The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription.
The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides -2264 and -2495 in the 5'-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site. (+info)
High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.
Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species. (+info)
Multiple cis-acting regulatory regions are required for restricted spatio-temporal Hoxa5 gene expression.
Genetic analyses have revealed the essential role of the murine Hoxa5 gene for the correct specification of the cervical and upper thoracic region of the skeleton, and for the normal organogenesis and function of the respiratory tract, both structures expressing Hoxa5 during embryogenesis. To understand how the expression domains of the Hoxa5 gene are established during development, we have analyzed the cis-acting control regions mediating Hoxa5 gene expression using a transgenic approach. Four transcripts are derived from the Hoxa5 locus. The shortest and most abundant one displays a specific spatio-temporal profile of expression at earlier stages and in more anterior structures along the embryonic axis than the larger forms. We established that an 11.1 kilobase pair (kb) genomic fragment, extending from position -3.8 kb to +7.3 kb relative to Hoxa5 transcription initiation site, was sufficient to reproduce the temporal expression and substantially reconstitute the spatial pattern of the major Hoxa5 transcript. By deletion analyses, we identified a 2.1 kb fragment located downstream of the Hoxa5 gene that possesses mesodermal enhancer activity. Overall, the findings demonstrate that cis-acting regulatory elements essential for the correct expression of the major Hoxa5 transcript are located both upstream and downstream of the Hoxa5 coding sequences. (+info)
Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.
The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo. (+info)
Transcription of the stationary-phase-associated hspX gene of Mycobacterium tuberculosis is inversely related to synthesis of the 16-kilodalton protein.
The 16-kDa protein, an alpha-crystallin homologue, is one of the most abundant proteins in stationary-phase Mycobacterium tuberculosis. Here, transcription and translation of the hspX gene, which encodes the 16-kDa protein, have been investigated by Northern blotting analysis, primer extension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a microaerophilic stationary-phase model. Two transcripts of about 2.5 and 1.1 kb were demonstrated by Northern blot analysis and hybridized to the hspX gene probe. Primer extension analysis revealed that the transcription start site is located 33 nucleotides upstream of the hspX gene start codon. The cellular level of the hspX mRNA was maximum in log-phase bacilli and was markedly reduced after 20 days in unagitated culture, when the organisms had entered the stationary phase. A third transcript of 0.5 kb was detected 0.6 kb downstream of the hspX gene; this transcript has a transcriptional pattern completely different from that of the 1.1- and 2.5-kb products, suggesting that there may be another gene in this region. In contrast to the high level of hspX mRNA in log-phase bacilli, 16-kDa protein synthesis was low in log-phase bacteria and rose to its maximum after 20 days. In both log-phase and stationary-phase bacteria the mRNA was unstable, with a half-life of 2 min, which indicated that the transcript stability was growth rate independent and not a general means for controlling the gene expression. However, the cellular content of 16-kDa protein, while low in log-phase bacteria, rose to a maximum at 10 days and remained at this high level for up to 50 days, which indicates that this protein is a stable molecule with a low turnover rate. These data suggest that the regulation of hspX expression during entry into and maintenance of stationary phase involves translation initiation efficiency and protein stability as potential mechanisms. (+info)
Tight correlation between inhibition of DNA repair in vitro and transcription factor IIIA binding in a 5S ribosomal RNA gene.
UV-induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)-5S rDNA complex and Xenopus oocyte nuclear extracts. TFIIIA alters CPD formation primarily in the transcribed strand of the 50 bp internal control region (ICR) of 5S rDNA. During NER in vitro, CPD removal is reduced at most sites in both strands of the ICR when TFIIIA is bound. Efficient repair occurs just outside the TFIIIA-binding site (within 10 bp), and in the absence of 5S rRNA transcription. Interestingly, three CPD sites within the ICR [+56, +75 (transcribed strand) and +73 (non-transcribed strand)] are repaired rapidly when TFIIIA is bound, while CPDs within approximately 5 bases of these sites are repaired much more slowly. CPDs at these three sites may partially displace TFIIIA, thereby enabling rapid repair. However, TFIIIA is not completely displaced during NER, at least at sites outside the ICR, even though the NER complex could be sterically hindered by TFIIIA. Such inefficient repair of transcription factor binding sites could increase mutation frequency in regulatory regions of genes. (+info)
Transcription factors CCAAT/enhancer-binding protein beta and nuclear factor-Y bind to discrete regulatory elements in the very low density lipoprotein receptor promoter.
Expression of the very low density lipoprotein receptor (VLDL-R) is barely detectable in liver, but occurs in adipose tissue, skeletal muscle, heart, and placenta, where it is postulated to supply triglyceride to tissues that utilize fatty acids. To investigate its tissue-specific expression, cell lines were transfected with luciferase reporter gene constructs driven by the 5'-flanking region of the VLDL-R gene. Transcriptional activity of a 4.2-kb promoter fragment was 5-fold higher in BeWo placental cells than in Huh-7 hepatoma cells, consistent with relative endogenous expression of the VLDL-R. By deletion analysis, DNase I protection assays and site-directed mutagenesis, two regulatory elements were essential for maximal promoter activity in BeWo cells: footprint site D (-856 to -830) and an inverted CCAAT box (-703 to -707). Mutation of either element reduced promoter activity by 60% in BeWo cells, but had little effect in Huh-7 cells, suggesting that these elements direct cell-type specific transcription. Electrophoretic mobility-shift assays with BeWo nuclear extracts revealed that the inverted CCAAT box binds transcription factor NF-Y, and site D binds CCAAT/enhancer-binding protein b (C/EBPbeta) and minor amounts of C/EBPalpha and C/EBPdelta. Overexpression of a dominant negative NF-YA vector confirmed involvement of NF-Y in the regulation of the VLDL-receptor gene through the CCAAT box. However overexpression of C/EBP could not stimulate transcription from the VLDL-receptor promoter nor from site D fused to a heterologous promoter, suggesting that the simultaneous binding of an accessory factor(s) may be necessary for C/EBP transactivation via the D site. (+info)
Structural and functional characterization of the mouse Sox9 promoter: implications for campomelic dysplasia.
Mutations in SOX9 cause campomelic dysplasia (CD), a dominant skeletal dysmorphology and XY sex reversal syndrome. The CD phenotype is sensitive to dosage and expression levels of SOX9. Sox9 is expressed during chondrocyte differentiation and is up-regulated in male and down-regulated in female genital ridges during sex differentiation. In order to study the sex- and tissue-specific regulation of Sox9, we have defined the transcription start site and characterized the mouse Sox9 promoter region. The Sox9 proximal promoter shows moderately high nucleotide similarity between mouse and human. Transient transfection experiments using various deletion constructs of the 6.8 kb upstream region of mouse Sox9 fused to a luciferase reporter showed that the interval between 193 and 73 bp from the transcription start site is essential for maximal promoter activity in cell lines and in primary male and female gonadal somatic cells and liver cells isolated from 13.5 d.p.c. mouse embryos. This minimal promoter region was shown by DNase I hypersensitive site assay to be in an 'open' state of chromatin structure in gonads of both sexes, but not in the liver. Promoter activity was higher in testis than in ovary and liver, but deletion of the region from -193 to -73 bp abolished this difference. We conclude that the proximal promoter region is in part responsible for the sex- and tissue-specific expression of the Sox9 gene and that more distal positive and negative elements contribute to its regulation in vivo, consistent with the observation that translocations upstream from SOX9 can result in campomelic dysplasia. (+info)