Am J Physiol Gastrointest Liver Physiol. 2012.

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.

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Figures

Fig. 1.

Ligand-induced chromatin recruitment of Set7/9 to the promoters of FXR-target genes and the associated histone H3 activation mark. Chromatin immunoprecipitation (ChIP) analysis of hepatoma cells (Huh-7) showed recruitment of FXR, Set7/9, CARM1, and the H3K4 monomethylation mark of histone H3 to the endogenous short heterodimer partner (SHP; A) and human bile salt export pump (BSEP; B) promoters in the presence of the farnesoid X receptor (FXR)-ligand (GW4064). Schematic representation of the primers used for ChIP and the location of the sites on the SHP and BSEP promoters are shown below representative gels.

Fig. 2.

The effect of short interfering RNAs (siRNA) knockdown of Set7/9 expression on ligand-induced occupancy of Set7/9 and FXR and on H3K4 monomethylation at the endogenous BSEP and SHP promoters. siRNAs were used to reduce cellular Set7/9 levels with a nonsilencing siRNA used as control. Treatment of Huh-7 cells with siRNA against Set7/9 markedly reduced ligand-induced occupancy of Set7/9 and FXR at the SHP (A) and BSEP (B) promoters and decreased the H3K4 methylation mark, as assessed by real-time quantitative PCR. Each independent ChIP data point is the average of normalized values from PCR runs in duplicate wells. Each histogram is the average mean ± SE from independent ChIP experiments of n = 3. *P < 0.05, #P < 0.001.

Fig. 3.

Inhibition of ligand-induced FXR target gene expression in Set7/9-silenced Huh-7 cells. Set7/9 expression was markedly and specifically reduced compared with control in RNAi-transfected cells at both the protein (A) and mRNA level (B). C: SHP and BSEP mRNAs declined significantly, as assessed by real-time quantitative PCR. *P < 0.05, #P < 0.001.

Fig. 4.

Effect of Set7/9 overexpression on endogenous BSEP mRNA levels in Huh-7 cells. Huh-7 cells were cotransfected with FXR/RXRα and Set7/9. BSEP mRNA was measured by real-time PCR. Overexpression of Set7/9 together with FXRwt led to a significant increase in BSEP mRNA levels in Huh-7 cells. *P < 0.05, #P < 0.001.

Fig. 5.

FXR and Set7/9 occupy the same genomic locus at the SHP and BSEP promoters. An electromobility shift assay was done using nuclear extracts prepared from Huh-7 cells transfected with FXR, RXRα, and Set7/9 (A). Specific binding of FXR/RXRα to the FXRE was enhanced by expression of Set7/9 (lanes 4 and 5). In contrast, specific binding was significantly reduced in cells expressing the FXR K206R mutant (lane 3) or a Set7/9 mutant (lane 2) that is deficient in methyltransferase activity. Competition analysis was performed with unlabeled 25-fold molar excess of wild-type (lane 6) or mutant FXRE (lane 7). Sp, specific complex; NSp, nonspecific complex. Sequential chromatin immunoprecipitation (ChIP Re-ChIP) confirms that FXR and Set7/9 occupy the same genomic site (FXRE) in vivo at both the SHP and BSEP loci (B and C). ChIP-ReChIP was done using chromatin prepared from Huh-7 cells transfected with FXR, RXRα, and Set7/9 or the FXR K206 mutant or the Set7/9 mutant. B: PCR results using the SHP primer that includes the FXR binding site. C: PCR results using the BSEP primer that include FXR binding site. Lane numbers are the same to indicate that the DNA is from the same chromatin sample. Top: results PCR performed on an aliquot of DNA removed from the experiment after the first ChIP assay. Bottom: PCR results on DNA from chromatin samples after both ChIP steps. Chromatin samples subjected to the first ChIP using FXR antibody (top, lane 2) were then subjected to a second ChIP with a Set7/9 antibody (bottom, lane 2). Mouse IgG and no antibody controls were also performed from the first ChIP using FXR antibody (lane 2). RXR, retinoic X receptor; IP, immunoprecipitation; Mu, mutant; WT, wild type.

Fig. 6.

Methylation of FXR in vitro by Set7/9 and identification of the lysine methylation site. A: schematic representation of FXR indicating domains conserved in the nuclear hormone receptor superfamily: activation function-1 (AF-1), DNA binding domain (DBD), hinge region (hinge), and ligand-binding domain (LBD). The conserved methylation motif is underlined. The sequence methylated by Set7/9 in various known substrates is indicated to the right. *Target lysine residue. B: strong histone methyltransferase activity of a GST-Set7/9 preparation in vitro is indicated by methylation of recombinant histone H3. GST and BSA were negative control substrates. C: methylation of GST-FXR in an in vitro reaction. GST-Set7/9 (3 μg) was incubated with the GST-fused FXRwt and FXRmutants in the presence of [3H-methyl]-SAM. 3H signal of methylated FXR in the reaction mixture was detected by 10% SDS-PAGE and autoradiography. Recombinant Set7/9 methylated FXR. Mutation of the putative methylation site at K206 (K206R) to an arginine completely abrogated methylation of FXR by Set7/9. FXR was still methylated with mutation of an adjacent, irrelevant lysine (K204) to an arginine. GST lacked Set7/9 activity. bottom: Coomassie blue staining of the gel showing the protein substrates. D: methylation of various GST fused FXR fragments. The hinge domain but not the AF1, DBD, or LBD was methylated by Set7/9. Bottom: Coomassie blue staining of the gel shows the protein substrates.

Fig. 7.

Methylation of FXR in vivo by Set7/9. A: FXR immunoprecipitated (or IgG-incubated control) from Huh-7 cells total lysate was immunoblotted with the pan-methyllysine (αMe-K) antibody, and methylated FXR was detected by Western blot analysis. The methylation of FXR was abrogated in cells subjected to Set7/9 siRNA-mediated silencing. B: Huh-7 cells were cotransfected with plasmids encoding FXR (wild-type or K206Rmut) and Set7/9, and the Western blot FXR immunoprecipitate was probed with anti-αMe-K and anti-FXR antibodies. Immunoprecipitates from the transfected cells were then probed with the αMe-K antibody. Wild-type but not the K206R mutant was methylated when Set7/9 was expressed in these cells. Wild-type FXR transfected cells without Set7/9 cotransfection, or control, nontransfected cells were also analyzed. IB, immunoblotting.

Fig. 8.

Set7/9 associates with FXR in vivo_. A_: When FXR was immunoprecipitated from a whole cell lysate of Huh-7 cells, association was demonstrated with Set7/9 on Western blot analysis with a Set7/9 antibody. The input lysate was at 1% of the lysate used for immunoprecipitation. B: FXR-GFP also coimmunoprecipitated with Set7/9 from a Huh-7 cell lysate when Set7/9 was first immunoprecipitated, run on a Western blot analysis and probed with a GFP antibody. Huh-7 cells were transiently transfected with the FXR-GFP construct 1 day before collecting the cells. C: GST pull-down assays using various fragments of FXR and K206Rmut with in vitro translated Set7/9 protein was carried out as described in the

materials and methods

. FXR hinge domain bound to Set7/9, but binding was absent when the GST control and other domains were used in the assay. With use of the FXR K206R mut there was weak binding when compared with FXRwt. RLU, relative light unit.

Fig. 9.

Mammalian two-hybrid analysis of the interaction between FXR and Set7/9. A: mammalian two hybrid analysis: The association between pBIND- FXRwt, different domains of the FXR and pACT-Set7/9, is shown. A 2- to 3-fold increase in reporter gene activity was observed with the FXR hinge region and full length constructs. No significant increase in activity was seen with the remaining constructs. B: mammalian two hybrid analysis between pBIND- FXRwt, K206Rmut, and pACT-Set7/9 in the absence/presence of 1 μM GW4064 and the ensuing increase in reporter gene activity over empty vector are shown. Note the increased reporter gene activity when Set7/9 was cotransfected with the FXRwt but not with expression the 206R FXRmut (*P < 0.05, #P < 0.001 compared with FXRwt only).

Fig. 10.

Synergistic activation of SHP and BSEP by FXR and Set7/9. A: Huh-7 cells transfected with indicated plasmids were treated with FXR ligand GW 4064 24 h after transfection, and reporter assays were performed. The mean and mean ± SE are shown (n = 3). Set7/9 expression significantly increased SHP reporter gene activity, which was not observed with expression of the K206R FXR mutant or the Set7/9 mutant deficient in methyltransferase activity. # P < 0.001. B: Huh-7 cells were cotransfected with indicated reporter plasmids and expression plasmids for Set7/9 or the Set7/9 mutant and FXR WT or the FXR mutant, as indicated. Set7/9 expression significantly increased BSEP reporter gene activity, which was not observed with expression of the K206R FXR mutant or the Set7/9 mutant deficient in methyltransferase activity. #P < 0.001. C: CV-1 cells in 12-well plates were transfected with 3XFXRE-TK-Luc along with wild-type FXR, RXRα, and Set7/9 or the K206R mutant (Fig. 9_A_). 3XFXRE-TK-Luc was constructed by cloning 3 copies of the BSEP FXRE upstream of the minimal thymidine kinase (TK) promoter and the luciferase opening reading frame. Expression of the FXR-RXRα heterodimer along with Set7/9 in CV-1 cells led to a marked increase in expression of the _3X_FXRE-TK-LUC activity in the presence of GW4064. There was no activity of the promoter observed in the absence of FXR/RXRα. Set7/9 lysine methyltransferase failed to augment _3X_FXRE-TK-LUC activity with expression of the K206R mutant FXR and RXRα in the presence of ligand. *P < 0.05 compared with transfection in the absence of FXR /RXRα or with mutant FXR. #P < 0.001. D: effect of increasing amount of Set7/9 in CV-1 cells. CV-1 cells were transfected with FXR, RXRα, and increasing amounts of full-length Set7/9 cDNA plasmid. FXR transactivation of BSEP promoter was monitored by luciferase activity. Significant (*P < 0.05 compared with activity in cells without Set7/9 plasmid cotransfection) dose-dependent stimulation of promoter activity was seen with 0.8 to 2.2 μg of Set7/9 cDNA. *P < 0.05 compared with transfection in the absence FXR /RXRα. #P < 0.001, Pr, BSEP promoter; F/R, FXR /RXRα.

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Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes - PubMed (original) (raw)