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PubMed2002

Solution structure of the first HMG box domain in human upstream binding factor.

Human upstream binding factor is a nucleolar transcription factor involved in transcription by RNA polymerase I. It contains six HMG box domains; the HMG box is a minor groove DNA-binding domain that has been found in hundreds of proteins with different functions. Among the six HMG box domains in hUBF, the first one can bind to the ribosomal promoter specifically by itself and is essential for the whole protein's DNA binding specificity. Here we report the three-dimensional structure of this first HMG box free in solution determined by multidimensional NMR using (13)C,(15)N-labeled protein. Like the previously determined HMG box structures, hUBF HMG box 1 adopts a twisted L-shape consisting of three alpha-helices: helix 1 (17-30) and helix 2 (38-51) pack onto each other to form the short arm, while helix 3 (57-76) is associated with an extended strand N-terminal to helix 1 and forms the long arm. A cluster of conserved residues, in particular the aromatic residues F21, Y49, and Y60, is important to maintain the fold. The short arm is rigid due to extensive hydrophobic interaction between helix 1 and helix 2, while the long arm is less rigid.

Amino Acid Sequence↗

PubMed1990

A downstream initiation element required for efficient TATA box binding and in vitro function of TFIID.

The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells. In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint. One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site. We have now shown by transfection that both elements are required for efficient transcription in cultured cells. A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator. Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro. In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region. The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription. These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

Base Sequence↗

PubMed2003

An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm.

T-box genes encode transcription factors that play critical roles in generating the vertebrate body plan. In many developmental fields, multiple T-box genes are expressed in overlapping domains, establishing broad regions in which different combinations of T-box genes are coexpressed. Here we demonstrate that three T-box genes expressed in the zebrafish mesoderm, no tail, spadetail, and tbx6, operate as a network of interacting genes to regulate region-specific gene expression and developmental fate. Loss-of-function and gain-of-function genetic analyses reveal three kinds of interactions among the T-box genes: combinatorial interactions that generate new regulatory functions, additive contributions to common developmental pathways, and competitive antagonism governing downstream gene expression. We propose that T-box genes, like Hox genes, often function within gene networks comprised of related family members.

Animals↗

PubMed1992

Functional binding of the "TATA" box binding component of transcription factor TFIID to the -30 region of TATA-less promoters.

Many viral and cellular promoters transcribed in higher eukaryotes by RNA polymerase II lack obvious A+T-rich sequences, called "TATA" boxes, that bind the transcription factor TFIID. One such TATA-less promoter, the simian virus 40 major late promoter, contains a genetically important sequence element 30 base pairs upstream of its transcription initiation site that has no obvious sequence similarity to a TATA box. We show here that the cloned human TATA box-binding protein, hTFIID tau, functionally binds to this upstream sequence element, although with an affinity one-sixth of that to which it binds the TATA box of the adenovirus type 2 major late promoter. Analysis of point mutations in the -30 element of the simian virus 40 major late promoter shows that the affinity of binding correlates with the efficiency of transcription from this promoter. Furthermore, this element has genetic properties similar to those of a TATA box. (i) It directs RNA polymerase II to initiate transcription approximately 30 base pairs downstream of its location, and (ii) inactivation of this element results in increased heterogeneity in the sites of transcription initiation. All of five other TATA-less promoters tested were found to contain a sequence approximately 30 base pairs upstream of their major transcription initiation sites to which hTFIID tau binds. We conclude that many, if not all, TATA-less promoters differ from TATA box-containing promoters simply in the affinity of their -30 regions for binding of TFIID, with functional binding of TFIID supported in part by other nearby sequence elements of the promoter.

Base Sequence↗

PubMed1999

High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex.

The high mobility group (HMG) box domain has defined a family of proteins, mostly transcription factors, that specifically interacts with DNA on the minor groove and sharply bends it. The founding member of the family, HMG1, does not specifically recognize regular B-DNA but is recruited to DNA by interaction with other transcription factors and TATA box-binding protein (TBP). However, conflicting effects of HMG1 on transcription have been reported. We show that the interaction between HMG1 and TBP is species-specific. This interaction in turn affects the interaction of TBP with transcription factor (TF) IIB and is competed by TFIIA. A primary binding site was mapped to the H2' alpha-helix in the highly conserved core domain of human TBP. On HMG1, the primary binding site was only in the HMG box A, and HMG box A was also sufficient to interact with native TFIID. Both HMG boxes efficiently repressed transcription in vitro as fusions to the Gal4-DNA binding domain. Additionally, HMG box B showed a weak level of activation at very low amounts. These results suggest a general involvement of HMG1 at the early stages of polymerase II transcription that may result in subtle activation or repression of individual genes.

Carrier Proteins↗

PubMed2004

Human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer activity is inhibited by transcriptional factors, from the upstream stimulating factors family, via E-box and newly identified initiator element (Inr) in FSH-R non-expressing cells.

To localize the regulatory elements in the human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer and to determine the role of upstream stimulatingfactors (USFs) in these elements, we transiently transfected constructs of FSH-R promoter/enhancer in pGL3 luciferase reporter plasmids into Chinese hamster ovary cells and the activities were determined by measuring luciferase luminescence of the cell lysates. The 5'-flanking regions of the human FSH-R gene from nt -1485 to -1 with respect to the gene translation start site were amplified by polymerase chain reaction (PCR) and subcloned in pGL3. Deletion mutants were created using PCR or restriction enzyme digestion. Mutation in the E-box sequence from nt -124 to -119 (E-box 3), in the construct from -224 to nt -1 or in the Inr element, which encompasses the transcriptional start site at nt -99, resulted in a substantial reduction in the human FSH-R promoter/enhancer activity. Overexpression of upstream stimulating factor-1 (USF1) suppresses the activity of the human FSH-R promoter/enhancer via Inr and E-box elements. Upstream stimulating factor-2 (USF2) decreases FSH-R promoter/enhancer activity by acting on E-box 3. The results indicate that E-box 3 and the Inr element are important elements of the human FSH-R promoter/ enhancer. USF family members inhibit FSH-R gene activity by acting via these elements. USF1 and USF2 suppress human FSH-R promoter/enhancer activity by acting on E-box 3. USF1 also decreases activity by interacting with the Inr element.

Animals↗

PubMed2003

The comparative genomics of T-box genes.

T-box genes are defined by the presence of a conserved sequence, the so-called T-box; this codes for the T-domain, which is involved in DNA-binding and protein dimerisation. Members of this gene family have been found in all metazoans, from diploblasts to humans, and mutations in T-box gene family members in humans have been linked to several congenital disorders. Sequencing of the complete genomes of a range of invertebrate and vertebrate species has allowed the classification of individual T-box genes into five subfamilies: Brachyury, T-brain1, Tbx1, Tbx2 and Tbx6. This review will largely focus on T-box genes identified in organisms whose genomes have been fully sequenced, emphasising how comparative studies of the T-box gene family will help to reveal the roles of these genes during development and in the adult.

Animals↗

PubMed1991

Upstream box/TATA box order is the major determinant of the direction of transcription.

Mammalian gene promoters for transcription by RNA polymerase II are typically organized in the following order: upstream sequence motif(s)/TATA box/initiation site. Here we report studies in which the order, orientation and DNA sequences of these three elements are varied to determine how these affect polarity of transcription. We have constructed promoters with an 'octamer' upstream sequence ATTTGCAT (or its complement ATGCAAAT) in combination with several different TATA boxes and initiation (cap) sites, and tested these promoters in transfection experiments with cultured cells. TATA boxes derived from the adenovirus major late promoter (TATAAAA), immunoglobulin kappa light chain (TTATATA) and heavy chain (TAAATATA) promoter functioned equally well or even better when inverted. Only the beta-globin TATA box (CATAAAA) was poorly active when inverted. In addition, a symmetrical TATA box (TATATATA) derived from a casein gene was very active. Our results suggest that the asymmetry of most TATA boxes (consensus TATAAAA) is not a primary determinant of the polarity of transcription. We also found that the initiation (cap) site, which usually consists of an adenine embedded in a pyrimidine-rich region (PyPyCAPyPyPyPyPy), was permissive towards sequence alterations; even a randomly composed sequence worked well. However, an inverted, hence purine-rich, cap site reduced transcript levels to 1/7th, as did an oligo G sequence. Irrespective of the presence of a cap site, the configuration: 'TATA box/octamer' yielded a strong leftward, rather than rightward transcription. From this, we conclude that the polarity of transcription is primarily determined by the linear order of an upstream sequence relative to a TATA box, rather than by the individual orientations of either of these two elements.

Adenoviridae↗

PubMed2006

Dead-box proteins: a family affair--active and passive players in RNP-remodeling.

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.

Amino Acid Motifs↗

PubMed1989

Boxing-related ocular injuries in the United States Army, 1980 to 1985.

Boxing is a controversial sport in which neurologic and ocular injuries can be particularly devastating. Currently, competitive boxing is promoted throughout the military. Ocular injuries attributable to military boxing were studied as a subset of a larger study of boxing-related injuries. Ocular injuries occurred in 5% (22/401) of soldiers hospitalized for boxing-related trauma. The 22 patients with eye injuries spent an average of 8.1 days in the hospital and 17.8 days unfit for duty. Ocular injuries, on average, resulted in a longer hospitalization and disability than all other boxing injuries. One soldier was blind in one eye after complications from a ruptured globe.

Athletic Injuries↗

PubMed2004

CCAAT/enhancer-binding protein beta (nuclear factor for interleukin 6) transactivates the human MDR1 gene by interaction with an inverted CCAAT box in human cancer cells.

We investigated the mechanisms of MDR1 gene activation by CCAAT/enhancer binding protein beta (C/EBPbeta, or nuclear factor for interleukin 6) in human cancer cells. Transfection of the breast cancer cell line MCF-7 and its doxorubicin-selected variant MCF-7/ADR by either C/EBPbeta or C/EBPbeta-LIP (a dominant-negative form of C/EBPbeta) confirmed their roles in the activation or repression of the endogenous, chromosomally embedded MDR1 gene. Cotransfection experiments with promoter constructs revealed a C/EBPbeta interaction on the MDR1 promoter via the region within -128 to -75. Deletions within the putative AP-1 box (-123 to -111) increased MDR1 promoter activity when stimulated by C/EBPbeta, suggesting that the AP-1 site negatively regulates MDR1 activation by C/EBPbeta. Mutations within the inverted CCAAT box (Y box) (-82 to -73) abolished the C/EBPbeta-stimulated MDR1 promoter activity, indicating that the Y box is required for MDR1 activation by C/EBPbeta. Chromatin immunoprecipitation (ChIP) revealed that C/EBPbeta precipitates a transcription complex containing C/EBPbeta, the MDR1 promoter sequences (-250 to +54), and the hBrm protein. In conclusion, alteration of expression or function of C/EBPbeta plays an important role in MDR1 gene regulation. C/EBPbeta activates the endogenous MDR1 gene of MCF-7 cells, and this activation was associated with a novel C/EBPbeta interaction region within the proximal MDR1 promoter (-128 to -75). The mechanisms of MDR1 activation by C/EBPbeta include C/EBPbeta binding of the chromatin of the MDR1 gene and interactions of C/EBPbeta with the Y box and Y box-associated proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Two distinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcription.

The rabbit uteroglobin gene is expressed in a variety of epithelial cell types like the lung Clara cells and the glandular and luminal epithelial cells of the endometrium. Expression in Clara cells is on a high constitutive level, whereas expression in the rabbit endometrium is under tight hormonal control. One important element of the rabbit uteroglobin gene mediating its efficient transcription in two epithelial cell lines from human endometrium (Ishikawa) and lung (NCI-H441) is its noncanonical TATA box (TACA). Here, we show that two factors (TATA core factor [TCF] and TATA palindrome factor [TPF]) different from the TATA-box binding protein bind to the DNA major groove at two adjacent sites within the uteroglobin TATA-box region and that one of them (TCF) is specifically expressed in cell lines derived from uteroglobin-expressing tissues. The binding sites for TCF and TPF, respectively, are both required for efficient transcription in Ishikawa and NCI-H441 cells. Mutation of the TACA box, which we show is a poor TATA box in functional terms, to a canonical TATA motif does not affect TCF and TPF binding. Therefore, we suggest that the function of the unusual cytosine could be to reduce rabbit uteroglobin expression in cells lacking TCF and that the interaction of TATA-box binding protein with the weak TACA site is facilitated in TCF- and TPF-positive cells.

Animals↗

PubMed1996

Binding of YY1 to a site overlapping a weak TATA box is essential for transcription from the uteroglobin promoter in endometrial cells.

The gene for rabbit uteroglobin codes for a small calcium-, steroid-, and biphenyl metabolite-binding homodimeric protein which is expressed in a variety of epithelial cell types such as Clara cells (lung) and the glandular and luminal cells of the endometrium. One important region mediating its efficient transcription in a human endometrium-derived cell line, Ishikawa, is centered around a noncanonical TATA box. Two factors, TATA core factor (TCF), expressed in cell lines derived from uteroglobin-expressing tissues, and the ubiquitously expressed TATA palindrome factor, bind to the DNA major groove at two adjacent sites within this region. Here, we report the identification of the TATA palindrome factor as the transcription/initiation factor YY1 by microsequencing of the biochemically purified factor from HeLa cells. The binding site for YY1 within the uteroglobin gene is unique in its sequence and its location overlapping a weak TATA box (TACA). Binding of YY1 was required for efficient transcription in TCF-positive Ishikawa cells, which responded only weakly to a change of TACA to TATA, although in vitro binding affinity for the TATA-box-binding protein increased by 1 order of magnitude. In contrast, in CV-1 cells, lacking TCF, binding of YY1 was not required for transcription in the context of a wild-type TACA box, whereas a change from TACA to TATA led to significantly increased reporter gene expression. DNA binding data exclude a role of YY1 in stabilizing the interaction of the TATA-box-binding protein with the uteroglobin promoter. We conclude that cell lines derived from uteroglobin-expressing tissues overcome the weak TATA box with the help of auxiliary factors, one of them being YY1.

Adenoviridae↗

PubMed1999

Sport medicine and the ethics of boxing.

In the light of medical evidence of the health risks associated with boxing, a watchful agnostic position among sport physicians is no longer justifiable. The normal activity in a boxing match places the athletes at risk of head injury, some of which may be difficult to detect and impossible to repair. This suggests that sport physicians and others expert in the prevention and diagnosis of such injuries should take a public stand against boxing, as other medical associations have. Although there is a need for continuing research into the health risks, doctors can in the interim take steps to increase public awareness of these risks. Sport physicians in particular can make a strong public statement by also ending their professional involvement with boxing. This need not be interpreted as paternalism; doctors are qualified neither to make laws nor to restrict private behaviour. Sport physicians are, however, well equipped to advise those who do make laws and those who choose to engage in boxing. In the end, because this stance against boxing will probably reduce the number of brain injuries in certain athletes, autonomy will be preserved, rather than restricted.

Boxing↗

PubMed2006

An E box in the exon 1 promoter regulates insulin-like growth factor-I expression in differentiating muscle cells.

Insulin-like growth factor (IGF)-I expression is subject to complex temporal and spatial regulation. Endocrine synthesis occurs in the liver, where transcription is initiated from promoters located in either exon 1 (P1) or in exon 2 (P2), whereas local transcription is mainly initiated from P1. IGF-I is expressed in a range of tissues and, in particular, is an important regulator of skeletal muscle mass, although the mechanisms of tissue-specific regulation remain to be fully characterized. Gene regulation in skeletal muscle is associated with the E box DNA element (5'-CANNTG-3') recognized by myogenic regulatory factors (MRFs), such as MyoD1. Transcription element profiling identified a hypothetical myogenic E box (sequence 5'-CAGCTG-3') within P1, immediately upstream of the major muscle transcriptional start site, and we sought to test its activity in differentiating C2C12 myoblasts. We found P1-driven IGF-I mRNA expression to be associated with myogenic differentiation and, moreover, that a single base-pair mutation in the E box specifically reduced expression in myofibers. A synthetic enhancer construct containing a triplet repeat of the E box was active in muscle cells and strongly induced in myofibers. The capacity of a double-stranded IGF-I E box probe (but not one bearing a single-base pair alteration) to bind C2C12 nuclear lysates increased with myogenesis, and a transactivation assay demonstrated that the E box was recognized by E protein-MRF heterodimers. Mechanisms of tissue-specific gene activation are of increasing biological interest, and we have identified a cis-element able to direct muscle-specific IGF-I gene expression.

Animals↗

PubMed2006

Frequency distribution of TATA Box and extension sequences on human promoters.

BACKGROUND: TATA box is one of the most important transcription factor binding sites. But the exact sequences of TATA box are still not very clear. RESULTS: In this study, we conduct a dedicated analysis on the frequency distribution of TATA Box and its extension sequences on human promoters. Sixteen TATA elements derived from the TATA Box motif, TATAWAWN, are classified into three distribution patterns: peak, bottom-peak, and bottom. Fourteen TATA extension sequences are predicted to be the new TATA Box elements due to their high motif factors, which indicate their statistical significance. Statistical analysis on the promoters of mice, zebrafish and drosophila melanogaster verifies seven of these elements. It is also observed that the distribution of TATA elements on the promoters of housekeeping genes are very similar with their distribution on the promoters of tissue specific genes in human. CONCLUSION: The dedicated statistical analysis on TATA box and its extension sequences yields new TATA elements. The statistical significance of these elements has been verified on random data sets by calculating their p values.

Base Sequence↗

PubMed1996

A Y-box protein is a suppressor factor that decreases thyrotropin receptor gene expression.

The decanucleotides in a tandem repeat, -162 to -140 bp, are suppressor elements that decrease TSH receptor (TSHR) gene expression by different mechanisms. A factor(s) interacting with the 3'-decanucleotide compete for proteins that bind the cAMP response element, -139 to -132 bp, a constitutive enhancer necessary for efficient TSHR expression. The 5'-decanucleotide is in a CT-rich, S1 nuclease-sensitive region of the promoter; its suppressor activity has been related to its ability to bind a nonthyroid-specific protein to its coding strand. In this report we clone a complementary DNA encoding a single strand DNA-binding protein that forms a specific protein-DNA complex with the coding strand of the 5'- but not the 3'-decanucleotide and not with the 5'-decanucleotide noncoding or double strand. We show, by cotransfection with TSHR promoter-chloramphenicol acetyltransferase chimeras, that the protein is a suppressor that regulates the function of the 5'- but not the 3'-decanucleotide. The protein is a Y-box protein that was previously cloned as an enhancer factor from the rat liver; it is, however, 95% identical to human YB-1, which suppresses major histocompatibility class II gene expression, and to human nuclease-sensitive element protein-1, a Y-box protein identified by its ability to bind single strand, CT-rich, nuclease-sensitive elements of genes that, like the TSHR, have GC-rich promoters. Unexpectedly, the Y-box protein binds two other sites in the minimal TSHR promoter in a single strand-specific fashion and acts a suppressor at each of these sites. One is associated with the insulin response element of the minimal TSHR promoter and is not in an overtly CT-rich region. The other is located 3' to the cAMP response element in a region termed the S-box, -120 to -113 bp, because of its homology to the S-box of the major histocompatibility class II promoter; this site is in a CT-rich area and, as in the class II promoter, is linked to cAMP-induced gene suppression. A conserved CCTC sequence in each site is important for the binding and suppressor function of the Y-box protein.

Amino Acid Sequence↗

PubMed1998

Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development.

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

Animals↗