Deletion analysis of the mouse alpha 1 (III) collagen promoter (original) (raw)
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Identification of the promoter and first exon of the mouse α1(III) collagen gene
Journal of Biological Chemistry
We have identified the promoter-proximal exon of the mouse alpha 1 (III) collagen gene using a synthetic oligonucleotide as a hybridization probe and have determined the DNA sequence of this exon and 380 base pairs 5' to it. The exact start site of transcription was localized with a primer extension experiment. The region upstream of the start of transcription shows only scattered homologies with the analogous sequences in the alpha 1(I) and alpha 2(I) mouse collagen genes although these genes are often co-expressed and co-regulated. The most striking homology with the type I gene is seen around the start of translation. This region contains an inverted repeat which could form a stem-loop structure with a calculated delta G of -30 kcal in the type III collagen mRNA. When compared to the alpha 1(I) and alpha 2(I) signal peptides, the signal peptide of mouse alpha 1(III) collagen presents less homology than when these segments are compared to each other.
Structure of the rat collagen IV promoter
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1996
We have isolated a 1.6 kb clone from a rat genomic library which contains the bidirectional collagen IV promoter, flanked by exons coding for the a I(IV) and a2(IV) collagen chains. There are at least two transcription start sites within both the o~ I(IV) and ~ 2(IV) collagen genes. Rat mesangial cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing segments of the promoter and 5' flanking region, in both the c~ I(IV) and c~2(IV) orientations. Our results suggest that transcriptional efficiency of the bidirectional promoter is more efficient in the a 2(IV) direction than in the o~ l(IV) direction.
European Journal of Biochemistry, 1997
Cis-acting regions regulating transcription of the a1 (VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivn in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'-flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the al(V1) mRNA, these results suggest the presence of an activator(s) between-0.2 and-0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct pl.4CAT and p4.OCAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount al(V1) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region-4.O/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region-6S-7.5 on al(V1) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgenekhromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the a1 (VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.
Identification of Two Repressor Elements in the Mouse α2(I) Collagen Promoter
Archives of Biochemistry and Biophysics, 1999
We recently identified three areas of Sp1 binding located between ؊568 and ؊453 of the 5 flanking region of the murine ␣ 2 (I) collagen promoter which are necessary for optimal activity. We now identify two additional regions of Sp1 binding located at ؊371 to ؊351 (region 4) and at ؊690 to ؊613 (region 5), which when mutated increased promoter activity in transfected rat hepatic stellate cells indicating they contain negative regulatory elements. AP-2 bound to region 4 while YY1 bound most strongly to region 5. AP-2 decreased Sp1 binding to region 4 and had a dual effect on Sp1 binding to region 5 decreasing and increasing Sp1 binding at low and high concentrations of AP-2, respectively. YY1 enhanced Sp1 binding to both regions. AP-2 inhibited or enhanced the stimulatory effect of a transfected Sp1 expression vector on the ␣ 2 (I) collagen promoter in Drosophila cells at low or high AP-2 expression, respectively. YY1 enhanced or inhibited the activation of the promoter by low or high Sp1 expression, respectively. This study identifies two negative regulatory elements in the murine ␣ 2 (I) collagen promoter and shows that AP-2 and YY1 interact with Sp1 at these sites and can inhibit the activating action of Sp1.
2000
Type Icollagenisexpressedina varietyof connectivetissuecellsand itstranscriptional regula- tionishighlycomplex becauseoftheinfluenceof numerous developmental,environmental,and hor- monal factors.To investigate themolecularbasisfor one aspectofthiscomplex regulation,theexpression ofal(I)collagen(COLIA1) gene inosseoustissues, we fuseda 3.6-kbDNA fragmentbetween bases -3,521 and +115 oftheratCOLIA1 promoter,and threedeletionmutants,to thechloramphenicol acetyltransferase (CAT) marker gene.The expression oftheseCoICAT transgeneswas measured instably transfectedosteoblasticcelllinesROS 17/2.8,Py-la, and MC3T3-El and threefibroblastic linesNIH-3T3, Rat-1,and EL2 .Deletionofthedistal1.2-kbfragment ofthefull-length CoICAT 3.6constructreducedthe promoter activity7-to 30-foldintheosteoblastic cell lines,twofoldinEL2
The Journal of cell biology, 1996
To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue ...
Journal of Biological Chemistry, 1999
Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5flanking region of the ␣1(VI) collagen gene, mapping at ؊4.6, ؊4.4, ؊2.5, and ؊0.1 kilobase (kb) from the RNA start site. The site at ؊2.5 kb was independent from, whereas the other three sites could be related to, ␣1(VI) mRNA expression. The site at ؊0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides ؊104 and ؊73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at ؊0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (
Genomics, 1996
ple triple-helix domains and interruptions) within the The mouse gene for the a1 chain of type XVIII collacollagen superfamily. They are characterized by a large gen (Col18a1) is more than 102 kb and consists of 43 N-terminal globular domain, a highly interrupted triexons. Type XVIII collagen transcripts encode polyple helix, and a large C-terminal globular domain with peptides that differ with respect to three variant Nfour conserved cysteines terminal noncollagenous domains that are 301 (NC1Characterization of genomic clones revealed that the 1995; . Both collagens also contain three variant NC1 domains result from the use of two several potential attachment sites for serine-linked alternative promoters, separated by a distance of 50 glycosaminoglycans and asparagine-linked oligosackb. The upstream promoter, promoter 1, directs the charides, suggesting that these collagens may be extensynthesis of the NC1-301 domain in conjunction with sively glycosylated. The function of collagen types XV exons 1 and 2, whereas the downstream promoter, proand XVIII is currently unknown, but type XVIII collamoter 2, directs that of the NC1-517 and NC1-764 dogen was recently immunolocalized in the basement mains in conjunction with exon 3, with the latter two membrane zone (Muragaki et al., 1995). variants differing with respect to alternative splicing Elucidation of the complete primary structure of the of the exon 3 sequences. Exons 4-9 encode a portion mouse a1(XVIII) collagen chain and of a partial strucof the NC1 domain shared by all three polypeptide ture of the human counterpart have revealed a polypepvariants, and exons 9-43 encode the common collagetide consisting of 11 noncollagenous domains (NC1nous and C-terminal noncollagenous sequences. The NC11) 2 and 10 collagenous domains (COL1-COL10) marked differences previously observed in the expres- . Type XVIII collasion of variant type XVIII collagen transcripts in mouse tissues thus result from tissue-specific use of gen transcripts were found to be heterogeneous at their these two promoters. ᭧ 1996 Academic Press, Inc. 5 ends, encoding three variant N-terminal sequences covering the ensuing 1315-, 1527-, or 1774-residue collagen chains Rehn and Pihlajaniemi, untranslated region of the 5 end of the mRNA; COL, collagenous domain; NC, noncollagenous domain; STS, sequence-tagged 90220 Oulu, Finland.
Cell Type-specific Transcription of the α1(VI) Collagen Gene
Journal of Biological Chemistry, 1999
Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5flanking region of the ␣1(VI) collagen gene, mapping at ؊4.6, ؊4.4, ؊2.5, and ؊0.1 kilobase (kb) from the RNA start site. The site at ؊2.5 kb was independent from, whereas the other three sites could be related to, ␣1(VI) mRNA expression. The site at ؊0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides ؊104 and ؊73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at ؊0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (