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PubMed1997

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein alpha gene.

During adipocyte differentiation, the expression of C/EBPalpha is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program.

3T3 Cells↗
PubMed2004

Genetic and biochemical analysis of cis regulatory elements within the keratinocyte enhancer region of the human papillomavirus type 31 upstream regulatory region during different stages of the viral life cycle.

Using linker scanning mutational analysis, we recently identified potential cis regulatory elements contained within the 5' upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the viral life cycle. For the present study, we extended the linker scanning mutational analysis to identify potential cis elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the viral life cycle. The linker scanning mutational analysis identified viral cis elements located in the KE region that regulate transcription in the presence and absence of any viral gene products or viral DNA replication and determine the role of host tissue differentiation on viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same cis regulatory elements at different stages of the HPV differentiation-dependent life cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify cis regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the viral life cycle. The variable recruitment of transcription factors to the same cis element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.

Base Sequence↗
PubMed1989

Transcriptional regulation by estrogen of episomal prolactin gene regulatory elements.

As a first step in defining the role of chromatin structure in steroid-regulated gene transcription, we have established a steroid-responsive minichromosome system that contains the 5' upstream regulatory region of the rat PRL gene (PRL) from -10 to -1960-basepairs fused to the antibiotic resistance gene, Tn5. The hybrid gene was inserted into a bovine papilloma virus (BPV) vector and then transfected into GH3 cells. Southern analysis of total genomic DNA revealed that the PRL-Tn5-BPV DNA existed in the cells as unrearranged episomes or minichromosomes at a level of 25-100 copies/cell. We monitored the estrogen responsiveness of the minichromosome-based PRL regulatory regions by measuring Tn5 mRNA levels. Treatment of GH3 cells for 48 h with 10 nM 17 beta-estradiol (E2) increased Tn5 mRNA levels 3- to 6-fold over those in untreated cells. Concurrently, endogenous PRL mRNA levels were induced 8- to 15-fold. Using nuclear run-on assays, it was found that E2 increased PRL-Tn5 transcription rates approximately 3-fold over those in untreated cells. The induced transcription was mediated through the PRL elements and not through any other cis-acting elements within the minichromosome. The PRL elements that contain a functional enhancer are located 3' downstream of the BPV early gene promoters in the minichromosome. However, there was no detectable effect of the PRL enhancer on BPV early gene transcription. Thus, we have established a minichromosome system containing the transcriptional regulatory elements of the rat PRL gene that responds to E2 in a manner very similar to the endogenous rat PRL gene.

Animals↗
PubMed2003

[Effects of Mycobacterium tuberculosis infection on the transcriptional expression of human macrophage gene encoding ion channels and related regulatory elements].

Expression microarray was employed in this study to investigate whether the ion channels and their regulatory elements encoding genes participate in the immune response to Mycobacterium tuberculosis infection. The results of a virulent strain were compared with those of the clinically isolated strains. The data demonstrate that K(+), Na(+), Ca(2+) and Cl(-) channels and their regulatory elements, such as the G protein, receptor and second messenger, protein kinase and protein phosphatase were involved in the immune reaction. The clinical strain affected more types of ion channels and respective regulatory elements. The data provides clues for further scrutiny into the role of ion channels and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.

Gene Expression Regulation↗
PubMed2001

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Animals↗
PubMed1995

Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts.

Hepatitis B virus S transcripts contain a region, known as the posttranscriptional regulatory element (PRE), that activates their transport from the nucleus to the cytoplasm. J. Huang and T. J. Liang (Mol. Cell. Biol. 13:7476-7486, 1993) have shown that this element can partially substitute for the human immunodeficiency virus Rev-response element (RRE) in a reporter plasmid that is dependent on the RRE and Rev protein for expression and concluded that PRE exhibits Rev-RRE-like functions by inhibiting splicing. However, we have obtained additional data which indicate that the PRE functions in a novel manner that is not dependent on inhibition of splicing. Unlike Rev-RRE, the PRE functions independently of splice donor and acceptor sites and can activate cytoplasmic expression of an intronless (so-called prespliced) beta-globin transcript. Conversely, a heterologous intron can substitute for the PRE in increasing cytoplasmic expression of hepatitis B virus S transcripts. In addition, the host nuclear factor, YL2 (p32), which enhances Rev-RRE function has no effect on PRE-dependent gene expression. Since S transcripts are not normally known to be spliced and since RNA splicing and cytoplasmic transport are tightly linked processes in higher eucaryotic cells, we conclude that the PRE functions in cis to allow the export of nuclear transcripts that do not interact efficiently with the splicing pathway and hence are normally not exported well from the nucleus. Such elements are critical for the life cycle of viruses, such as hepatitis B virus, which undergo reverse transcription during replication.

Base Sequence↗
PubMed1994

Positive and negative regulatory elements are involved in transcriptional control of the rat glucokinase gene in the insulin producing cell line HIT M2.2.2.

Nested deletion mutants of the 5' flanking region of the beta-cell transcription unit of the rat glucokinase gene (r beta GK) were fused to the CAT-reporter gene. Transient expression studies in HIT M2.2.2 and BHK21 cells revealed a distal (upstream of -359) and a proximal promoter region (between -278/-49) harbouring positive and negative regulatory elements. DNaseI footprinting revealed three protected areas between nucleotides -190 and -60. DNA-elements playing a crucial role in transcriptional control of the insulin genes (IEB- and CT-motifs) have been detected within the proximal promoter region and contribute to beta-cell specific gene regulation. 3' deletion analysis revealed that DNA-elements located downstream from transcription initiation sites (up to +123) contribute to transcriptional regulation.

Animals↗
PubMed2006

A rare missense mutation in a type 2 diabetes patient decreases the transcriptional activity of human sterol regulatory element binding protein-1.

Sterol regulatory element binding protein 1 (SREBP-1) transcription factors play a key role in energy homeostasis by regulating genes involved in both carbohydrate and lipid metabolism, and in adipocyte differentiation. The 5' end of the mRNA-encoding SREBP-1 exists in two forms, designated 1a and 1c. The divergence results from the use of two transcription start sites that produce two separate 5' exons, each of which is spliced to a common exon 2. Mutations in the sterol regulatory element binding protein gene (SREBF)-1 may contribute to insulin resistance states. However, the variants described to date do not affect the SREBP function. In this study, we investigated the functional consequences of a novel missense mutation common to both SREBP-1 isoforms identified in a Spanish type 2 diabetic patient (c.677C>T, SREBP-1a p.T226M; c.605C>T, SREBP-1c p.T202M). Using reporter gene analysis and electrophoretic mobility shift assays, we found that this variant impairs the transcriptional activity and reduces DNA binding ability despite its comparable protein stability to the wild-type SREBP-1. This decreased activity impairs the expression of known downstream targets, such as the LDL receptor and fatty acid synthase genes. Our findings suggest that the threonine residue and/or surrounding region play an important role in the SREBP-1 function.

Animals↗
PubMed2003

The time of appearance of the C. elegans let-7 microRNA is transcriptionally controlled utilizing a temporal regulatory element in its promoter.

MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Here, we show that, like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. We also find that the nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. We speculate that these heterochronic genes regulate let-7 expression through its TRE.

Animals↗
PubMed2004

Surface plasmon resonance-based sensors to identify cis-regulatory elements.

In eukaryotes, transcription is regulated by multiprotein complexes binding to specific regions of genomic DNA, called cis-regulatory elements. Comprehensive identification of these elements is an important goal of functional genomics. Hence, it is of practical interest to develop a high-throughput assay to identify cis-regulatory elements. Toward that goal, we demonstrate that a surface plasmon resonance-based assay can identify whether a specific region of DNA binds to proteins present in raw nuclear lysate. Specifically, we immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter to the Texas Instruments Spreeta, a surface plasmon resonance sensor. As a control, in a separate experiment, we immobilized a similar piece of DNA that differed by only a single base pair. We observed a significant difference in surface plasmon resonance signal when these two probes were exposed to raw nuclear lysate from NIH/3T3 cells. Using a luciferase-reporter vector transfected into live NIH/3T3 cells, we measured a significant difference in transcriptional activity between the two pieces of DNA. We conclude that a surface plasmon resonance-based sensor is capable of identifying physiologically significant cis-regulatory elements.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Multiple ocs-like elements required for efficient transcription of the mannopine synthase gene of T-DNA in maize protoplasts.

Regulatory elements controlling transcriptional activity of the mannopine synthase 2' promoter (mas 2') were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between -305 and -290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5' deletions downstream of -153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position -325 increased activity by up to 7.5-fold. Insertion of the -325 to -275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5'-deletion mutants. Restoration of activity was not obtained with mutants deleted past position -127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (-306 to -275; mas 1' element), d (-127 to -108), and e (-82 to -39; mas 2' element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2' promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2' transcription.

Agrobacterium tumefaciens↗
PubMed1987

The vitamin D-binding protein gene contains conserved nucleotide sequences that respond to heavy metal, adipocyte and mitotic signals.

Characterization of the 5'-flanking region of the DBP genomic sequence is reported. Nucleotide sequences that serve as cis-regulatory elements for transcription in other genes have been found and include metal regulatory elements, viral enhancers, adipocyte and mitotic signals. The promoter region of DBP is not homologous to the 5'-flanking region of the albumin and alpha-fetoprotein genes despite the strong protein homology and evolutionary relationship among the three proteins.

Adipose Tissue↗
PubMed1994

Three distinct regions within the constitutive activation domain of cAMP regulatory element-binding protein (CREB) are required for transcription activation.

The transcription factor cAMP regulatory element-binding protein (CREB) mediates both constitutive and cAMP-induced gene expression through distinct, independently acting domains. The constitutive activation domain (CAD) (amino acids (aa) 165-252) encompasses and overlaps exon 9 of the CREB gene (E9, aa 180-243). In the present study, deletion of either the CAD or exon 9 from CREB-GAL4 (CRG) reduced constitutive activity to less than 2-fold, without affecting kinase inducible activity. However, fusion of the CAD to the GAL4 DNA binding domain (CAD-G4) stimulated transcription, whereas fusion of exon 9 sequences did not. Deletion of the amino-terminal flanking region of exon 9 (aa 165-180), but not COOH-terminal flanking sequences (aa 243-252), decreased constitutive activation in either the CAD-G4 or CRG background. Deletion of the previously characterized glutamine-rich region (Q3, aa 218-252) or of a region containing a hydrophobic cluster of amino acids (HC, aa 180-218) also reduced constitutive activation by either CAD-G4 or CRG. No single mutation of hydrophobic residues within HC impaired activity of the CAD, but double and triple mutations did, suggesting that multiple weak interactions are involved in function of the HC region. Thus, exon 9 of the CREB gene is necessary but not sufficient for constitutive activation. The CAD requires three distinct regions for function, suggesting that CREB may interact with multiple targets in the RNA polymerase II complex.

Amino Acid Sequence↗
PubMed2006

HOX11L2/TLX3 is transcriptionally activated through T-cell regulatory elements downstream of BCL11B as a result of the t(5;14)(q35;q32).

The t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation.

Cell Differentiation↗
PubMed1998

Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements.

We analyzed the expression of mouse DMAHP / Six5 (the myotonic dystrophy-associated homeodomain protein gene) during embryogenesis and in various tissues by northern blotting. Expression was observed as early as embryonic day 7 (E7) and continued to E17. Abundant expression was observed in neonatal heart and skeletal muscle with potential links to the phenotype of myotonic dystrophy. The transcription initiation sites of the gene were analyzed in mouse E11 and E15 embryos and in adult skeletal and heart muscle. Three major transcription initiation sites were identified, the proximal site was specific to the early E11 embryo, while the other two were common among the heart and skeletal muscle and E11 and E15 embryos. All transcription initiation sites were downstream of the corresponding CTG repeat locus of the mouse gene (-1195), excluding a possible inclusion of the CUG repeat sequence in mRNA leading to abnormal splicing or to translation of aberrant protein. For analysis of the regulatory elements in the promoter region, we used P19 embryonal carcinoma cells which abundantly express mouse DMAHP / Six5. Multiple positive and negative elements were identified in the promoter region. All positive elements were Sp1/Sp3 binding sites and one of the negative elements was a novel factor binding site. The transcription initiation sites and regulatory elements are conserved between human and mouse DMAHP.

Animals↗
PubMed1982

Expression of the ROAM mutations in Saccharomyces cerevisiae: involvement of trans-acting regulatory elements and relation with the Ty1 transcription.

The regulatory mutations in Saccharomyces cerevisiae designated cargA + Oh, cargB + Oh, and durOh are alterations in the control regions of the respective structural genes. The alteration causing the cargA + Oh mutation has been shown to be an insertion of a Ty1 element in the 5' noncoding region of the CAR1 ( cargA ) locus. All three mutations cause overproduction of their corresponding gene products and belong to the ROAM family of mutations (Regulated Overproducing Allele responding to Mating signals) in yeast. The amount of overproduction in ROAM mutants is determined, at least in part, by signals that control mating functions in yeast. We report the identification of two genetic loci that regulate Oh mutant gene expression but that do not affect mating ability. These loci are defined by the recessive roc mutations ( ROAM mutation control) that reduce the amount of overproduction caused by the cargA + Oh, cargB + Oh, and durOh mutations. RNAs homologous to CAR1 ( cargA ), DUR1 ,2 and Ty1 DNA probes were analyzed by the Northern hybridization technique. In comparison with wild-type strains, cargA + Oh and durOh mutant strains grown on ammonia medium contain increased amounts of CAR1 and DUR1 ,2 RNA. This RNA overproduction is diminished in MATa/MAT alpha diploid strains as well as in haploid strains that also carry the ste7 mutation which prevents mating or that carry either of the roc1 or roc2 mutant alleles. The amount of RNA homologous to Ty1 DNA is also reduced in ste7 , roc1 , and roc2 mutant strains. This reduction is not observed in a strain with the ste5 mutation, which prevents mating but has no effect on overproduction of ROAM mutant gene products.(ABSTRACT TRUNCATED AT 250 WORDS)

Allophanate Hydrolase↗
PubMed1998

Structure and expression of the human Na,K-ATPase beta 2-subunit gene.

We cloned and characterized the human Na,K-ATPase beta 2-subunit gene. The gene encompasses over 8 kb at chromosome 17 in the human genome and is composed of seven exons. Primer extension analysis identified a major transcription initiation site 529 bases upstream of the translation start site. The 5'-flanking region of the gene harbors a potential TATA sequence, located 94 bases upstream of the transcription initiation site and a number of potential promoter and regulatory elements, among them a Sp1 site, at position -120. A functional Sp1 site has also been found in the rat Na,K-ATPase beta 2-subunit gene (Kawakami, K., Watanabe, Y., Araki, M., Nagano, K., 1993). Sp1 binds to the adhesion molecule on glia regulatory element that functions as a positive transcription regulatory element in astrocytes. (J. Neurosci. Res. 35, 138-146). Putative AATAAA and TG sequences were found at positions 7018 and 7068, respectively. These signals delimit the origin of the the poly(A) tail and mark the end of the sequence that completes the 3'-UT downstream sequence of the human cDNA. An Alu repetitive sequence is located between positions 5961 and 6274. The gene is expressed as a single mRNA species, of 3.36 kb, which is present in cerebrum, cerebellum, kidney and heart, being more abundant in neural tissues. Structural analyses of this and other of the P-type ATPase beta subunit genes reveal that they evolved from a common ancestor.

Animals↗