Transcription factor TFIIB sites important for interaction with promoter-bound TFIID.

Transcription initiation factor TFIIB recruits RNA polymerase II to the promoter subsequent to interaction with a preformed TFIID-promoter complex. The domains of TFIIB required for binding to the TFIID-promoter complex and for transcription initiation have been determined. The carboxyl-terminal two-thirds of TFIIB, which contains two direct repeats and two basic residue repeats, is sufficient for interaction with the TFIID-promoter complex. An extra 84-residue amino-terminal region, with no obvious known structural motifs, is required for basal transcription activity. Basic residues within the second basic repeat of TFIIB are necessary for stable interaction with the TFIID-promoter complex, whereas the basic character of the first basic repeat is not. Functional roles of other potential structural motifs are discussed in light of the present study.

Unliganded thyroid hormone receptor alpha can target TATA-binding protein for transcriptional repression.

Unliganded human thyroid hormone receptor alpha (hTR alpha) can repress transcription by inhibiting the formation of a functional preinitiation complex (PIC) on promoters bearing thyroid hormone receptor (TR)-binding elements. Here we demonstrate that hTR alpha directly contacts the TATA-binding protein (TBP) and that preincubation of hTR alpha with TBP completely alleviates TR-mediated repression in vitro. Using stepwise preassembled PICs, we show that hTR alpha targets either the TBP/TFIIA or the TBP/TFIIA/TFIIB steps of PIC assembly for repression. We also show that the repression domain of hTR alpha maps to the C-terminal ligand-binding region and that direct TR-TBP interactions can be inhibited by thyroid hormone. Together, these results suggest a model in which unliganded hTR alpha contacts promoter-bound TBP and interferes with later steps in the initiation of transcription.

The Rpb6 subunit of fission yeast RNA polymerase II is a contact target of the transcription elongation factor TFIIS.

The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeast Schizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathione S-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

Identification of an enhancer responsible for tumor marker gene expression by means of transgenic rats.

The glutathione transferase P (GST-P) gene is known for its specific expression during chemical hepatocarcinogenesis of the rat and is used as a tumor marker for hepatocellular carcinoma. We have shown recently that the upstream 2.9-kb region of the GST-P gene is sufficient for conferring tumor-specific expression of the gene in vivo (S. Morimura et al., Proc. Natl. Acad. Sci. USA, 90: 2065-2068, 1993). To further identify crucial sequence elements regulating the unique expression of this gene, we have established six independent lines of transgenic rats bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase coding region and analyzed changes of the chloramphenicol acetyltransferase activity during the course of chemical hepatocarcinogenesis. We demonstrate here that the enhancer, glutathione transferase P enhancer I, that is located 2.5 kb upstream of the GST-P gene is required and sufficient for its tumor-specific expression of the gene among other controlling elements. This approach to transgene expression could be used to define other enhancers, the activity of which is dependent on cellular changes such as carcinogenesis, development, and differentiation.

Structure of the core promoter of human and mouse ribosomal RNA gene. Asymmetry of species-specific transcription.

In vitro transcription of the ribosomal RNA gene (rDNA) shows a remarkable species specificity such that human and mouse rDNA cannot use heterologous extracts of each other. The region that is responsible for this specificity has been studied using human-mouse chimeric genes and characteristic structures of both core promoters are presented. When the mouse sequence is substituted by the corresponding human sequence from upstream, the promoter activity in the mouse extract begins to decline at nucleotide -32 or -30, decreasing gradually and is lost completely at -19. A similar gradual decrease was noted for the 3' side substitution, which started at nucleotide -14 and was completed when up to the nucleotide -22 mouse position was replaced by the corresponding sequence from human. Thus, in the mouse rDNA core promoter, the sequence that is involved in species specificity resides only in a stretch encompassing the non-conserved region between the distal conserved sequence (DCS) and the proximal conserved sequence (PCS), plus two altered nucleotides in the PCS. When human rDNA is transcribed with human cell extract, the mouse sequence cannot substitute for the human sequence within the region from nucleotide -43 to +17 without affecting promoter activity significantly. This asymmetry of species specificity is due to the presence of nucleotides -43, +1 and +17, which are sensitive to change in only the human core promoter. The difference in the 5' border is ascribed to the species specificity of a transcription factor TFID, which recognizes this region. But the large difference of the 3' border is apparently due to another factor, possibly RNA polymerase I itself, because this region is not recognized by TFID in either human or mouse. Mammalian rDNA core promoter appears to consist of a tandem mosaic in which three evolutionarily conserved sequences alternate with non-conserved sequences having certain functionally important nucleotides. Not only non-conserved sequences and non-conserved nucleotides in conserved sequences, but also the spacings between the three conserved regions, play a crucial role in species specificity.

Insulin resistance in a patient with diabetes mellitus associated with Turner's syndrome.

We evaluated insulin resistance and assessed the effect of gliclazide on insulin resistance in a patient with diabetes mellitus associated with Turner's syndrome. Insulin-induced glucose metabolism markedly decreased compared with 12 healthy subjects. The insulin dose-response curve of this patient shifted to the right and down, and recovered somewhat after the administration of gliclazide. This patient had exhibited marked insulin resistance, which seemed to be caused by a defect at the receptor and/or post-receptor levels. Gliclazide reduced her insulin resistance, which suggests that this agent is suitable for treating the insulin resistance in diabetic patients with Turner's syndrome.

[Chronic myelomonocytic leukemia (CMMoL) with systemic lymph node swelling and paroxysmal nocturnal hemoglobinuria (PNH)-like complication].

A 37-year-old male was diagnosed as having chronic myelomonocytic leukemia (CMMoL) with chief complaint of systemic lymph node swelling. On admission, his peripheral blood revealed mild anemia and mild thrombocytopenia with giant platelets, and monocytosis (1480/microliters). NAP score was low. Serum lysozyme increased. The bone marrow showed normal cellularity consisting of 4% myeloblasts and 14.4% promyelocytes, and a few myeloid cells were positive for double staining by alpha-naphthyl butyrate and naphthol ASD chloroacetate esterase. Biopsied specimens of the cervical lymph node showed infiltration of monocytoid cells, which were positive for lysozyme staining, into interfollicular tissue. As for chromosome variation, 21 large satellite was observed in all dividing cells from his bone marrow and peripheral blood. Furthermore, hemolytic anemia with hemoglobinuria developed during his course. Sugar water test was positive, but Ham test negative. Coombs test and Donath-Landsteiner reaction were negative. Abnormal hemoglobin, spherocyte and fragmentation were not found. Hemolysis disappeared about two months later. However, blastic crisis appeared and he died. We showed a case of CMMoL with 21 large satellite and paroxysmal nocturnal hemoglobinuria (PNH)-like complication. Satellite have usually been reported as asymptomatic, and thus this chromosome variant and CMMoL may have been coincidentally observed.

Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity.

The protein TFIIB is a general transcription initiation factor that interacts with a promoter complex (D.DNA) containing the TATA-binding subunit (TFIID tau, or TBP) of TFIID to facilitate subsequent interaction with RNA polymerase II (ref. 2) through the associated TFIIF (ref. 3). The potential bridging function of TFIIB raises the possibility of two structural domains and emphasizes the importance of TFIIB structure-function studies for a further understanding of preinitiation complex assembly and function. Here we show that human TFIIB (refs 5,6) is comprised of functionally distinct N- and C-terminal domains. The C-terminal domain, containing the direct repeats and associated basic regions, is necessary and sufficient for interaction with the D.DNA complex. By contrast, the N-terminal domain that is dispensable for formation of the TFIID tau-TFIIB-promoter (D.B.DNA) complex is required for subsequent events leading to basal transcription initiation. On the basis of these results, we discuss structural and functional similarities between TFIIB and TFIID tau, which have similar structural organization and motifs.

Regulation of receptor activator of NF-kappaB ligand-induced tartrate-resistant acid phosphatase gene expression by PU.1-interacting protein/interferon regulatory factor-4. Synergism with microphthalmia transcription factor.

The receptor activator of NF-kappaB ligand induces the expression of tartrate-resistant acid phosphatase (TRAP) and transcription factor, PU.1-interacting protein (Pip), during osteoclastogenesis. In this paper, we have examined the role of transcription factors in the regulation of TRAP gene expression employing reporter constructs containing the promoter region of TRAP gene. Transient transfection of RAW264 cells with sequential 5'-deletions of mouse TRAP gene promoter-luciferase fusion constructs indicated that at least two sites are required for the full promoter activity. Deletion and site-directed mutation studies revealed that M-box and interferon regulatory factor element sites are critical for TPAP gene expression in the cell, suggesting that microphthalmia transcription factor (MITF) and Pip could induce the gene expression independently. Moreover, the overexpression of MITF and Pip functionally stimulated TRAP promoter activity in a synergistic manner. Analysis of the deletion mutants of Pip protein indicated that both N-terminal DNA-binding and C-terminal regulatory domains are indispensable to the promoter-enhancing activity. Subcellular localization of green fluorescence protein-fused Pip and its mutant proteins indicated that the C-terminal domain is required for the translocation of Pip into the nucleus. These results suggest that Pip regulates and acts synergistically with MITF to induce the promoter activity of TRAP gene.

The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1.

The protein TFIID is a general transcription factor which initiates preinitiation complex assembly through direct interaction with the TATA promoter element. It is a multisubunit complex containing a small TATA-binding polypeptide (TBP) and other TBP-associated factors (TAFs) ranging in size from about 30-250K (refs 7-10). Although native TFIID can mediate both activator-independent (basal) and activator-dependent transcription in reconstituted systems, TBP itself can mediate only basal transcription, even in cases where TBP or the general factor TFIIB are known to interact directly with transcriptional activators. TFIID subunits other than TBP must therefore be essential cofactors, and thus potential targets for activators, consistent with earlier demonstrations that activators interact with TFIID (refs 3, 5, 16, 17). Here we show that the 250K subunit of TFIID is identical to a gene product previously implicated in progression through the late G1 phase of the cell cycle. Part of p250 may thus serve a specific function in the activation of a subset of genes important for cell cycle progression.

Regulation of mouse UBF gene by multiple growth-related control elements.

Transcription of the mouse Upstream Binding Factor (mUBF) gene, that encodes one of the essential transcription factors for ribosomal DNA transcription, starts from several nucleotides. Neither typical TATA-box nor CCAAT-box is found upstream of the transcription initiation site. In the promoter region, there are eight GC-boxes, eight AP-2 binding consensus sequences, four cAMP response elements, and several serum response element equivalent sequences. These elements appear to play a positive role for the regulation of the mUBF gene as a whole. Among serum response elements, members located between -1182 and -343 are indeed responsive to serum, suggesting their important role in the high expression of mUBF under cell growth conditions.

Cloning and structural analysis of cDNA and the gene for mouse transcription factor UBF.

The gene and protein structure of the mouse UBF (mUBF), a transcription factor for mouse ribosomal RNA gene, have been determined by cDNA and genomic clones. The unique mUBF gene consists of 21 exons spanning over 13 kb. Two mRNAs coding for mUBF1 and mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same region as mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminal non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained both molecular species, although mUBF2 was the predominant form of UBF. The mRNA of mUBF1 was expressed one half of the mUBF2 in quiescent mouse fibroblasts but reached the same amount in growing state.

Sequence of general transcription factor TFIIB and relationships to other initiation factors.

Transcription factor TFIIB is a ubiquitous factor required for transcription initiation by RNA polymerase II. Previous studies have suggested that TFIIB serves as a bridge between the "TATA"-binding factor (TFIID) and RNA polymerase II during preinitiation complex assembly and, more recently, that TFIIB can be a target of acidic activators. We have purified TFIIB to homogeneity, shown that activity resides in a 33-kDa polypeptide, and obtained cDNAs encoding functional TFIIB. TFIIB contains a region with amino acid sequence similarity to a highly conserved region of prokaryotic sigma factors. This is consistent with analogous functions for these factors in promoter recognition by RNA polymerases and with similar findings for TFIID, TFIIE, and TFIIF/RAP30. Like TFIID, TFIIB contains both a large imperfect repeat that could contribute an element of symmetry to the folded protein and clusters of basic residues that could interact with acidic activator domains. These findings argue for a common origin of TFIIB, TFIID, and other general transcription factors and for the evolutionary segregation of complementary functions.

A case of vasculitic cholecystitis associated with Schönlein-Henoch purpura in an adult.

A case of Schönlein-Henoch Purpura (SHP) in a 32 year-old female, showing gastrointestinal manifestations including acute vasculitic cholecystitis was reported. In the course of hospitalization urgent laparotomy was performed because of the severe abdominal pain. The gallbladder was inflamed with a brownish-red edematous wall and subserosal hemorrhage, and was resected. Histological examination of the resected gallbladder specimen revealed leucocytoclastic vasculitis. The patient was treated with prednisolone postoperatively, and symptoms abated over two weeks. Acute cholecystitis with SHP is extremely rare, and as far as the authors know this is the second case of this disorder documented by histological examination. Patients with acute abdomen associated with SHP should be managed with consideration of the complications of acute cholecystitis.

The fission yeast rpa17+ gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae.

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called alpha-related subunits which are shared only by polymerases I and III. The alpha-related subunits show limited homology with the alpha-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of alpha-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17 kDa which can functionally replace AC19 - an alpha-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae - and was thus named rpa17+. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17+ shows that the gene is essential for cell growth. Sequence comparison with other alpha-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the alpha-related subunits.

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III.

Eukaryotic RNA polymerases I and III share two distinct alpha-related subunits that show limited homology to the alpha subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of alpha-related subunits in eukaryotes, we have previously identified the alpha-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42+, which encodes the alpha-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42+ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42+ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct alpha-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.

Sequence-specific binding of a transcription factor TFID to the promoter region of mouse ribosomal RNA gene.

The binding sites of TFID, a species-dependent transcription factor, on the mouse ribosomal RNA gene (rDNA) have been analyzed by DNase I footprinting using partially purified TFID. The region -12 to -140 spanning over the upper half of the core promoter (-12 to -40) and an upstream sequence (-40 to -140) was protected. Human fraction D could not protect corresponding regions of mouse rDNA indicating that the protection was indeed caused by TFID. This was corroborated by a competition experiment using point mutants having different affinities to TFID. The analysis with deletion mutants of upstream sequence together with the competition data indicates that the binding of TFID to the core sequence is required for the binding of TFID or some co-purified proteins to the upstream sequence, while the presence of upstream sequence stabilizes the TFID binding to the core sequence. The pattern of protection of the upstream sequence suggests that at least a part of the upstream binding does not require a specific DNA sequence there but rather is caused by protein-protein interaction involving TFID bound with the core sequence. These protection patterns did not change with TFID highly purified by sequence-specific DNA affinity chromatography.