Interaction between the Retinoid X Receptor and Transcription Factor IIB Is Ligand-dependent in Vivo

Abstract

The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR␤ (mRXR␤) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR␤ with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR␤ (amino acids (aa) 254 -389) and two regions in the carboxyl region of TFIIB (aa 178 -201 and aa 238 -271). Furthermore, the ⌬390 -410 mRXR␤ mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR␤ with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.Nuclear hormone receptors are ligand-dependent transcription factors that regulate numerous cellular functions, including growth, development, differentiation, reproduction, and metabolism (Refs. 1-3 and references therein). Ligand binding to nuclear hormone receptors leads to assembly of a functional preinitiation complex (PIC) 1 and subsequent RNA transcription in vitro (4, 5). Activated transcription of RNA polymerase II (RNAPII) genes requires binding of transcription factor IID (TFIID) to the TATA element, which is facilitated by TFIIA, and stepwise assembly of the following transcription factors: TFIIB, RNAPII/TFIIF, TFIIE, and TFIIH (reviewed in Refs. 6 and 7). TFIID includes the TATA binding protein (TBP) associated factors (TAFs) (Ref.7, and references therein). Interaction of nuclear receptors with the basal transcription complex may occur directly (8 -14) or in association with TAFs (15) or other cofactors (Refs. 16 -25; reviewed in Refs. 26 and 27). Ligand binding to the receptor results in recruitment of factors that facilitate assembly of an activated complex (28 -30) or, conversely, the release of repressor molecules, such as SMRT (16) or N-CoR (17, 18) that inhibit transcription (16 -18, 31-32). A significant advance was the demonstration that TFIIB interacts with the estrogen receptor (10), progesterone receptor (10), chicken ovalbumin upstream promoter-transcription factor (10), thyroid hormone receptor (TR) (11, 31), and vitamin D receptor (VDR) (12)(13)(14). The interaction of nuclear hormone receptors with TFIIB is significant since recruitment of TFIIB to the PIC by transcriptional activat...

Vitamin D Analogue-Specific Recruitment of Vitamin D Receptor Coactivators

“…(3,(7)(8)(9)(10) Coactivator recruitment and gene transactivation by NHRs is mediated through a highly conserved motif in the ligand-binding domain (LBD), the activation function-2 (AF-2) domain. Coactivators that interact with NHRs in a ligand-and AF-2-dependent manner include steroid receptor coactivator-1 (SRC-1, NcoA-1, or p160), glucocorticoid receptor interacting protein-1 (GRIP1, TIF2, NcoA-2, or SRC-2), and receptor-associated coactivator-3 (RAC3, pCIP, ACTR, AIB1, SRC-3, or TRAM-1).…”

Interactions of SKIP/NCoA-62, TFIIB, and Retinoid X Receptor with Vitamin D Receptor Helix H10 Residues

“…Residues Gln-380, Asp-384, and Arg-386 are in the ninth heptad repeat region, which is highly conserved among the nuclear receptors. Consistent with this conservation, TFIIB interacts with RXR␤ and TR␤ (14,28), and RXR␣ contacts PPAR␥ and RAR␣ (29,30) through heptad 9 residues analogous to mVDR Gln-380, Asp-384, and Arg-386. By extension, therefore, SKIP/NCoA-62 may also interact with nuclear receptors other than VDR via the helix H10 interface.…”

“…Full-length wild type cDNA was similarly mutated to produce mutant AX3. Constructs pACTIISKIP (amino acids 145-536) (12), pAC-TIITFIIB (14), pACTII␤-RXR␣, pGAD424-GRIP1 (nucleotides 204 -4878), pGAD10-RAC3 (nucleotides 1289 -3698), pSG5GRIP1, and pSG5RAC3 (15) have been described.…”

“…Immunoblot Analysis-Lysates were prepared from diploid yeast strains (14) and mammalian cells (17). Equal amounts of protein were resolved by SDS-PAGE (10%), blotted, and probed with 9A7 antibody (Affinity Bioreagents Inc.) to detect VDR and anti-HA tag antibody (Roche Molecular Biochemicals) to detect HA-SKIP.…”

“…Haploid SFY526 and Y187 strains were mated and diploid strains selected with tryptophan and leucine. Six independent colonies from each diploid strain were tested in ligand assays as described previously (14). Cell lysate protein content was determined (Bio-Rad), and ␤-galactosidase activity was assayed by chemiluminescence (Tropix).…”