Regulatory elements controlling pituitary-specific expression of the human prolactin gene

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

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Intestinal Apolipoprotein A-IV Gene Transcription Is Controlled by Two Hormone-Responsive Elements: A Role for Hepatic Nuclear Factor-4 Isoforms

Molecular Endocrinology ◽

10.1210/me.2004-0462 ◽

2005 ◽

Vol 19 (9) ◽

pp. 2320-2334 ◽

Cited By ~ 22

Author(s):

Amena Archer ◽

Dominique Sauvaget ◽

Valérie Chauffeton ◽

Pierre-Etienne Bouchet ◽

Jean Chambaz ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Distal Region ◽

Proximal Region ◽

Dominant Negative ◽

Specific Expression ◽

Nuclear Factors ◽

Dominant Negative Form ◽

Responsive Element ◽

Hormone Responsive Element

Abstract In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4α and γ. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4α repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4α and γ functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

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Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1488 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1488-1499 ◽

Cited By ~ 34

Author(s):

H J Roth ◽

G C Das ◽

J Piatigorsky

Keyword(s):

Transcription Factors ◽

Hela Cell ◽

Dna Sequences ◽

Nuclear Extract ◽

Regulatory Elements ◽

Dnase I ◽

Flanking Sequence ◽

Mobility Shift ◽

Promoter Elements ◽

Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

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Characterization of oocyte-expressed GDF9 gene in buffalo and mapping of its TSS and putative regulatory elements

Zygote ◽

10.1017/s0967199411000712 ◽

2012 ◽

Vol 21 (2) ◽

pp. 115-124 ◽

Cited By ~ 1

Author(s):

B. Roy ◽

S. Rajput ◽

S. Raghav ◽

P. Kumar ◽

A. Verma ◽

...

Keyword(s):

Core Promoter ◽

Regulatory Elements ◽

Vital Role ◽

Minimal Promoter ◽

Base Pairs ◽

Specific Expression ◽

Oocyte Competence ◽

Tata Element ◽

Putative Transcription Factor ◽

E Boxes

SummaryIn spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at –57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at –23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at –113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5′ flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at –520 and –718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the –3471 and –203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity.

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cDNA structure, tissue-specific expression, and chromosomal localization of the murine band 7.2b gene

Blood ◽

10.1182/blood.v86.1.359.bloodjournal861359 ◽

1995 ◽

Vol 86 (1) ◽

pp. 359-365 ◽

Cited By ~ 17

Author(s):

PG Gallagher ◽

M Romana ◽

JH Lieman ◽

DC Ward

Keyword(s):

Alpha Helix ◽

Distal Region ◽

Proximal Region ◽

Erythrocyte Membranes ◽

Chromosome 2 ◽

Specific Expression ◽

Tissue Specific ◽

Reading Frame ◽

Tissue Specific Expression ◽

Membrane Spanning

Band 7.2b is an integral phosphoprotein absent from the erythrocyte membranes of patients with hydrocytosis, an autosomal, dominantly inherited, hemolytic anemia characterized by stomatocytic red blood cells with abnormal permeability to Na+ and K+. The role of this protein in the erythrocyte membrane is not well understood. To gain additional insight into the structure and function of this protein, we have cloned the murine band 7.2b cDNA and studied its tissue-specific expression. 2,873 bp of cDNA with an open reading frame of 852 bp were isolated. This fragment encodes a protein of 284 amino acids with a predicted molecular weight of 31 kD. The band 7.2b gene had a wide pattern of expression, with high levels of mRNA in heart, liver, skeletal muscle, and testis and low levels in lung, brain, and spleen. Using fluorescent in situ hybridization, the murine band 7.2b gene was mapped to chromosome 2, at the border of the distal region of 2B and proximal region of C1, syntenic to 9q33-q34, the location of the human homologue. Models of the predicted protein structure showed a short NH2- terminal head, a strongly hydrophobic 28-amino acid stretch presumably encoding a single membrane-spanning domain, and a large domain composed of beta sheet and alpha helix. Database searching showed no significant homology of other known proteins to murine or human band 7.2b.

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Dissociation of the AT-specific bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM from TpA sites in DNA

Biochemical Journal ◽

10.1042/bj3060015 ◽

1995 ◽

Vol 306 (1) ◽

pp. 15-19 ◽

Cited By ~ 4

Author(s):

M C Fletcher ◽

R K Olsen ◽

K R Fox

Keyword(s):

Dissociation Rate ◽

Dnase I ◽

Time Dependent ◽

Base Pairs ◽

Unusual Structure ◽

Dnase I Footprinting ◽

Calf Thymus ◽

Stability Of Complexes ◽

Dna Fragment ◽

The Stability

We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.

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Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.6003 ◽

1990 ◽

Vol 10 (11) ◽

pp. 6003-6012 ◽

Cited By ~ 13

Author(s):

M L Smith ◽

P J Mitchell ◽

G F Crouse

Keyword(s):

Promoter Region ◽

Transcriptional Control ◽

Cpg Island ◽

Housekeeping Genes ◽

Regulatory Elements ◽

Dnase I ◽

Control Mechanisms ◽

Cpg Dinucleotides ◽

Dnase I Footprinting ◽

Chloramphenicol Acetyltransferase Gene

The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements. The region contains four 48-bp repeats, each of which contains an Sp1-binding site. Another gene, Rep-3 (formerly designated Rep-1), shares the same general promoter region with Dhfr, being transcribed in the direction opposite that of Dhfr. Both genes appear to be housekeeping genes and are expressed at relatively low levels in all tissues. The 5' termini of the major Dhfr transcripts are separated from the 5' termini of the Rep-3 transcripts by approximately 140 bp. This curious structural arrangement suggested that the two genes might share common regulatory elements. To investigate the promoter sequences driving bidirectional transcription, a series of promoter mutations was constructed. These mutations were assayed by a replicating minigene system and by promoter fusions to the chloramphenicol acetyltransferase gene. Linker-scanning mutations that spanned the four repeats produced a variety of mRNA transcript phenotypes. The effects were primarily quantitative, generally reducing the abundance of transcripts for one or both genes. Some mutations affected Dhfr in a qualitative manner, such as by changing the startpoint of one of the major Dhfr transcripts or changing the relative abundance of the two major Dhfr transcripts. Additionally, protein transcription factors that bind to sequences in the mouse Dhfr/Rep-3 major promoter region, potentially affecting expression of either or both genes, were investigated by DNase I footprinting. The results indicate that multiple protein-DNA interactions occur in this region, reflecting potentially complex transcriptional control mechanisms that might modulate expression of either or both genes under different physiological conditions.

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Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1488-1499.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1488-1499

Author(s):

H J Roth ◽

G C Das ◽

J Piatigorsky

Keyword(s):

Transcription Factors ◽

Hela Cell ◽

Dna Sequences ◽

Nuclear Extract ◽

Regulatory Elements ◽

Dnase I ◽

Flanking Sequence ◽

Mobility Shift ◽

Promoter Elements ◽

Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

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Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides

Biochemical Journal ◽

10.1042/bj3010569 ◽

1994 ◽

Vol 301 (2) ◽

pp. 569-575 ◽

Cited By ~ 17

Author(s):

E Washbrook ◽

K R Fox

Keyword(s):

Metal Ion ◽

Dimethyl Sulphate ◽

Dnase I ◽

Target Sequence ◽

Base Pairs ◽

Helix Formation ◽

Dnase I Footprinting ◽

Triple Helix Formation ◽

Triple Helices ◽

Dna Triple Helix

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.

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Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2059 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2059-2069 ◽

Cited By ~ 16

Author(s):

B Kemper ◽

P D Jackson ◽

G Felsenfeld

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Globin Gene ◽

Regulatory Elements ◽

Dnase I ◽

Specific Factor ◽

Mobility Shift ◽

Protein Binding Sites ◽

Dnase I Footprinting ◽

Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

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