Cis-acting Transcriptional Elements Research Articles

Importance of the CArG box in regulation of β-actin-encoding genes

The β-actin-encoding gene ( Act) in carp is regulated by several cis-acting regulatory elements including the evolutionarily conserved CC(A/T) 6GG ( CArG box or serum-response element) sequences positioned in the promoter region between the CAAT and TATA boxes and in the first intron. To address the roles of the two CArG boxes on gene expression, we replaced them with linker sequences. The CArG box in the proximal promoter was not required for promoter activity in tissue-cultured cells, but was required in conjunction with a second CArG box in the first intron to give full expression in transgenic embryos. Likewise, the geometry of cis-acting transcriptional elements in the proximal promoter was more important for expression of transgenic constructs in developing embryos than in tissue-cultured fibroblasts. Mobility-shift and exonuclease mapping experiments indicated that the same or similar protein factors bind around the two CArG boxes, suggesting that interactions between the promoter and the first intron are involved in Act regulation.

A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the kappa B enhancer element

The immunoglobulin kappa light chain enhancer, kappa B, is an important cis-acting transcriptional element. kappa B binds a number of proteins including the members of the ubiquitous NF-kappa B family of transcription factors. Agarose beads coupled to a double-stranded oligonucleotide containing the kappa B motif were used to isolate a 65-kDa predominantly nuclear phosphoprotein. Southwestern blot analysis demonstrated that this phosphoprotein can bind the kappa B element directly and specifically. This kappa B-associated protein was phosphorylated in vivo and in vitro by a nuclear serine/threonine kinase(s) which, in a number of different cell lines, appeared to be stimulated in response to interleukin-1 alpha and lipopolysaccharide treatment. In the B cell lines 70Z/3 and CH12 LX2B, and the T cell line EL-4 6.1 C10 the activity of the kappa B-associated kinase(s) correlated with the binding activity of nuclear NF-kappa B displayed in a gel shift assay. In vitro, the 65-kDa protein was phosphorylated in the absence of exogenously added kinase. The 65-kDa phosphoprotein and the kinase activity remained associated following sequential anion-exchange and hydrophobic interaction chromatography. These results suggest that the kappa B-associated phosphoprotein is either autophosphorylated or is phosphorylated by a closely associated kinase(s). Stimulation of a nuclear protein kinase which is closely associated with a sequence-specific DNA may reflect a novel mechanism by which growth factors regulate gene expression.

The v-src inducible gene 9E3/pCEF4 is regulated by both its promoter upstream sequence and its 3' untranslated region.

The 9E3/pCEF4 mRNA is strongly induced in Rous sarcoma virus-transformed chicken embryo fibroblasts when compared to untransformed cells. To identify cis-acting transcriptional elements that confer inducibility by v-src, we isolated the 9E3 promoter upstream region. We found that 1.53 kilobases upstream of the transcriptional start site, when placed in front of a reporter gene, conferred a small degree of inducibility by v-src, in both transient and stable transfections. Two potential AP-1 sites were identified in the 9E3 promoter. AP-1 elements have been implicated previously in mediating a transcriptional response to v-src in fibroblast cell lines. These elements alone do not confer a significant inducibility by v-src in primary chicken embryo fibroblasts. Since the 9E3 mRNA is stabilized in transformed cells, we replaced the 3' untranslated region of the reporter gene with the 9E3 3' untranslated region and found this construct to be strongly responsive to stimulation by v-src. In addition, the 9E3 3' untranslated region increased the response to serum and the tumor promoter phorbol 12-myristate 13-acetate. This suggests that a posttranscriptional mechanism plays a major role in the induction of 9E3 expression.

A specific cis-acting element regulates in vitro transcription of sex-dependent mouse steroid 16 alpha-hydroxylase (C-P450(16 alpha)) gene.

The mouse steroid 16 alpha-hydroxylase (C-P450(16 alpha)) family consists of structurally similar genes with very divergent expressions (Wong, G., Itakura, T., Kawajiri, K., Skow, L., and Negishi, M. (1989) J. Biol. Chem. 264, 2920-2927). Of five family members, the C-P450(16 alpha) gene exhibits a male-specific expression whereas the P450cb gene is equally expressed in both males and females although the level of expression is much lower than that of the C-P450(16 alpha) gene. By DNase I footprinting and in vitro transcription of the C-P450(16 alpha) gene, we determined two cis-acting transcription elements. SDI (sex difference information) in the -84/-102 region from the transcription start site is one of the two cis-acting transcription elements, which is specific to the sex-specific C-P450(16 alpha) gene and confers a high transcription level of this gene. We substituted the nucleotide(s) in the SDI sequence to the corresponding base(s) in the P450cb gene promoter and tested each substituted gene promoter by in vitro transcription. The results indicate that the function of SDI as the transcription element depends most critically on the nucleotides at positions -100 to -98. Another cis-acting transcription element, CTE (common transcription element), is located between -44 and -68 in both C-P450(16 alpha) and P450cb genes. A column chromatography of nuclear extract indicates that the proteins bound to SDI and CTE are different. We speculate therefore that SDI is a specific and necessary element involved in the male-specific C-P450(16 alpha) gene transcription. The sequence comparisons of the corresponding regions of SDI and CTE among the mouse and the closely related rat P450 genes suggest that SDI is a newly evolved cis-acting element in the sex-specific C-P450(16 alpha) gene which arose through gene duplication and selective nucleotide substitution.

Loss of a DNA-protein complex correlates with extinguished major histocompatibility complex class II expression in a human B cell.

An E beta DNA protein complex termed complex A, whose binding activity has recently been shown to correlate with both constitutive and regulated class II expression in murine cell lines, is also present in a human B cell, Raji. The DNA involved in complex A, which includes three previously defined transcriptional motifs, W, X, and Y, is a cis-acting transcription element in Raji cells. Both complex A binding activity and transcriptional activity of its target sequence are absent in an Ia- mutant subclone of Raji, RJ 2.2.5. This cell line, whose defect is complemented by a locus on mouse chromosome 16, reexpresses both class II and complex A upon transfection with mouse genomic DNA. We suggest that factors that form complex A or that regulate complex A formation account for the molecular lesion in this cell line.

The presence of a DNA binding complex correlates with E beta class II MHC gene expression.

The class II major histocompatibility complex (MHC, Ia) antigens are a family of membrane proteins whose expression is strictly regulated. They have a limited tissue distribution and their expression is regulated both developmentally and in response to external stimuli. Here we report the identification of a DNA binding protein complex (termed complex A) within the murine E beta MHC gene, which is restricted to cells that express Ia antigens. Complex A binding activity is developmentally regulated in cells of the B lineage in accordance with class II expression and is responsive to two different Ia-inducing lymphokines, interferon-gamma in macrophages and interleukin-4 in pre-B cells. The DNA target sequence in complex A includes three previously defined transcriptional motifs W, X and Y, and acts as a cis-acting transcription element. Complex A is present both in cells that are constitutive for class II MHC expression and in cells that have been induced for class II MHC expression. These results suggest that complex A may play a critical role in the regulation of class II MHC gene expression.

Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro

SV40 DNA replication in vivo is greatly stimulated by cis-acting transcriptional elements. We studied a model viral chromosome containing a single binding site for the cellular transcriptional activator, nuclear factor I (NF-ICTF), located adjacent to the replication origin. The presence of the NF-I recognition site increased replication efficiency over 20-fold in vivo. Purified NF-I had little effect on the replication efficiency in the standard SV40 cell-free system when the template was introduced as naked DNA. However, NF-I specifically prevented the repression of DNA replication that occurred when the template was preassembled into chromatin. Our data support a model in which the binding of a transcriptional activator perturbs the local distribution of nucleosomes, thereby increasing the accessibility of the origin region.

Regulatory elements controlling the basal and drug-inducible expression of glutathione S-transferase Ya subunit gene.

The synthesis of the glutathione S-transferase Ya subunit is induced in the mammalian liver by chemicals such as phenobarbital and 3-methylcholanthrene. To study the mechanism of this induction, the 5'-flanking region of a mouse glutathione S-transferase Ya subunit gene was fused to the structural gene for chloramphenicol acetyltransferase. The fusion gene was introduced into hepatoma cells for the assay of the expressed acetyltransferase activity. At least two cis-regulatory elements were identified in the 5'-flanking region of the Ya gene: one, responsible for the basal level of expression, is present in the sequence up to -0.2 kb; another, responsible for the inducible expression by aromatic compounds such as beta-naphthoflavone and 3-methylcholanthrene, is located in the sequence from -0.2 kb to -1.6 kb. The inducible element was functional only in cells with normal aromatic compound receptors, and it retained responsiveness to beta-naphthoflavone when transfected into homologous (mouse) or heterologous (rat, human) hepatoma cells. A 150-bp region upstream from the transcription initiation site of the mouse Ya gene was investigated for cis-acting transcriptional elements that are recognized by specific DNA-binding proteins. We show by DNase I foot-printing assays using extracts from liver nuclei that the Ya gene promoter contains, in addition to the TATA and CCAAT boxes, a more distal element that binds a protein which is probably related to the family of nuclear factor 1 (NF1).

Purification and properties of the Rous sarcoma virus internal enhancer binding factor.

The internal enhancer binding factor (IBF) that specifically binds sequences within the gag gene internal enhancer of Rous sarcoma virus Schmidt-Ruppin A was purified to near homogeneity from BHK cells. The polypeptides that constituted IBF DNA-binding activity were identified by sodium dodecyl sulfate-polyacrylamide gel analysis. As isolated from BHK cells, IBF consisted of two different but related polypeptides. One (IBF alpha) had a molecular weight of 40,000; the other (IBF beta) had a molecular weight of 20,000 and appeared to be a proteolytic product of IBF alpha. The site within the gag gene to which IBF bounds in vitro (internal enhancer site 2; nucleotides 856 to 878 of the Rous sarcoma virus genome) were demonstrated to function as a cis-acting transcriptional stimulatory element both in vivo and in vitro. By using HeLa cell nuclear transcription extracts, purified IBF was found to function as a trans-acting transcription factor that stimulated transcription in vitro. Purified IBF was also demonstrated to be very similar to EBP20 (K. Carlberg, T. A. Ryden, and K. Beemon, J. Virol. 62:1617-1624, 1988), and it may well belong to the same family of DNA-binding proteins.

Purification and Properties of the Rous Sarcoma Virus Internal Enhancer Binding Factor

The internal enhancer binding factor (IBF) that specifically binds sequences within the gag gene internal enhancer of Rous sarcoma virus Schmidt-Ruppin A was purified to near homogeneity from BHK cells. The polypeptides that constituted IBF DNA-binding activity were identified by sodium dodecyl sulfate-polyacrylamide gel analysis. As isolated from BHK cells, IBF consisted of two different but related polypeptides. One (IBF alpha) had a molecular weight of 40,000; the other (IBF beta) had a molecular weight of 20,000 and appeared to be a proteolytic product of IBF alpha. The site within the gag gene to which IBF bounds in vitro (internal enhancer site 2; nucleotides 856 to 878 of the Rous sarcoma virus genome) were demonstrated to function as a cis-acting transcriptional stimulatory element both in vivo and in vitro. By using HeLa cell nuclear transcription extracts, purified IBF was found to function as a trans-acting transcription factor that stimulated transcription in vitro. Purified IBF was also demonstrated to be very similar to EBP20 (K. Carlberg, T. A. Ryden, and K. Beemon, J. Virol. 62:1617-1624, 1988), and it may well belong to the same family of DNA-binding proteins.

A cis-acting transcription element of the c-myc gene can assume an H-DNA conformation.

I have used chemical probes and an oligonucleotide-association assay to determine the structure of a nuclease-sensitive, c-myc DNA region. I find that this DNA region can form a triplex-single stranded conformer in vitro--the H-DNA conformer. This DNA region has been shown previously to be a positive, cis-acting transcription element of the c-myc gene and to bind nuclear factors, including a base-paired ribonucleoprotein. Therefore, H-DNA may be a functionally important in vivo topoisomer where the H-DNA and B-DNA conformers have different transcriptional activities.

Growth Factors Regulate Transin Gene Expression by c- fos -Dependent and c- fos -Independent Pathways

The rapid induction of the proto-oncogene c-fos by growth factors and other bioactive agents, and the recent evidence that the c-fos protein (Fos) is associated with transcriptional complexes, suggests that Fos may represent an integral part of an intracellular messenger pathway that triggers changes in gene expression and ultimately phenotypic alterations. This report examines the role of c-fos in growth factor stimulation of transin, a matrix-degrading secreted metalloproteinase. Platelet-derived growth factor (PDGF) stimulation of transin RNA was blocked by a selective reduction in Fos synthesis with antisense c-fos mRNA, whereas epidermal growth factor (EGF) stimulation of transin occurred despite an equivalent inhibition of Fos levels. The stimulatory effect of both PDGF and EGF on transin transcription involved factors recognizing the sequence TGAGTCA, which is found in the transin promoter and is reported to be a binding site for the transcriptional factor Jun/AP-1 and for associated Fos and Fos-related complexes. Thus both Fos-dependent and Fos-independent pathways exist for growth factor regulation of gene expression, and both effects may be mediated through the same cis-acting transcription element.

Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis.

The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors. We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3. These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes. Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes. HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other. Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1. We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter. However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences. Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription. Alternatively, TF-X1 may not be a DNA-binding protein at all.

Evidence for positive and negative regulation in the promoter of the chicken δ1-crystallin gene

We investigated the role of sequences flanking the transcription initiation site of the δ1-crystallin gene in transient transfection assays of primary embryonic chicken lens epithelial cells or fibroblasts. Varying lengths of the 5′ flanking sequence of the δ1-crystallin gene (containing some untranslated sequence from exon 1) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene in the pSVOCAT plasmid. A plasmid carrying the bacterial β-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter was used as an internal control. Standardized results showed that the sequence located between −120 to −43 exhibited strong promoter activity; however, the promoter activity was markedly reduced (20-fold) when the upstream sequence between −603 and −120 was included in the construct. The δ1-crystallin promoter displayed little lens preference. This upstream sequence did not reduce the activity of the Simian virus 40 (SV40) early promoter (with or without its enhancer) or the Herpes thymidine kinase promoter in transfection tests, indicating some specificity in its effect. Evidence for a δ1-crystallin negative trans-acting factor was provided by competition experiments. Our data raise the possibility that expression of the δ1-crystallin gene involves a negative cis-acting transcription element, a speculation which may deserve further attention in view of the gradual decrease in δ-crystallin synthesis in the developing lens.

Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and that the particular combination of domains used is dependent on the availability, in kind or amount, of trans-acting, transcription-modulating factors present in each cell type. Thus, multiple myogenic factors that vary qualitatively and quantitatively may be responsible for the different and complex modulatory programs of actin gene expression observed during in vivo muscle differentiation.

Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

Transfection into myogenic and nonmyogenic cell lines was used to investigate the transcriptional regulation of the human alpha-skeletal actin gene. We demonstrated that 1,300 base pairs of the 5'-flanking region directed high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated mouse C2C12 and rat L8 myotubes but not in mouse nonmuscle L.TK- and HuT-12 cells. Unidirectional 5' deletion analysis and heterologous promoter stimulation experiments demonstrated that at least three transcription-regulating subdomains lie in this 1,300-base-pair region. A proximal cis-acting transcriptional element located between positions -153 and -87 relative to the start of transcription at +1 was both sufficient and necessary for muscle-specific expression and developmental regulation during myogenesis in the two myogenic cell systems. The region 3' of position -87 interacted with factors present in both myogenic and fibroblastic cells and appeared to define, or to be a major component of, the basal promoter. In C2C12 myotubes, but not in L8 myotubes, a distal sequence domain between positions -1300 and -626 and the proximal sequence domain between positions -153 and -87 each induced transcription about 10-fold and synergistically increased CAT expression 100-fold over levels achieved by the sequences 3' of position -87. Furthermore, these cis-acting elements independently and synergistically modulated an enhancerless, heterologous simian virus 40 promoter in a tissue-specific manner. DNA fragments which included the proximal domain displayed classical enhancerlike properties. The central region between positions -626 and -153, although required in neither cell line, had a positive, two- to threefold, additive role in augmenting expression in L8 cells but not in C2C12 cells. This suggests that certain elements between positions -1300 and -153 appear to be differentially utilized for maximal expression in different myogenic cells and that the particular combination of domains used is dependent on the availability, in kind or amount, of trans-acting, transcription-modulating factors present in each cell type. Thus, multiple myogenic factors that vary qualitatively and quantitatively may be responsible for the different and complex modulatory programs of actin gene expression observed during in vivo muscle differentiation.