Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 32 (6), 1957-66

Multiple Domains of the Receptor-Interacting Protein 140 Contribute to Transcription Inhibition

Affiliations

Multiple Domains of the Receptor-Interacting Protein 140 Contribute to Transcription Inhibition

Audrey Castet et al. Nucleic Acids Res.

Abstract

In this study, we have investigated the role of C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) in the repressive activity of the nuclear receptor cofactor Receptor-Interacting Protein 140 (RIP140). We have defined the interaction of both CtBP1 and CtBP2 with RIP140 and delineated two motifs (PIDLS and PINLS) differentially required for in vitro interaction. Using different approaches (titration of endogenous CtBPs, mutagenesis and transfection in CtBP knock-out cells), we find that recruitment of CtBPs only partially explains the negative regulation exerted by RIP140. We then demonstrate that RIP140 associates in vitro not only with class I HDACs but also with class II enzymes such as HDAC5. This interaction mainly involves the N-terminus of RIP140 (residues 27-199) and two domains of HDAC5. Moreover, the two proteins functionally interfere in transfection experiments, and confocal microscopy indicates that they co-localize in the nucleus. Interestingly, using the specific HDAC inhibitor trichostatin A, we show that HDAC activity is dispensable for active transrepression by RIP140. Finally, we demonstrate that the C-terminal region of RIP140 contains two additional silencing domains and confers strong active transrepression independently of HDAC activity and CtBPs. Altogether, these data indicate that transcriptional inhibition by the cofactor RIP140 involves complex mechanisms relying on multiple domains and partners.

Figures

Figure 1
Figure 1
Intrinsic repression activity of RIP140. HeLa cells were transiently transfected with the L8G5-Luc reporter construct (0.4 µg) together with the expression vector for LexA-VP16 (0.2 µg) and increasing concentrations of Gal4, Gal-RIP or Gal-SHP (0.25, 0.5 and 1 µg). Luciferase activity was quantified as indicated in Materials and Methods. Results are expressed relative to control and are the mean (±SD) of three independent experiments.
Figure 2
Figure 2
In vitro interaction of RIP140 with CtBPs. (A) GST pull-down assays were carried out as described in Materials and Methods using bacterially expressed GST–CtBP1 or GST–CtBP2 proteins to retain 35S-labelled RIP140. (B) GST–RIP140 proteins containing different regions of the molecule, i.e. GST–RIP(27–439), GST–RIP(429–739) or GST–RIP(683–1158), were tested for their interaction with 35S-labelled CtBP1 and CtBP2. (C) GST pull-down assays were performed to precipitate 35S-labelled CtBP1 with the GST–RIP(429–582) protein containing the wild-type or mutated sequences for the PIDLS (mutPIDLS), PINLS (mutPINLS) or both (mutPID/NLS) sites. (D) GST–CtBP1 or GST–CtBP2 proteins were used to pull-down radiolabelled RIP140 either wild type or mutated for the PIDLS (mutPIDLS), PINLS (mutPINLS) or both sites (mutPID/NLS). Results are expressed as a percentage of the binding of the wild-type RIP140.
Figure 3
Figure 3
Role of CtBPs in transcriptional repression by RIP140. (A) A western blot experiment using an antibody specific for CtBP1 and CtBP2 was performed as described in Materials and Methods. Cell lines from various tissues were analysed. MCF-7 and T47-D are derived from ERα-positive breast carcinoma, whereas MDA-MB231, MDA-MB435 or MDA-MB 436 are from ERα-negative breast carcinoma. Ishikawa (Ishi), RL-95 and HEC1A are from endometrium, HeLa cells from a cervix carcinoma, and CV-1 and COS-1 from African green monkey kidney. (B) Transient transfections of HeLa cells were performed using Lipofectamine 2000 reagent as described in Materials and Methods. The ERE-βGlob-Luc reporter (125 ng) and ERα (50 ng) expression plasmid were transfected together with either the vectors encoding RIP140 wild type, mutated for the PIDLS and PINLS sites (mutPID/NLS), or the empty vector only (750 ng). Relative luciferase activity was measured 24 h after the treatment of cells with 17β-estradiol (E2, 10–8 M) or vehicle alone and is expressed as a percentage of control in the presence of E2. (C) HeLa cells were transiently transfected with the 17M5βGlob-Luc reporter construct (1 µg) together with 1.5 µg of Gal-RIP wild type (black box) or mutated for the two motifs PIDLS and PINLS (Gal-RIPmutPID/NLS, grey box) versus empty vector alone (white box). Luciferase activity was measured 48 h after transfection and is expressed as a percentage of control with Gal4 alone. Results are the mean (±SD) of three values obtained in three independent experiments. (D) HeLa cells were transiently transfected with the pSV40G5E1BLuc reporter (1 µg) together with 1 µg of Gal-RIP plasmid (black boxes) or empty vector (white boxes) in the presence or absence of the pcDNA3-dl1119 plasmid (2.5 µg) allowing the expression of the E1A exon 2 (E1A-CID). Results are expressed as relative luciferase activity (% of control) and are the mean (±SD) of six values from two independent experiments.
Figure 4
Figure 4
Role of CtBPs in transcriptional repression by RIP140. (A) MEFs (either CtBP+/– or CtBP–/–) were transiently transfected with the ERE-βGlob-Luc reporter (0.5 µg) together with the ERα (0.2 µg) and RIP140 (1.8 µg) expression plasmid (black boxes) or empty vector (white boxes) in the presence of 17β-estradiol (E2) or 4-hydroxytamoxifen (OHTam, 10–8 M). Results are expressed as raw luciferase activity and are the mean (±SD) of six values from two independent experiments. The level of expression of CtBPs assessed by western blot analysis in the two types of MEFs is also shown. (B) Empty vector (1 µg), Gal-RIP full-length construct and its central part comprised between residues 429 and 739 were transiently transfected in CtBP+/– (white boxes) or CtBP–/– (black boxes) MEFs together with the L8G5-Luc reporter and the LexA-VP16 activator plasmids. Cells were transfected using Lipofectamine 2000 reagent. Results are expressed as relative luciferase activity (percentage of control Gal4) and are the mean of three values. (C) Transient transfections were realized in CtBP–/– MEFs using Lipofectamine 2000 reagent. Gal4, Gal-RIP or Gal-RIP(429–739) expression plasmids (1 µg) have been transfected together with the L8G5-Luc reporter and the LexA-VP16 activator plasmids in the presence or absence of CtBP1 or CtBP2. Luciferase activity which represents the mean (±SD) of three values was measured 48 h after the transfection and is expressed as a percentage of each control without RIP140.
Figure 5
Figure 5
RIP140 recruits both class I and II HDACs. (A) The GST–HDAC1 and GST–HDAC2 fusion proteins were tested for their ability to interact with radiolabelled RIP140 in GST pull-down experiments. (B and C) Various bacterially expressed GST–RIP140 mutants were analysed for their interaction with [35S]HDAC5. (D) Interaction between different GST–HDAC5 mutants and 35S-labelled RIP140, either full-length or C- or N-terminus deleted. Inputs represent 10% of the material used in the assays.
Figure 6
Figure 6
RIP140 and HDAC5 co-localize and functionally interact. (A) CFP–HDAC5 and YFP–RIP140 expression plasmids were co-transfected in COS-1 cells using Lipofectamine 2000 reagent. Fluorescence signals were detected independently or simultaneously (merge) with a Leica sp2 confocal microscope. (B) HeLa cells were transiently transfected with various expressing plasmids encoding Gal-HDAC5 domains (0.25 µg), together with the L8G5-Luc reporter and the LexA-VP16 activator plasmids, in the presence or absence of the expression vector for RIP140 (1.5 µg).
Figure 7
Figure 7
HDAC inhibition does not relieve RIP140 transcription repression. (A) GST pull-down assays were performed as described in Materials and Methods by incubating GST–HDAC1, 2 and 5 (encompassing residues 674–1113) or GST alone, together with radiolabelled RIP140 in the presence or absence of TSA (5 mg/ml). (B) HeLa cells were transiently transfected with the L8G5-Luc reporter construct together with the expression vector for LexA-VP16, Gal-RIP, Gal-RIP(27–199), Gal-RIP(429–739) or empty vector Gal4 alone (2 µg). Cells were then treated or not by TSA (500 ng/ml) for 24 h and luciferase activity was quantified as indicated in Materials and Methods. Results are expressed as relative luciferase activity and are the mean (±SD) of three values. (C) HeLa cells were transfected with the ERE-βGlob-Luc or ERE-TK-Luc reporter constructs (0.5 µg) together with the ERα-encoding plasmid and the expression vector for RIP140 (black boxes) or empty vector alone (white boxes). Luciferase activity was quantified as indicated in Materials and Methods after 24 h of stimulation by estradiol together with increasing concentrations of TSA. Results are expressed as raw luciferase activity and are the mean (±SD) of three values.
Figure 8
Figure 8
Two independent domains of the C-terminal region of RIP140 mediate transcriptional repression. (A) HeLa cells were transfected with the L8G5-Luc reporter and LexA-VP16, together with increasing amounts of Gal-RIP, Gal-RIP(753–1158) or empty Gal4 vectors (0.5, 1 and 2 µg). Luciferase activity was quantified 48 h after transfection, and the results, expressed as a percentage of control, are the mean (±SD) of three values. (B) MCF-7 cells (black boxes) and Jurkat T cells (grey boxes) were transfected with the pSV40G5E1BLuc reporter construct (1 µg) together with 1.5 µg of Gal-RIP, Gal-RIP(753–1158) mutant or Gal4 vector alone. Results are expressed as relative luciferase activity (% of control Gal4) and are the mean (±SD) of six values. (C) Gal-RIP(753–1158) or empty vector was transfected together with the L8G5 reporter and the LexA-VP16 in either CtBP+/– or CtBP–/– cells, using Lipofectamine 2000 reagent (left panel) or in HeLa cells (right panel). Relative luciferase activity was measured 24 h after TSA treatment (500 ng/ml) in HeLa cells and 48 h after transfection for MEFs. Results are expressed as a percentage of Gal4 activity. (D) HeLa cells were transiently transfected with the 17M5βGlob-Luc reporter construct (1 µg) together with the indicated Gal-RIP mutants (3 µg). Results are expressed as relative luciferase activity (% of control Gal4) and are the mean (±SD) of three values.
Figure 9
Figure 9
Schematic representation of the different silencing domains identified in RIP140. The position of each domain is indicated with the corresponding amino acid coordinates. The preferential binding of HDAC and CtBP on domains 1 and 2 is also shown, together with their sensitivity to TSA.

Similar articles

See all similar articles

Cited by 27 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback