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...
Tcm (total cataract with microphthalmia) is an autosomal dominant mouse eye mutation. Heterozygous Tcm/+ mice are born with several eye malformations including microphthalmia, retinal and iris dysplasia, total lens cataract, and ventral coloboma. The Tcm mutation was previously mapped to a 26-Mb region on Chr 4 between D4Mit235 and D4Mit106. In this study, we characterize the Tcm/ Tcm homozygous mutant and find they are viable but severely microphthalmic. The developing eye in the Tcm/Tcm homozygote shows defects during early eye development, before formation of the optic cup. Further genetic mapping reduced the Tcm critical region to a 1.3-Mb region bordered by SNPs rs3666764 and rs3713818. This critical region contains two known genes (Asph and Gfd6) and three predicted genes, all of which are positional candidates for Tcm. Sequence analysis of Tcm genomic DNA revealed no mutations in the coding regions and splice site junctions of the five candidate genes. These results indicate that the causitive Tcm mutation falls within a noncoding regulatory region of one of the five candidate genes or in an undescribed gene.
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