scholarly journals Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation

2017 ◽  
Vol 29 (4) ◽  
pp. 843-853 ◽  
Author(s):  
Jenna E. Gallegos ◽  
Alan B. Rose
Keyword(s):  
Dna Sequences ◽  
Proximal Promoter ◽  
Transcript Initiation

Tc, an unusual promoter element required for constitutive transcription of the yeast HIS3 gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4447-4455
Author(s):  
S Mahadevan ◽  
K Struhl
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Base Pair ◽  
Point Mutations ◽  
Proximal Promoter ◽  
Promoter Element ◽  
Tata Element ◽  
Typical Sequence ◽  
His3 Gene ◽  
Transcriptional Stimulation

Tc is the proximal promoter element required for constitutive his3 transcription that occurs in the absence of the canonical TATA element (TR) and is initiated from the +1 site. The TC element, unlike TR, does not respond to transcriptional stimulation by the GCN4 or GAL4 activator protein. Analysis of deletion, substitution, and point mutations indicates that Tc mapped between nucleotides -54 and -83 and is a sequence-dependent element because it could not be functionally replaced by other DNA sequences. However, in contrast to the behavior of typical promoter elements, it was surprisingly difficult to eliminate Tc function by base pair substitutions. Of 15 derivatives averaging four substitutions in the Tc region and representing 40% of all possible single changes, only 1 inactivated the Tc element. Moreover, the phenotypes of mutant and hybrid elements indicated that inactivation of Tc required multiple changes. The spacing between Tc and the initiation region could be varied over a 30-base-pair range without significantly affecting the level of transcription from the +1 site. From these results, we consider it possible that Tc may not interact with TFIID or some other typical sequence-specific transcription factor, but instead might influence transcription, either directly or indirectly, by its DNA structure.

A 3-Bp Deletion Between Transcription Factor Binding Motifs In the HBS1L-MYB Intergenic Region on Chromosome 6q23 Is Associated with HbF Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1013-1013
Author(s):  
John J. Farrell ◽  
Richard M. Sherva ◽  
Zhi-yi Chen ◽  
Luo Hong-yuan ◽  
Banjamin F. Chu ◽  
...  
Keyword(s):  
Hong Kong ◽  
Transcription Factors ◽  
Dna Sequences ◽  
Globin Gene ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Deletion Polymorphism ◽  
Functional Motif ◽  
A Genome

Abstract Abstract 1013 More than 3% of Chinese in Hong Kong are heterozygous carriers of β-thalassemia. Homozygotes or compound heterozygotes for β-thalassemia are usually severely ill and require monthly transfusions. Increased production of fetal hemoglobin (HbF) can modulate the disease severity by compensating for the shortfall of HbA caused by the β-thalassemia mutations. HbF level in adults varies and is regulated as a multigenic trait. Three major HbF quantitative trait loci (QTL) have been identified: the C/T SNP also known as the Xmn I site at the Gγ-globin gene promoter, the BCL11A polymorphism on chromosome 2p16, and the HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23. The functional motif for each of these 3 QTLs responsible for their effects upon HbF is not known. We undertook a genome-wide association study (GWAS), using Illumina Human 610-Quad BeadChip array, on 619 Chinese β-thalassemia heterozygotes from Hong Kong. In this population, the variance in HbF due to HMIP is 13.5%, significantly higher than that due to BCL11A polymorphism (6.4%). We used 1,000 Genomes Project data, SNP imputation, comparisons of association results across populations, predicted binding of transcription factors, and phylogenetic conservation to identify the functional variant in HMIP. Based on these lines of evidence, a hitherto unreported association between HbF expression and a 3-bp deletion on chromosome 6q23 was found. In 335 Chinese β-thalassemia heterozygotes, the 3-bp deletion polymorphism is in complete linkage disequilibrium with rs9399137, the SNP found in multiple GWAS to be most significantly associated with HbF (P=1.4E-24 in the Chinese cohort GWAS). Flanking this deletion are conserved binding sites for TAL1/SCL1, E47, GATA, and RUNX1/AML1, which are essential erythropoiesis-related transcription factors. The 3-bp deletion changes the normal DNA binding configuration of these transcription factors and spatial configuration for DNA-protein binding and/or protein-protein interactions. Furthermore, this 3-bp deletion polymorphism resides within a likely erythroid distal regulatory region manifested by DNase I hypersensitivity and GATA-1 binding (Wahlberg et al, Blood 114:1254, 2009). We hypothesized that a 61-bp fragment of DNA that encompasses the site of the 3-bp deletion polymorphism might have enhancer-like activity. When ligated to the Gγ-globin gene 1.4 kb proximal promoter linked to a luciferase reporter gene, the 61-bp fragment of DNA enhances the Gγ-globin gene promoter activity by more than 3-fold after transient transfection into K562 cells. A 58-bp fragment of DNA that includes the 3-bp deletion has 60% more enhancer-like activity than the 61-bp fragment without the deletion. These findings suggest that this 3-bp deletion polymorphism is most likely the functional motif accounting for HMIP modulation of HbF. Further studies are needed to identify target genes for this enhancer-like activity mediated by the DNA sequences encompassing the 3-bp deletion polymorphism in HMIP. These studies also suggest that this experimental approach could be used to identify functional motifs in other genotype-phenotype association studies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements

1998 ◽  
Vol 330 (1) ◽  
pp. 335-343 ◽  
Author(s):  
M. Bahaa FADEL ◽  
C. Stephane BOUTET ◽  
Thomas QUERTERMOUS
Keyword(s):  
Gene Expression ◽  
Endothelial Cell ◽  
Dna Binding ◽  
Protein Binding ◽  
Dna Sequences ◽  
Molecular Basis ◽  
Proximal Promoter ◽  
Specific Gene ◽  
Specific Gene Expression

To investigate the molecular basis of endothelial cell-specific gene expression, we have examined the DNA sequences and the cognate DNA-binding proteins that mediate transcription of the murine tie2/tek gene. Reporter transfection experiments conformed with earlier findings in transgenic mice, indicating that the upstream promoter of Tie2/Tek is capable of activating transcription in an endothelial cell-specific fashion. These experiments have also allowed the identification of a single upstream inhibitory region (region I) and two positive regulatory regions (regions U and A) in the proximal promoter. Electrophoretic mobility-shift assays have allowed further characterization of three novel DNA-binding sequences associated with these regions and have provided preliminary characterization of the protein factors binding to these elements. Two of the elements (U and A) confer increased transcription on a heterologous promoter, with element U functioning in an endothelial-cell-selective manner. By employing embryonic endothelial-like yolk sac cells in parallel with adult-derived endothelial cells, we have identified differences in functional activity and protein binding that may reflect mechanisms for specifying developmental regulation of tie2/tek expression. Further study of the DNA and protein elements characterized in these experiments is likely to provide new insight into the molecular basis of developmental- and cell-specific gene expression in the endothelium.

scholarly journals Identification of cis-regulatory elements required for larval expression of the Drosophila melanogaster alcohol dehydrogenase gene.

Genetics ◽  
1990 ◽  
Vol 124 (3) ◽  
pp. 637-646 ◽  
Author(s):  
V Corbin ◽  
T Maniatis
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Dna Sequences ◽  
Regulatory Elements ◽  
Malpighian Tubules ◽  
Proximal Promoter ◽  
Start Site ◽  
Wild Type ◽  
Tissue Specific ◽  
Adh Gene

Abstract The Alcohol dehydrogenase (Adh) genes of two distantly related species, Drosophila melanogaster and Drosophila mulleri, display similar, but not identical, patterns of tissue-specific expression in larvae and adults. The regulatory DNA sequences necessary for wild-type Adh expression in D. mulleri larvae were previously reported. In this paper we present an analysis of the DNA sequences necessary for wild-type Adh expression in D. melanogaster larvae. We show that transcription from the proximal promoter of the melanogaster Adh gene is regulated by a far upstream enhancer and two or more elements near the transcription start site. The enhancer is tissue specific and stimulates transcription to high levels in fat body and to lower levels in midgut and malpighian tubules whether linked to the proximal promoter or to a heterologous promoter. The enhancer activity localized to at least two discrete regions dispersed over more than 1.7 kb of DNA. Deletion of any one of these subregions reduces Adh transcription in all three larval tissues. Similarly, two regions immediately upstream of the proximal promoter start site are necessary for wild-type transcription levels in all three tissues. Thus, each of the identified regulatory elements is sufficient for low levels of Adh gene expression in all three larval tissues, but maximal levels of expression requires the entire set.

scholarly journals Sequence-Specific Deoxyribonucleic Acid (DNA) Recognition by Steroidogenic Factor 1: A Helix at the Carboxy Terminus of the DNA Binding Domain Is Necessary for Complex Stability

2006 ◽  
Vol 20 (4) ◽  
pp. 831-843 ◽  
Author(s):  
Tanya H. Little ◽  
Yongbo Zhang ◽  
Christina K. Matulis ◽  
Jennifer Weck ◽  
Zhipeng Zhang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Hormone Receptors ◽  
Terminal Segment ◽  
Reproductive Hormones ◽  
Binding Domain ◽  
Proximal Promoter ◽  
Dna Binding Domain ◽  
Steroidogenic Factor 1 ◽  
The Core

Abstract Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-α gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

scholarly journals DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5).

1995 ◽  
Vol 15 (5) ◽  
pp. 2858-2871 ◽  
Author(s):  
T Czerny ◽  
M Busslinger
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Sea Urchin ◽  
Dna Sequences ◽  
Binding Specificity ◽  
Proximal Promoter ◽  
Paired Domain ◽  
Distal Enhancer ◽  
Sequence Recognition ◽  
Dna Binding Specificity

Pax-6 is known to be a key regulator of vertebrate eye development. We have now isolated cDNA for an invertebrate Pax-6 protein from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart with the paired domain and homeodomain being virtually identical. Consequently, we found that the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily only at one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, we were able to identify a short amino acid stretch in the N-terminal part of the paired domain which is responsible for these differences in DNA-binding specificity. Mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP resulted in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids were furthermore shown to discriminate between the Pax-6- and BSAP-specific nucleotide at the divergent position of the two consensus recognition sequences.

scholarly journals Unmethylated thyroglobulin promoter may be repressed by methylation of flanking DNA sequences

1994 ◽  
Vol 298 (3) ◽  
pp. 537-541 ◽  
Author(s):  
B Pichon ◽  
C Christophe-Hobertus ◽  
G Vassart ◽  
D Christophe
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Sequences ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Enhancer Element ◽  
Differentiated Cells ◽  
Gene Enhancer ◽  
Thyroglobulin Gene

The thyroglobulin gene, like many other tissue-specific genes, appears to be specifically less methylated in the differentiated cell type where it is transcribed. The thyroglobulin gene promoter elements themselves are highly CG-deficient and do not contain any HpaII/MspI sites. In this study, using DNA constructs that were methylated in vitro with HpaII or MspI methylases, we show that DNA methylation of vector sequences is sufficient to repress the activity of the thyroglobulin gene promoter in transient transfection experiments. Reporter-gene expression from a plasmid containing only the proximal thyroglobulin gene promoter is sensitive to DNA methylation even in fully differentiated thyrocytes. Transcription from methylated plasmids containing the thyroglobulin gene enhancer and proximal promoter is also clearly reduced when the transfected cells are maintained under less-differentiated conditions. These results indicate that DNA methylation can influence, from a distance, the activity of an unmodified promoter. Our results also agree with the view that loss of DNA methylation does not constitute a prerequisite for thyroglobulin gene expression in differentiated thyrocytes, where the thyroglobulin gene enhancer and promoter are activated. However, the production of thyroglobulin transcripts could be severely impaired when this activation is not maximal, as is the case in less-differentiated cells or when the enhancer element is lacking. We suggest that DNA methylation helps to maintain the thyroglobulin gene in an inactive state unless all of the conditions required for its expression are fulfilled, and that the thyroid-specific demethylation events are a consequence of the activation state of the gene.

scholarly journals Tc, an unusual promoter element required for constitutive transcription of the yeast HIS3 gene.

1990 ◽  
Vol 10 (9) ◽  
pp. 4447-4455 ◽  
Author(s):  
S Mahadevan ◽  
K Struhl
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Base Pair ◽  
Point Mutations ◽  
Proximal Promoter ◽  
Promoter Element ◽  
Tata Element ◽  
Typical Sequence ◽  
His3 Gene ◽  
Transcriptional Stimulation

Tc is the proximal promoter element required for constitutive his3 transcription that occurs in the absence of the canonical TATA element (TR) and is initiated from the +1 site. The TC element, unlike TR, does not respond to transcriptional stimulation by the GCN4 or GAL4 activator protein. Analysis of deletion, substitution, and point mutations indicates that Tc mapped between nucleotides -54 and -83 and is a sequence-dependent element because it could not be functionally replaced by other DNA sequences. However, in contrast to the behavior of typical promoter elements, it was surprisingly difficult to eliminate Tc function by base pair substitutions. Of 15 derivatives averaging four substitutions in the Tc region and representing 40% of all possible single changes, only 1 inactivated the Tc element. Moreover, the phenotypes of mutant and hybrid elements indicated that inactivation of Tc required multiple changes. The spacing between Tc and the initiation region could be varied over a 30-base-pair range without significantly affecting the level of transcription from the +1 site. From these results, we consider it possible that Tc may not interact with TFIID or some other typical sequence-specific transcription factor, but instead might influence transcription, either directly or indirectly, by its DNA structure.

scholarly journals c-Myb Binds to a Sequence in the Proximal Region of the RAG-2 Promoter and Is Essential for Promoter Activity in T-Lineage Cells

2000 ◽  
Vol 20 (24) ◽  
pp. 9203-9211 ◽  
Author(s):  
Qian-Fei Wang ◽  
Josh Lauring ◽  
Mark S. Schlissel
Keyword(s):  
T Cells ◽  
Dna Sequences ◽  
Promoter Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Distinct Region ◽  
Promoter Construct ◽  
Promoter Dna ◽  
B And T Lymphocytes

ABSTRACT The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG-2 promoter-reporter construct in cotransfection assays and that this transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site.

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