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. 2009 Mar 20;284(12):8127-35.
doi: 10.1074/jbc.M808815200. Epub 2009 Jan 13.

SUG-1 plays proteolytic and non-proteolytic roles in the control of retinoic acid target genes via its interaction with SRC-3

Christine Ferry  1 , Maurizio Gianni, Sébastien Lalevée, Nathalie Bruck, Jean-Luc Plassat, Ivan Raska Jr, Enrico Garattini, Cécile Rochette-Egly
Affiliations

SUG-1 plays proteolytic and non-proteolytic roles in the control of retinoic acid target genes via its interaction with SRC-3

Christine Ferry et al. J Biol Chem. .

Abstract

Nuclear retinoic acid receptor alpha (RARalpha) activates gene expression through dynamic interactions with coregulatory protein complexes, the assembly of which is directed by the ligand and the AF-2 domain of RARalpha. Then RARalpha and its coactivator SRC-3 are degraded by the proteasome. Recently it has emerged that the proteasome also plays a key role in RARalpha-mediated transcription. Here we show that SUG-1, one of the six ATPases of the 19 S regulatory complex of the 26 S proteasome, interacts with SRC-3, is recruited at the promoters of retinoic acid (RA) target genes, and thereby participates to their transcription. In addition, SUG-1 also mediates the proteasomal degradation of SRC-3. However, when present in excess amounts, SUG-1 blocks the activation of RARalpha target genes and the degradation of RARalpha that occurs in response to RA, via its ability to interfere with the recruitment of SRC-3 and other coregulators at the AF-2 domain of RARalpha. We propose a model in which the ratio between SUG-1 and SRC-3 is crucial for the control of RARalpha functioning. This study provides new insights into how SUG-1 has a unique role in linking the transcription and degradation processes via its ability to interact with SRC-3.

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Figures

FIGURE 1.
FIGURE 1.
SUG-1 interacts with SRC-3 and contributes to RA-induced degradation of SRC-3. A, in MCF7 and HeLa cells, MG132 reverses the RA-induced degradation of SRC-3 and RARα (16 h). B, COS-1 cells were transfected with the FLAG-SRC-3 vector in the absence or presence of SUG-1 and treated or not with RA (1 h). Nuclear extracts were immunoprecipitated with FLAG antibodies and analyzed by immunoblotting. The two upper panels correspond to aliquots (10%) of unprecipitated extracts. C, in MCF7 cells, SUG-1 interacts with SRC-3 in coimmunoprecipitation experiments performed with SRC-3 antibodies. D, immobilized GST and GST-SUG-1 proteins were incubated with extracts from COS-1 cells overexpressing SRC-3. Bound SRC-3 was analyzed by immunoblotting. Lane 1 corresponds to 5% of the loaded material. E, MCF7 and HeLa cells were transfected with control or SUG-1 SMARTpool siRNA (50 nm) and RA-treated for 16 h. Knockdown of SUG-1 was analyzed by immunoblotting as well as the expression and degradation of SRC-3 and RARα. *, a nonspecific band recognized by the RARα antibodies.
FIGURE 2.
FIGURE 2.
Knockdown of SUG-1 inhibits the activation of RA target genes. A, the RA-induced expression of the endogenous Cyp26A1 gene was measured by qRT-PCR in MCF7 cells transfected with control, SUG-1, SRC-3, or SRC-2 SMARTpool siRNAs. The results are an average of two experiments that agreed within 15%. B, the efficiency and specificity of the knockdown was checked by immunoblotting. C, in MCF7 cells, knockdown of SRC-3 with SMARTpool siRNAs does not affect the RA-induced degradation of RARα. D, MCF7 cell knockdown for SUG-1, SRC-3, or SRC-2, was cotransfected with a DR5-tk-Luc reporter gene, treated with RA, and analyzed for Luciferase activity. The values are the mean ± S.D. of triplicate experiments. E, in HeLa cells, knockdown of SUG-1 with SMARTpool siRNA blocks the RA-induced expression of the endogenous RARα2 gene, measured by qRT-PCR. The values are the mean ± S.D. of triplicate experiments.
FIGURE 3.
FIGURE 3.
In vivo, SUG-1 is co-recruited with SRC-3 to the promoter of RARα target genes. A, schematic representation of the promoter regions of the Cyp26A1 gene with the primer pairs used for their amplification. B and C, kinetic ChIP experiments performed with RA-treated MCF7 cells and determining the recruitment of RARα, SRC-3, and SUG-1 to the R1 and R2 regions. Values are expressed as -fold enrichment relative to untreated cells and correspond to a representative experiment among 3. D and E, Re-ChIP experiments performed with the indicated antibodies, showing that, at 60 min following RA addition, DNA-bound SRC-3 and RARα are associated with SUG-1. Values are expressed as -fold enrichment relative to control Re-ChIP experiments and are the mean ± S.D. of triplicate experiments. F, ChIP-Western experiments performed with MCF7 cells treated or not with RA for 60 min. The complexes immunoprecipitated with SRC-3 antibodies were analyzed by immunoblotting as indicated. Lane 1 corresponds to aliquots (10%) of unprecipitated extracts. G, kinetic ChIP experiments determining the recruitment of RARα, SRC-3, and SUG-1 to the RARβ2 promoter as in A. The promoter with the DR5 RARE is also shown.
FIGURE 4.
FIGURE 4.
In MCF7 cells, overexpression of SUG-1 blocks the transcription of RARα target genes and the degradation of RARα. A, MCF7 cells were transfected with a vector encoding SUG-1 and were RA-treated. SRC-3 and RARα degradation as well as the efficiency of SUG-1 overexpression were analyzed by immunoblotting. B, overexpression of SUG-1 blocks the RA-induced expression of a DR5-tk luciferase reporter gene. The values are the mean ± S.D. of triplicate experiments. The efficiency of SUG-1 overexpression is shown in the right panel. C and D, overexpression of SUG-1 reduces the RA-induced expression of Cyp26A1, as assessed by qRT-PCR. The results are the average of three experiments that agreed within 15%. E and F, in MCF7 cell knockdown for SUG-1, re-expression of SUG-1 in excess amounts blocks the degradation of RARα and the expression of a DR5-tk-Luc reporter gene. The values are the mean ± S.D. of three independent experiments.
FIGURE 5.
FIGURE 5.
SUG-1 interferes with SRC-3 for binding to RARα. A, SUG-1 and SRC-3 interact with the same helix 12 of RARα. Nuclear extracts from COS-1 cells transfected with an expression vector for RARα (WT or ΔH12) along with SUG-1 or FLAG-SRC-3 were RA-treated (1 h) and immunoprecipitated with RARα monoclonal antibodies, followed by immunoblotting. Lanes 1 and 4 correspond to 5% of the amount of immunoprecipitated extracts. B and C, overexpression of SUG-1 blocks the interaction of RARα with FLAG-SRC-3 in cotransfected COS cells. Coimmunoprecipitations were performed with either RARα (B) or FLAG (C) antibodies and analyzed by immunoblotting. The upper panels correspond to aliquots (10%) of unprecipitated extracts.
FIGURE 6.
FIGURE 6.
Overexpression of SRC-3 reverses the inhibitory effects of SUG-1 on RARα degradation and RARα-mediated transcription. A, in COS-1 cells cotransfected with RARα and SUG-1 expression vectors and RA-treated for 1 h, overexpression of SRC-3 reverses the interaction of SUG-1 with RARα. Nuclear extracts were immunoprecipitated with RARα monoclonal antibodies and analyzed by immunoblotting. The upper panels correspond to aliquots of unprecipitated extracts. B and C, COS-1 cells were cotransfected with the RARα vector and the DR5-tk-LUC reporter construct, along with SUG-1 and/or FLAG-SRC-3 and RA-treated. Extracts were analyzed by immunoblotting (B) and for luciferase activity (C). The results are the mean ± S.D. of at least three independent experiments. D and E, the same as in B and C but with SRC-1 instead of FLAG-SRC-3.
FIGURE 7.
FIGURE 7.
Model recapitulating the roles played by SUG-1 in the control of RARα target genes. A, in response to RA, SUG-1 interacts with SRC-3 and thereby fine-tunes RARα-mediated transcription. It also contributes to the proteasomal degradation of SRC-3. B, when present in excess amounts, SUG-1 accelerates the degradation of SRC-3. It also blocks the AF-2 domain of RARα and impedes the recruitment of SRC-3 and of the transcription and degradation machineries.
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