Transcriptional corepressor SHP recruits SIRT1 histone deacetylase to inhibit LRH-1 transactivation

Abstract

Orphan nuclear receptor Small Heterodimer Partner (SHP; NR0B2) is a transcriptional corepressor of a wide variety of nuclear receptors (NRs). Here, we report that SHP recruits SIRT1, a class III histone deacetylase, in an NR-specific manner to inhibit transcriptional activity. SHP interacts and co-localizes specifically with SIRT1 in vivo and inhibition of SIRT1 activity leads to a recovery from the intrinsic repressive activity of SHP but not of DAX1. Furthermore, we observed that SIRT1 does not deacetylate SHP or LRH1. However, inhibition of either SIRT1 or SHP significantly diminished the repressive effect of SHP on LRH1 transactivity. LRH1-mediated activation of CYP7A1 and SHP gene transcription was significantly repressed by both SHP and SIRT1 whereas inhibition of SIRT1 activity by inhibitors or dominant negative SIRT1 or knockdown of SHP led to a significant release of this inhibitory effect. ChIP assays revealed that SHP recruits SIRT1 on LRH1 target gene promoters and SIRT1 deacetylated template-dependent histone H3 and H4 to inhibit transcription of LRH1 target genes. Finally, we demonstrated that inhibition of SIRT1 activity significantly reversed SHP-mediated inhibition of bile-acid synthesis by LRH1 overexpression, thereby suggesting a novel mechanism of SHP-mediated inhibition of LRH1-dependent bile-acid homeostasis via recruitment of SIRT1 histone deacetylase protein.

Figure 1.

Utilization of differential repressive mechanism by SHP. Reporter assays (A–C) were performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with indicated reporter luciferase vectors (100 ng each) and expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with RA (1 µM; panel A, right) or E2 (10 nM; panel B, right) for 12 h followed by TSA (100 nM) or NAM (20 µM) treatments for further 12 h in the absence or presence of RA or E2. Cells were treated only with TSA or NAM (panel A, B; left, middle) for 12 h prior to the measurement of luciferase activity. (D) Effect of siRNAs of siSIRT1 and siHDAC1 on the expression of SIRT1 and HDAC1 respectively. HepG2 cells were transfected with pSuper- siSIRT1 or siHDAC1 or pSuper [control (–)] and cells were collected for RNA isolation, by RT–PCR analysis, 72 h post-transfection. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments.

Figure 2.

SHP interacts and colocalizes with SIRT1. (A and B) In vivo interaction of exogenous SHP with exogenous SIRT1, SIRT6 and SIRT7 (A). 293T cells were cotransfected with Flag–SIRT1 (panel A, left), Flag–SIRT6 or Flag–SIRT7 (panel A, right) and pEBG–SHP (GST–SHP) or with pEBG (GST) alone. The complex formation (GST purification) and the amount of Flag–SIRT1, Flag–SIRT6 or Flag–SIRT7 used for in vivo binding assay (cell lysate) were determined by western blot using indicated antibodies. In vivo interaction of endogenous SIRT1 with endogenous SHP (B). Co-immunoprecipitation assays were performed with cell extracts from HepG2 cells (panel B, left) and C57BL/6J mouse liver tissue extracts (panel B, right). Endogenous SIRT1 or endogenous SHP was immunoprecipitated with SHP or SIRT1 respectively, and were analyzed by western blot using indicated antibodies. (C) Colocalization of SHP with SIRT1. HeLa cells grown on coverslips in 12-well plates were transfected with expression vectors encoding GFP–SHP and Flag–SIRT1 (200 ng each). For the immunofluorescence of fixed cells, Flag–SIRT1 protein was immunostained with mouse monoclonal anti-Flag antibody and visualized with dye Alexa Fluor 488-conjugated anti-mouse antibody. The cell images were captured under 400× magnifications. Data is representative of atleast three independently performed experiments.

Figure 3.

Interaction domains of SIRT1 and SHP. Schematic representation of structure of SIRT1 (A, top) and SHP protein (B, top) with amino-acid numbers indicated. HepG2 cells were co-transfected with Flag–SIRT1 mutants and pEBG alone or pEBG–SHP (A and C) or with pEBG alone or pEBG–SHP mutants and Flag–SIRT1 (B and D) as indicated. Protein interactions were examined via in vivo GST pull-down assay. The top and middle panels (GST purification) show GST beads-precipitated Flag–SIRT1 and GST fusions, respectively. The bottom panel shows the protein expression levels of Flag–SIRT1 in cell lysates. Data is representative of atleast three independently performed experiments.

Figure 4.

SHP recruits SIRT1 to inhibit LRH1 transactivation. Reporter assays (A, C and G) were performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with indicated expression vectors (200 ng each) and Gal4–Luc (A and C) or Sft4–Luc (G) luciferase reporter vector (100 ng). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with NAM (20 µM), resveratrol (100 nM) or piceatannol (20 µM) for 12 h prior to the measurement of luficerase activity. Data is representative of atleast three independently performed experiments and shown as mean ± SD; *P < 0.05 using Student’s t-test. (B, D and F) HepG2 cells were cotransfected with Flag–SHP and Myc–SIRT1 (B) or HA–LRH1 (D), HA–HNF4α (F) and Flag–SIRT1. Forty-eight hours post transfection, Flag–SHP, HA–LRH1 or HA–HNF4α were immunoprecipitated and analyzed by western blot analysis using indicated antibodies. Data is representative of atleast three independently performed experiments. (E) In vitro GST pull-down assays. ³⁵S-radiolabeled SIRT1 protein (upper panel) or ³⁵S-radiolabeled SHP protein (lower panel) were incubated with GST, or GST–LRH1 fusion proteins. The input lane represents 10% of the total volume of in vitro-translated proteins used for binding assay. Protein interactions were detected via autoradiography.

Figure 5.

Involvement of SHP and SIRT1 in the repression of LRH1 target genes. Reporter assay (A and B) was performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with luciferase constructs of human CYP7A1 (hCYP7A1–Luc) and human SHP (hSHP–Luc) gene promoters (100 ng each) and indicated expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with NAM (20 µM) or resveratrol (Resv, 100 nM) for 12 h prior to the measurement of luficerase activity. (C) HepG2 cells were transfected with pcDNA3 only (–) or pcDNA3–HA–LRH1 (400 ng each) or infected with indicated adenovirus vectors (50 MOI) for 36–72 h and cells extracts were for western blot analysis using indicated antibodies. (D and E) HepG2 cells were treated with indicated adenovirus vectors (50 MOI) for 36–72 h. Twelve hours prior to RNA extraction cells were treated with NAM (20 µM, lane 3 and 6) and total RNA isolated were used for RT–PCR (top) and qPCR (bottom) analysis of CYP7A1 and SHP gene expression. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments and shown as mean ± SD; *P < 0.05 using Student’s t-test.

Figure 6.

SHP recruits SIRT1 deacetylase to inhibit LRH1-mediated target gene activation. (A) The recruitment of SIRT1 by SHP on CYP7A1 (left) and SHP (right) gene promoters is associated with template-associated histone (H3 and H4) deacetylation. HepG2 cells were treated with indicated adenovirus vectors (50 MOI) for 36–72 h. Twelve hours prior to preparation of cell lysates for ChIP assay cells were treated with NAM (20 µM). Chromatin fragments were prepared and immumoprecipitated with the indicated specific antibodies. DNA fragments covering BARE-I and BARE-II element on CYP7A1 (left) and LRH1-binding regions on SHP promoter (right) were PCR-amplified as described in the ‘Materials and Methods’ section. (B) HepG2 cells were infected with adenovirus vectors as indicated for 36–72 h and for the last 12 h cells were treated with NAM (20 µM) as indicated. Media was collected for total bile synthesis using Sep-Pak cartridges as described in the ‘Materials and Methods’ section. Data is representative of atleast three independently performed experiments and shown as mean ± SD; *P < 0.05 using Student’s t-test. (C) HepG2 cells were infected with adenovirus vectors as indicated for 36-72 h and for the last 12 h cells were treated with BA (50 µM) as indicated and total RNA isolated were used for RT–PCR (top) and qPCR (bottom) analysis of CYP7A1 mRNA level. β-actin (ACTB) gene expression was shown as control. Data is representative of atleast three independently performed experiments and shown as mean ± SD; *P < 0.05 versus untreated, **P < 0.05 versus BA treatment and ^#P < 0.05 versus adenovirus infected lanes alone, using Student’s t-test. (D) Schematic representation of an auto-regulatory loop controlling the expression of SHP by LRH1 and inhibition of LRH1 activity by SHP itself via recruitment of SIRT1 histone deacetylase activity on target gene promoters of LRH1.