Cis-acting Promoter Sequences Research Articles

Identification of Cis-Acting Promoter Elements in Cold- and Dehydration-Induced Transcriptional Pathways in Arabidopsis, Rice, and Soybean

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.

Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice

Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the “classical” sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/ db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.

Regulation of the human tumor necrosis factor-α promoter by angiotensin II and lipopolysaccharide in cardiac fibroblasts: different cis-acting promoter sequences and transcriptional factors

We recently showed that angiotensin (ANG) II as well as mechanical stretch stimulated production of tumor necrosis factor (TNF) in cardiac fibroblasts. Presently, we examined the molecular mechanisms by which ANGII and lipopolysaccharide (LPS) upregulate TNF-α gene expression. In neonatal rat cardiac fibroblasts, increased transcription of TNF-α mRNA was detected as luciferase activity associated with activity of the TNF-α promoter. Progressive deletion from this promoter located the LPS-responsive region between –200 and –120 bp from the transcription initiation site, while the sequence between –120 and –70 bp was required for ANGII-induced expression. Next, we examined which cis–acting sequences in the TNF-α promoter region were essential for induction of TNF-α transcription. Competition analysis by electrophoretic mobility shift assay with and without specific antibodies showed that LPS increased binding of Sp1 and Sp3 to the Sp1–binding site, while Egr-1 was unimportant. With ANGII, binding of ATF-2/c–jun to the CRE site was required for TNF-α gene induction; neither Ets nor NF-κB was essential. Mutation analysis confirmed that response to LPS relied upon the Sp1 site in the TNF-α promoter, while the CRE-binding site was essential for stimulation by ANGII. We concluded that since TNF-α gene expression is transcriptionally activated by ANGII or LPS in cardiac fibroblasts via different cis–acting sequences in the TNF-α promoter and different transcriptional factors, mechanisms inducing TNF production differ between heart failure or cardiac hypertrophy and infectious disease.

12-O-tetradecanoyl-phorbol-13-acetate down-regulates the Huntingtin promoter at Sp1 sites.

We have studied the effects of the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Huntington's disease (HD) gene transcription in neuronal and non-neuronal cell lines, to investigate pathways regulating HD gene expression. TPA reduced transcription from the HD gene promoter in SK-N-SH (neuroblastoma) and HeLa cells but not in JEG3 (choriocarcinoma) cells. In SK-N-SH cells, the responsible cis-acting promoter sequences comprise the tandemly duplicated Sp1 sites in the region from -213 to -174, relative to the translation start site. The TPA-down-regulating region in HeLa cells was mapped to the sequence from -141 to -126. In conclusion, this demonstrates that HD gene transcription can be down-regulated in vitro in a cell-specific manner.

Regulation of IL-2 gene expression through CD28 is dependent on cooperativity between cis-acting promoter sequences

Regulation of IL-2 gene expression through CD28 is dependent on cooperativity between cis-acting promoter sequences

Regulation of IL-2 gene expression through CD28 is dependent on cooperativity between cis-acting promoter sequences

Regulation of IL-2 gene expression through CD28 is dependent on cooperativity between cis-acting promoter sequences

Avian Metallothioneins: Structure, Regulation and Evolution

This article reviews studies of the molecular biology of the avian metallothionein (MT) genes. Analysis of cloned genes and/or cDNAs from chicken, turkey, pheasant and quail suggests that each of these species possesses a very simple MT gene family. The MT from these birds is a cysteine-rich protein of 63 amino acids that shares extensive structural homology with the mammalian MTs, and, remarkably, the deduced amino acid sequence of the major metallothionein is identical in each of these birds. The chicken MT gene is inducible by dietary or injected metal ions [i.e., zinc (Zn) and copper (Cu)], bacterial lipopolysaccharide and oxidative stress. Furthermore, it is expressed during liver development. The turkey and chicken MT genes are identical in gross structure to other functional MT genes. They consist of three exons separated by two intervening sequences. Comparisons of the nucleotide sequences of the turkey and chicken MT genes revealed regions of exceptionally high sequence conservation, suggesting important functions such as splicing, polyadenylation and transcriptional activation. Structure-function studies of the chicken MT promoter using transient transfection assays and transgenic mice have revealed cis-acting promoter sequences involved in the induction of chicken MT gene expression by metals ions. A metal-responsive enhancer was located in the proximal 107-bp of the chicken MT promoter in a region highly conserved in both the turkey and chicken MT genes. Function of this enhancer element apparently requires cooperation of transcription factors interacting with an Sp1 binding site and a single palindromic metal-responsive element. In this regard, the structure of the proximal region of the chicken and turkey MT promoters is unique. Our current studies suggest that this enhancer regulates gene expression in a position-independent manner in transgenic mice.

Interaction of Ethanol with Inducers of Glucose-regulated Stress Proteins: ETHANOL POTENTIATES INDUCERS OF grp78 TRANSCRIPTION

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.

Possible intermediate steps in the evolution of a prokaryotic developmental system

Sigma factors (sigma) are transcription factors that operate global switches in gene expression in prokaryotes. They work by directing core RNA polymerase to specific cis-acting promoter sequences; each sigma has a cognate class of promoters with specific sequence characteristics. In Bacillus subtilis four different sigma factors have been implicated in the regulation of gene expression during spore formation, which is a simple differentiation system involving two cell types. In this review I show how the modern developmental system may have arisen from a primitive organism that used only two sigma factors, by a series of steps involving gene duplication and divergence. The increasing sophistication of eukaryotic developmental systems may reflect similar evolutionary processes.

Derepression of a mouse α-fetoprotein expression vector in COS-1 cells by amplification of specificcis-acting sequences of the AFP promoter

The existence of trans-acting regulatory factors has been demonstrated by in vivo competition with cis-acting sequences from both viral and eukaryotic genomes. Plasmids containing a functional SV40 origin of replication when transfected into permissive SV40 T-antigen producing COS-1 cells will amplify to high copy numbers (5,000 to 10,000) without inflicting toxic effects upon the host cell. This amplification vector (pSVori) has been used to amplify cis-acting regulatory elements which can act as competitors for positive and negative trans-acting factors in vivo. Using this amplification system we conducted experiments to determine whether amplification of alpha-fetoprotein (AFP) and albumin cis-acting promoter sequences could activate a corresponding co-transfected AFP-promoter-CAT or Alb-promoter-CAT expression vector in COS-1 cells. We used pMoMLV(-1009)AFPcat, or p(-308)Albcat-MoMLV as reporter genes and pSVori to amplify specific promoter sequences of the AFP or albumin promoter. Our experiments indicated that amplification of a region from -53 to -202 of the AFP promoter resulted in the activation of the pMoMLV(-1009)AFPcat and p(-308)Albcat-MoMLV expression vectors in COS-1 cells. Surprisingly, amplification of the albumin promoter sequences failed to activate either the pMoMLV(-1009)AFPcat or p(-308)Albcat-MoMLV plasmids.

Multiple Sequence Elements Are Required for Maximal in Vitro Transcription of a Human Histone H2B Gene

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.

Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene.

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.