The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathione S-transferase-TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region-the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (*18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, *30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a sixtransmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA⅐TBP⅐TATA complex formation and transcription activation in vivo. We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA ␣␤ reduced TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAF II 250) phosphorylated TFIIA on the ␤ subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF II 250 phosphorylation in vitro. The NH 2 -terminal kinase domain of TAF II 250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF II 250 interaction or tumor suppressor activity. TFIIA phosphorylation had little effect on the TFIIA⅐TBP⅐TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a ␥ subunit Y65A mutation strongly stimulated TFIIA⅐TBP⅐TATA complex formation. TFIIA-␥Y65A is defective for binding to the ␤-sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA⅐TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure. TFIIA1 was identified originally as an activity required for optimal in vitro transcription with HeLa cell nuclear extracts and various viral and cellular templates (1) (reviewed in Refs. 2-5). TFIIA was isolated as three polypeptides from human and Drosophila embryo extracts, and the two largest subunits are proteolytic cleavage products of a single open reading frame (6 -13). TFIIA could be purified as a TATA-binding protein (TBP)-associated factor, although it could also be found as a chromatographically separate complex from TBP (7). In addition to binding directly to TBP, TFIIA stabilized the binding of TBP with TATA DNA in electrophoretic mobility shift assays (EMSA) (14, 15). In transcription reactions reconstituted with partially purified general factors and coactivators, TFIIA is essential for activator-mediated transcription and has a weak stimulatory effect on basal transcription lacking exogenous activators. In transcription reactions reconstituted with recombinant and affinity-purified general factors and coactivato...