Cloning and Characterization of Mouse RIP140, a Corepressor for Nuclear Orphan Receptor TR2

Nuclear receptors regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (19, 45). Recently, different coactivators and corepressors for several nuclear receptors have been identified (16). For instance, the nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) have been shown to function as corepressors for retinoic acid (RA) receptor α (RARα), thyroid hormone receptors (T3Rs), and orphan receptor COUP-TFI (4, 15, 36). The newly identified nuclear protein Nab1 is a corepressor for the orphan receptor NGFI-A family (38), and SUN-CoR is able to potentiate transcriptional repression by T3Rs and RevErb (44). In the coactivator category, the transcriptional mediator/intermediary factor 2 and steroid receptor coactivator 1 (SRC-1) are known to mediate transcriptional activation of RAR, estrogen receptor (ER), retinoid X receptor (RXR), and T3Rs (8, 14, 33, 39, 42). With respect to the working mechanism of corepressors and coactivators, it has been demonstrated that their actions involve the alteration of chromatin structure, such as the acetylation status of histone proteins (13, 34, 37). The orphan receptor TR2 belongs to the superfamily of nuclear receptors (21, 31). This receptor gene is expressed most abundantly in the testes of adult animals, particularly the developing germ cell populations (22, 24, 25). During embryonic stages, TR2 expression is highest between embryonic day 8 (E8) and E12 (22). It is also known that this gene encodes two isoform receptors, one retaining the entire ligand binding domain (LBD) and the other truncated at the LBD, which exhibit differential expression patterns in developing testes (24). The biological function of the full-length TR2 has been examined in a variety of systems, such as the cellular RA binding protein I promoter containing a direct repeat 4 (DR4) element (6), a RAR-responsive element (RARE) of the DR5 type from RARβ (24, 30), a DR2 from the simian virus 40 (SV40) promoter (26), and a DR2 from the erythropoietin gene promoter (27). In all tested systems, the full-length TR2 consistently represses reporter gene expression in cell cultures supplemented with either regular serum or charcoal-depleted serum. In contrast, no specific biological activity has been detected for the LBD-truncated TR2 isoform in these reporter gene systems. To understand the molecular mechanisms of TR2 functions and identify the associate proteins for TR2, we performed yeast two-hybrid screening experiments using the LBD of TR2 as the bait. Based on the expression pattern of TR2, we constructed two cDNA libraries for the screening, one prepared from poly(A) RNA of mouse embryos at E11.5 and E12.5 and another from poly(A) RNA of adult mouse testes. By screening these libraries, we isolated and characterized one strongly interacting clone which appeared to encode the mouse homologue of the human receptor-interacting protein 140 (hRIP140), a coactivator for ligand-activated receptors such as RAR, ER, vitamin D receptor, T3Rβ1, and androgen receptor (3, 8, 18, 20, 29, 32). However, the cloned mouse RIP140 (mRIP140) functioned as a corepressor for mouse TR2 and exerted a trans-repressive activity when it was tethered to the DNA binding domain (BD) of GAL4. In this study, we report the cloning and characterization of mRIP140, demonstrate its interaction with TR2 both in vitro and in vivo, and provide evidence for a corepressor activity of RIP140 for orphan receptor TR2.