人皮肤成纤维细胞中α1(Ⅰ)前胶原基因转录调控研究

为寻找纤维化形成中调控人Ⅰ型前胶原基因高水平转录的启动序列及其DNA结合蛋白 ,以人皮肤成纤维细胞α1(Ⅰ )前胶原基因转录起始点上游 - 2 5kb至 + 4 2bp的片段为靶序列 ,采用PCR、基因重组、报告基因测活、细胞基因转染技术比较不同长短启动子活性 .凝胶滞留实验 (EM SA)研究高启动活性片段相应的DNA结合蛋白 .基因转染高活性转录因子识别序列至靶细胞 ,探讨前胶原基因激活阻断的新手段 .结果表明 ,- 2 4 83～ + 4 2bp、 - 2 6 8～ + 4 2bp序列具有强启动调控活性 ,而 - 10 5～ + 4 2bp片段启动活性最低 .EMSA对高启动活性小片段DNA结合蛋白的分析提示 ,- 2 6 8～ + 4 2bp序列中存在转录因子Ap 1、Sp 1、NF 1的特异结合位点 .转染高活性转录因子识别序列Ap 1、Sp 1至靶细胞可竞争性阻断胶原基因启动转录激活 .研究提示 ,人α1(Ⅰ )前胶原基因 - 2 4 83～ + 4 2bp、 - 2 6 8～ + 4 2bp片段有高启动活性 .转录因子Ap 1、Sp 1、NF 1与 - 2 6 8～ + 4 2bp序列中相应识别序列的结合与其基础高转录活性有关 .转染高活性转录因子识别序列Ap 1、Sp 1可从转录水平阻断胶原基因的激活     

Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein.

In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong heterologous promoters and therefore must be a direct autoregulatory response that cannot be explained by promoter competition effects. In gel mobility retardation assays with infected-cell nuclear extracts, a prominent and specific DNA-protein complex was formed with DNA fragments containing sequences from -108 to +30 in the IE175 promoter region. This activity was not present in mock-infected samples. Even stronger binding occurred with a fragment containing sequences from -128 to +120 in the IE110 promoter, but this second locus was not associated with any detectable response phenotype in cotransfection assays. Supershift experiments with an anti-IE175 monoclonal antibody confirmed the presence of the IE175 protein in both DNA-protein complexes. In the IE175 promoter, specific binding correlated closely with the presence of an intact autoregulatory signal near the cap site as judged by the loss of both activities in a 3'-deleted promoter fragment lacking sequences from -7 to +30. Insertion of a cloned 30-mer synthetic oligonucleotide sequence from positions -8 to +18 in IE175 restored both IE175 binding activity and the down-regulation phenotype. Direct shift-up assays with a similar 30-base-pair (bp) oligonucleotide containing 21 bp from positions -75 to -55 of IE110 (which encompasses a consensus ATCGTC motif) also produced a specific DNA-protein complex containing the IE175 protein. This ATCGTC motif proved to be a necessary component of both the IE110 and IE175 binding sites, but was insufficient on its own for complex formation. Finally, deletion of 2 bp from positions -3 and -4 within the ATCGTC sequence in the IE175 cap site region abolished both binding activity and the IE175-dependent autoregulation phenotype.     

Bovine Tyrosine Hydroxylase Gene-Promoter Regions Involved in Basal and Angiotensin II-Stimulated Expression in Nontransformed Adrenal Medullary Cells

Abstract: The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.     

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.     

alpha7 nicotinic receptor gene promoter polymorphisms in inbred mice affect expression in a cell type-specific fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.     

Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor kappaB (NF-kappaB) was previously shown to inhibit expression of murine alpha(1)(I) and human alpha(2)(I) collagen promoters. The present study identifies binding of NF-kappaB, present in nuclear extracts of stellate cells, to a region between -553 and -537 of the murine alpha(2)(I) collagen promoter. The NF-kappaB (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-kappaB-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor beta and tumor necrosis factor alpha, respectively, on promoter activity. Acetaldehyde increased IkappaB-alpha kinase activity and phosphorylated IkappaB-alpha, NF-kappaB nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-kappaB to the murine alpha(2)(I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-kappaB to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.