LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved.

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni.

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Regulation of XMyoDa transcription by the binding of XMEF2A to the TATA box

MEF2 is a $\underline{\rm m}$yocyte-specific $\underline{\rm e}$nhancer-binding $\underline{\rm f}$actor that binds a conserved DNA sequence, CTA(A/T)$\sb4$TAG. A MEF2 binding site in the XMyoDa promoter overlaps with the TATA box and is required for muscle specific expression. To examine the potential role of MEF2 in the regulation of MyoD transcription during early development, the appearance of MEF2 binding activity in developing Xenopus embryos was analyzed with the electrophoretic mobility shift assay. Two genes were isolated from a X. Laevis stage 24 cDNA library that encode factors that bind the XMyoDa TFIID/MEF2 site. Both genes are highly homologous to each other, belong to the MADS ($\underline{\rm M}$CM1-$\underline{\rm A}$rg80-agamous-$\underline{\rm d}$eficiens-$\underline{\rm S}$RF) protein family, and most highly related to the mammalian MEF2A gene, hence they are designated as XMEF2A1 and XMEF2A2. Proteins encoded by both cDNAs form specific complexes with the MEF2 binding site and show the same binding specificity as the endogenous MEF2 binding activity. XMEF2A transcripts accumulate preferentially in developing somites after the appearance of XMyoD transcripts. XMEF2 protein begins to accumulate in somites at tailbud stages. Transcriptional activation of XMyoD promoter by XMEF2A required only the MADS box and MEF2-specific domain when XMEF2A is bound at the TATA box. However, a different downstream transactivation domain was required when XMEF2A activates transcription through binding to multiple upstream sites. These results suggest that different activation mechanisms are involved, depending on where the factor is bound. Mutations in several basic amino acid clusters in the MADS box inhibit DNA binding suggesting these amino acids are essential for DNA binding. Mutation of Thr-20 and Ser-36 to the negatively charged amino acid residue, aspartic acid, abolish DNA binding. XMEF2A activity may be regulated by phosphorylation of these amino acids. A dominant negative mutant was made by mutating one of the basic amino acid clusters and deleting the downstream transactivation domain. In vivo roles of MEF2 in the regulation of MyoD transcription were investigated by overexpression of wild type MEF2 and dominant negative mutant of XMEF2A in animal caps and assaying for the effects on the level of expression of MyoD genes. Overexpression of MEF2 activates the transcription of endogenous MyoD gene family while expression of a dominant negative mutant reduces the level of transcription of XMRF4 and myogenin genes. These results suggest that MEF2 is downstream of MyoD and Myf5 and that MEF2 is involved in maintaining and amplifying expression of MyoD and Myf5. MEF2 is upstream of MRF4 and myogenin and plays a role in activating their expression. ^

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

The importin/karyopherin Kap114 mediates the nuclear import of TATA-binding protein

Two high copy suppressors of temperature-sensitive TATA-binding protein (TBP) mutants were isolated. One suppressor was TIF51A, which encodes eukaryotic translation initiation factor 5A. The other high copy suppressor, YGL241W, also known as KAP114, is one of 14 importin/karyopherin proteins in yeast. These proteins mediate the transport of specific macromolecules into and out of the nucleus. Cells lacking Kap114 partially mislocalize TBP to the cytoplasm. Kap114 binds TBP in vitro, and binding is disrupted in the presence of GTPγS. Therefore, Kap114 is an importer of TBP into the nucleus, but alternative import pathways must also exist.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP’s ability to occlude its DNA binding domain through dimerization. Using a “pull-down” based assay, we find that TBP dimers dissociate slowly (t½ = 6–10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.

Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells

The TATA-binding protein (TBP)-related factor TRF1, has been described in Drosophila and a related protein, TRF2, has been found in a variety of higher eukaryotes. We report that human (h)TRF2 is encoded by two mRNAs with common protein coding but distinct 5′ nontranslated regions. One mRNA is expressed ubiquitously (hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted expression pattern and is extremely abundant in testis. In addition, we show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2. Neither recombinant human (rh)TRF2 nor the native flag⋅hTRF2-TFIIA complex is able to replace TBP or TFIID in basal or activated transcription from various RNA polymerase II promoters. Instead, rhTRF2, but not the flag⋅hTRF2–TFIIA complex, moderately inhibits basal or activated transcription in the presence of rhTBP or flag⋅TFIID. This effect is either completely (TBP-mediated transcription) or partially (TFIID-mediated transcription) counteracted by addition of free TFIIA. Neither rhTRF2 nor flag⋅hTRF2–TFIIA has any effect on the repression of TFIID-mediated transcription by negative cofactor-2 (NC2) and neither substitutes for TBP in RNA polymerase III-mediated transcription.