Cis-acting Regulatory Regions Research Articles

Cis-acting regulatory regions in the long terminal repeat of simian foamy virus type 1.

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.

Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.

In French bean, the glycine-rich cell wall protein GRP 1.8 is specifically synthesized in the vascular tissue. To identify cis-acting sequences required for cell type-specific synthesis of GRP 1.8, expression patterns of fusion gene constructs were analyzed in transgenic tobacco. In these constructs, the uidA (beta-glucuronidase) gene was placed under control of 5' upstream deletions as well as internal deletions of the GRP 1.8 promoter. Four different cis-acting regulatory regions, SE1 and SE2 (stem elements), a negative regulatory element, and a root-specific element, were found to control the tissue-specific expression. Deletion of the negative regulatory element resulted in expression of the uidA gene in cell types other than vascular cells. The SE1 region was essential for expression in several cell types in the absence of further upstream regulatory sequences. Full-length promoters having insertions between the negative regulatory element and SE1 strongly expressed the gene in nonvascular cell types in stems and leaves. Thus, vascular-specific expression of the GRP 1.8 promoter is controlled by a complex set of positive and negative interactions between cis-acting regulatory regions. The disturbance of these interactions results in expression in additional cell types.

Expression of micF involved in porin synthesis in Escherichia coli: two distinct cis-acting elements respectively regulate micF expression positively and negatively

micF RNA, whose sequence is highly complementary to a 5'-portion of ompF mRNA, has been implicated in the osmoregulation and thermoregulation of the ompF porin gene in Escherichia coli. To define and characterize cis-acting regulatory regions upstream of the micF promoter, a series of deletions of the micF promoter fused to the lacZ gene were constructed. Two distinct regions, which function differently, were identified as cis-acting regulatory elements, namely, one responsible for OmpR-dependent activation and the other for OmpR-independent repression of micF expression. The former contains the OmpR-binding site, which simultaneously regulates both the genes, micF and ompC, in response to the medium osmolarity. The latter may be involved in an unknown regulatory process of micF expression.

Expression ofmicFinvolved in porin synthesis inEscherichia coli: two distinctcis-acting elements respectively regulatemicFexpression positively and negatively

micF RNA, whose sequence is highly complementary to a 5'-portion of ompF mRNA, has been implicated in the osmoregulation and thermoregulation of the ompF porin gene in Escherichia coli. To define and characterize cis-acting regulatory regions upstream of the micF promoter, a series of deletions of the micF promoter fused to the lacZ gene were constructed. Two distinct regions, which function differently, were identified as cis-acting regulatory elements, namely, one responsible for OmpR-dependent activation and the other for OmpR-independent repression of micF expression. The former contains the OmpR-binding site, which simultaneously regulates both the genes, micF and ompC, in response to the medium osmolarity. The latter may be involved in an unknown regulatory process of micF expression.

Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen

Bradyrhizobium japonicum expresses hydrogenase in microaerophilic free-living conditions in the presence of nickel. Plasmid-borne hup-lacZ transcriptional fusion constructs were used to study the regulation of the hydrogenase gene. The hydrogenase gene was transcriptionally induced under microaerobic conditions (0.1 to 3.0% partial pressure O2). The hydrogenase gene was not transcribed or was poorly transcribed in strictly anaerobic conditions or conditions above 3.0% O2. Hydrogen gas at levels as low as 0.1% partial pressure induced hydrogenase transcription, and a high level of transcription was maintained up to at least 10% H2 concentration. No transcription was observed in the absence of H2. Hydrogenase was regulated by H2, O2, and Ni when the 5'-upstream sequence was pared down to include base number -168. However, when the upstream sequence was pared down to base number -118, the regulatory response to O2, H2, and Ni levels was negated. Thus, a common cis-acting regulatory region localized within 50 bp is critical for the regulation of hydrogenase by hydrogen, oxygen, and nickel. As a control, the B. japonicum hemA gene which codes for delta-aminolevulinic acid synthase was also fused to the promoterless lacZ gene, and its regulation was tested in the presence of various concentrations of O2 and H2. hemA-lacZ transcription was not dependent on levels of Ni, O2, or H2. Two different hup-lacZ fusions were tested in a Hup- background, strain JH47; these hup-lacZ constructs in JH47 demonstrated dependency on nickel, O2, and H2, indicating that the hydrogenase protein itself is not a sensor for regulation by O2, H2, or nickel.

Transcription of the myxobacterial hemagglutinin gene is mediated by a sigma 54-like promoter and a cis-acting upstream regulatory region of DNA

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation of the fruiting bacterium Myxococcus xanthus. In this study, DNA sequences mediating the transcriptional regulation of mbhA have been identified. Examination of nucleotide sequences upstream of the start site for mbhA transcription has indicated a region of DNA that bears strong homology to the consensus sequence for promoters recognized by the sigma 54 holoenzyme form of RNA polymerase of Escherichia coli and other eubacteria. Deletion of this sequence completely abolished mbhA transcription. Additionally, a cis-acting DNA element, affecting the efficiency of mbhA transcription, has been mapped within a region of DNA 89 to 276 nucleotides upstream of the sigma 54-like sequence. Transposon insertions, mapping within the cis element, drastically reduced mbhA transcriptional activity. These observations suggest that transcription of mbhA requires a productive interaction between a form of RNA polymerase that recognizes a sigma 54-like sequence and a transcriptional activator that binds to DNA sequences upstream of the mbhA promoter.

Factors influencing transient expression in cytotoxic T cells following DEAE dextran-mediated gene transfer

A number of transfection protocols have been tested for the introduction of exogenous DNA into cytotoxic T cells. These included electroporation, lipofection, calcium phosphate coprecipitation, polybrene-assisted gene transfer, and DEAE dextran-mediated transfer. Only the latter gave significant and reproducible transfection efficiencies coupled with low toxicity. The DEAE dextran protocol was optimized for the transfection of a transcription reporter construct pRSVcat into a cloned cytotoxic cell line. Among the parameters investigated were cell density, amount of input DNA, concentration of DEAE dextran, DNA adsorption time, temperature, use of permeabilization and expression facilitators, and recovery time. The optimized protocol was then used to demonstrate the presence of cis-acting regulatory regions in the 5'-flanking sequences of two cytotoxic cell-specific serine protease genes and, in addition, was shown to be applicable to other cloned T-cell lines.

Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon

Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.

Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.

Editorial: Primary Erythrocytosis in Childhood

Regulation of erythropoiesis to provide the right number of circulating red cells for optimal oxygen transport is a complex process in which the hormone erythropoietin (EPO) plays a central role. Exciting new research is bringing into focus the nature of the biochemical processes that regulate EPO production and the interaction of EPO with target hematopoietic progenitor cells. An oxygen sensor in the adult kidney or fetal liver, thought to be a heme protein,' modulates EPO synthesis so as to maintain normal tissue oxygenation. As reflected in studies of hepatoma cell lines that respond to hypoxia by synthesizing EPO, in the liver the oxygen sensor is found in the same cell that produces EPO so that regulatory signals, which are as yet undefined, may exert their action locally rather than at a distance. In hepatomas, EPO gene expression is regulated at the mRNA transcriptional level and is the consequence of both increased EPO mRNA synthesis and diminished EPO mRNA degradation.* Cis-acting regulatory regions that serve as promotors or enhancers have been identified adjacent to the coding region of the EPO gene3 and the precise way in which these regions, as well as more distant transacting factors, govern EPO gene expression is under active investigation. In situ hybridization studies in the kidney have shown that EPO is produced in tubular epithelial cells or in peritubular interstitial cells.' However, little is known about the renal oxygen sensor, because an in vitro experimental system derived from renal tissue that secretes EPO is not available. EPO stimulates the proliferation and differentiation of erythroid progenitor cells in the fetal liver or postnatal bone marrow. These cells display only sparse numbers of EPO receptors (200-1000 per cell), which exhibit both high- and low-affinity binding of radiolabeled recombinant EPO. The EPO receptor gene has been cloned from murine hematopoietic tissue by D'Andrea et al.4 It consists of 507 amino acids, has a single membrane-spanning domain, and shares considerable homology with several other growth factor receptor proteins (GMCSF, IL-3, IL-4, IL-5, prolactin, and human growth h~rmone).~ Cross-linking studies that use radiolabeled EPO suggest that two EPO receptor proteins (MW 80 kda and 100 kda approximately) are found in erythroid progenitors, implyPediatric Hmiolosy and Oncolosy, B:iii-u, 1991

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

Evolutionary Conservation of Homeodomain-Binding Sites and Other Sequences Upstream and within the Major Transcription Unit of the Drosophila Segmentation Gene engrailed

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

The spatial and temporal expression pattern of sevenless is exclusively controlled by gene-internal elements.

The sevenless gene controls the fate of a single photoreceptor cell type in the developing eye of Drosophila. Its RNA and protein accumulate transiently in a subpopulation of cells in the developing eye imaginal disc. We have used P-element transformation with different minigenes and marker gene constructs to identify cis-acting regulatory regions required for the complex sevenless expression pattern. Our results indicate that the upstream region of the sevenless gene is devoid of any detectable regulatory elements and that sequences located 3' to the transcription start site are sufficient to promote the sevenless expression pattern. These gene-internal sequences function in both orientations on heterologous promoters, also when placed at the 3' end of a lacZ reporter gene.

The Epstein-Barr virus (EBV) early promoter DR contains a cis-acting element responsive to the EBV transactivator EB1 and an enhancer with constitutive and inducible activities

The Epstein-Barr Virus (EBV) DR promoter controlled the expression of the PstI repeat region IR4. This promoter was activated by the EBV trans-acting factor EB1, mainly at the transcriptional level, and the activation was mediated by the TATA box and two cis-acting regulatory regions, one proximal to the TATA box and one distal to the TATA box. The distal region had enhancer properties. In HeLa cells, it activated transcription from the herpes simplex virus type 1 thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene when located in inverted orientation upstream of the thymidine kinase promoter or downstream of the chloramphenicol acetyltransferase gene coding sequence. This enhancer also activated transcription from the simian virus 40 early upstream regulatory elements. These results indicate that the DR These results indicate that the DR enhancer can constitutively activate heterologous promoters in HeLa cells. However, the DR enhancer was not active in EBV genome-negative B cell lines, but it became active when these cells were infected by EBV and when the expression of the EBV early genes was induced by EB1. This suggests that an EBV early gene product induces the DR enhancer activity. The DR promoter TATA box-proximal cis-acting regulatory element contained EB1-responsive sequences.

Improvement and application of a promoter-probe vector bearing the PHO5 gene as the indicator marker in Saccharomyces cerevisiae

A promoter-probe plasmid vector, pVC701, in Saccharomyces cerevisiae having the repressible acid phosphatase (rAPase; EC 3.1.3.2) gene, PHO5, of S. cerevisiae as the selection marker (Hwang et al., Appl. Microbiol. Biotechnol., 28, 155–159, 1988) was improved by inserting multiple cloning sites in the 5′ non-transcribed region of PHO5 and by replacing a 93-bp sequence containing three ATG sequences out of the frame but directly flanked by the translation initiation codon ATG with a 5′-CACC-3′ sequence. The resulting YCp type plasmid, pVC727, is replicable in both S. cerevisiae and Escherichia coli hosts. The function of the vector was confirmed by inserting a DNA fragment bearing a promoter region of the GAP gene encoding glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae at the cloning site. Using the vector, we could analyse the cis-acting upstream regulatory region of the HIS5 gene of S. cerevisiae and also isolate Gcd − ( general control derepressed) mutants occurring in the trans-acting loci of the general amino acid control.

A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor.

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor.

Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression. Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression. One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site. Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences. The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells. A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed. The protein-binding site for the constitutive factor coincided with the block II element. Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex. The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

Localization of the cis-acting regulatory DNA sequences of the Myxococcus xanthus tps and ops genes

The cis-acting regulatory regions of the tps and ops genes of Myxococcus xanthus were localized by analyzing the expression of fusions of these genes with the lacZ gene. A 201-base-pair (bp) fragment of tps DNa extending 95 bp upstream (-95) from the transcriptional start was sufficient to direct developmentally regulated expression of fusion gene activity. The segment of tps DNA between -95 and -81 contained information necessary for developmental regulation. A segment of ops DNa extending upstream to -131 directed a very low level of ops-lacZ fusion expression, but the inclusion of DNA to -208 greatly increased the amount of developmentally regulated expression. M. xanthus DNA upstream from -108 in the tps gene and -311 in the ops gene was required for maximal expression of gene fusion activity. The upstream regulatory regions of both the tps and ops genes seem to be involved in positive transcriptional regulation. Two mutations, a deletion of 1 bp at -8 in the tps gene and a 3-bp substitution at -27 to -29 in the ops gene, greatly increased the level of vegetative expression of gene fusion activity, suggesting that both genes may also be subject to negative regulation in M. xanthus.

Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions.

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.

Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene.

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.