Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.     

Erythroid specific activation of the Xenopus laevis adult alpha-globin promoter in transient heterokaryons.

Insertion of 1.5 kb of the 5' flanking region of the adult alpha-globin gene of X. laevis in front of the CAT structural gene promotes synthesis of CAT in transiently transfected X. laevis kidney cells. Fusion of transiently transfected kidney cells with erythroblasts isolated from anaemic frogs stimulates CAT expression 3-4 fold in the resulting transient heterokaryons. The stimulation is specific for the alpha-globin promoter and is obtained after fusion with erythroid cells but not with hepatocytes or kidney cells. Stably transfected kidney cells express drastically reduced CAT activity as compared with transiently transfected cells. Nevertheless, fusion of stably transfected kidney cells with erythroblasts leads to a 10-17 fold stimulation of CAT expression. The experiments suggest that erythroid specific transacting factors stimulate expression of CAT controlled by the adult alpha-globin promoter.     

Nitric oxide regulation of gene transcription via soluble guanylate cyclase and type I cGMP-dependent protein kinase

Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax.

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells.

We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.     

Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures

To study the regulation of insulin gene expression by physiological regulators, primary cultures of rat islet cells were transfected with portions of the rat insulin I gene 5'-flanking sequence linked to the reporter gene chloramphenicol acetyltransferase (CAT). Incubation of the cells in increasing glucose concentrations led to a parallel increase in both CAT activity and CAT mRNA levels. Pretreatment of the cells with the beta-cell-specific toxin streptozotocin reduced CAT activity 97%. Beta-Cell-specific expression of CAT was also demonstrated by co-staining the transfected cells with antisera to both CAT and insulin. Experiments showing a reduction in the response to glucose in the presence of the calcium channel blocker verapamil suggest that calcium plays a role in the glucose response, possibly via regulation of factors interacting with this limited portion of the insulin gene.     

The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter.

We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytes

TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between -78 and -273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [(32)P]-labeled probe containing C/EBP response element equivalent of the -265 to -242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPalpha and an increase in that of C/EBPbeta among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPalpha, C/EBPbeta, or C/EBPdelta through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPalpha or C/EBPbeta. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells.     

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.