Purification of BBF, a DNA-binding protein recognizing a positive cis-acting element in the mouse alpha 1(III) collagen promoter.

A positive cis-acting element, the B element, located between -83 and -61 in the mouse alpha 1(III) collagen promoter, binds a factor present in nuclear extracts of NIH 3T3 fibroblasts and HeLa cells. We have purified this factor using ion exchange chromatography, sequence-specific DNA affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel fractionation. The DNA sequence used for the affinity chromatography was a single-base substitution in the B element that increased the stability of the B element-protein complex by 50%. Purification of the B element-binding factor (BBF) by DNA affinity chromatography resulted in the apparent loss of most or all of the DNA-binding activity of this factor. The DNA-binding activity could, however, be reconstituted by combining two chromatographic fractions: the high-salt eluate and the column flow-through. When the partially purified high-salt eluate was size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent renaturation of gel fractions from guanidine HCl, the purified BBF (apparent molecular weight of about 95,000) bound to the B element with high affinity. These results suggest that during DNA affinity purification of BBF a factor that inhibits BBF DNA binding was co-eluted with BBF. This inhibition of BBF DNA binding was reversed by the addition of the DNA affinity column flow-through. The binding of BBF to the B element of the mouse alpha 1(III) collagen promoter is therefore an apparently complex process involving interactions between BBF and other protein factors.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell- specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis- acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.     

Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.     

A combination of cis-acting elements is required to activate the pro-alpha 1(I) collagen promoter in tendon fibroblasts of transgenic mice

The genes encoding the two type I collagen chains are selectively activated in few cell types, including fibroblasts and osteoblasts. By generating transgenic mice, we have previously shown that the activity of the mouse pro-alpha1(I) promoter was controlled by separate cell-specific cis-acting elements. In particular, a sequence located between -3.2 and -2.3 kb was needed to induce expression of the reporter gene at high levels in tendon fibroblasts. In the present work, by using the same transgenic approach, we have identified two short elements in this sequence, named tendon-specific element (TSE) 1 and TSE2, that were necessary to direct reporter gene expression selectively in tendon fibroblasts. Gel shift assays showed that TSE1 and TSE2 bound proteins specifically present in nuclear extracts from tendon fibroblasts and that the sequence of TSE2 binding a tendon-specific protein corresponded to an E-box. Analysis of transgenic mice further indicated that TSE1 and TSE2 needed to cooperate not only with each other but also with other cis-acting elements of the proximal promoter to activate reporter gene expression in tendon fibroblasts. Similarly, it pointed out that the so-called osteoblast-specific element had to interact with downstream sequences to drive reporter gene expression in osteoblasts of transgenic mice. Thus, expression of the mouse pro-alpha1(I) collagen gene in tendon fibroblasts appears to be the result of a unique combination of different cis-acting elements, including TSE1 and TSE2.     

The complete primary structure of mouse alpha 2(IV) collagen. Alignment with mouse alpha 1(IV) collagen

We have determined the nucleotide and amino acid sequences of mouse alpha 2(IV) collagen which is 1707 amino acids long. The primary structure includes a putative 28-residue signal peptide and contains three distinct domains: 1) the 7 S domain (residues 29-171), which contains 5 cysteine and 8 lysine residues, is involved in the cross-linking and assembly of four collagen IV molecules; 2) the triple-helical domain (residues 172-1480), which has 24 sequence interruptions in the Gly-X-Y repeat up to 24 residues in length; and 3) the NC1 domain (residues 1481-1707), which is involved in the end-to-end assembly of collagen IV and is the most highly conserved domain of the protein. Alignment of the primary structure of the alpha 2(IV) chain with that of the alpha 1(IV) chain reported in the accompanying paper (Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317) suggests that a heterotrimeric collagen IV molecule contains 26 imperfections in the triple-helical domain. The proposed alignment is consistent with the physical data on the length and flexibility of collagen IV.     

Cooperation of two distinct cis-acting elements is necessary for the S phase-specific activation of the wheat histone H3 promoter

We have previously shown that the proximal promoter region (−185 to +57) of the wheat histone H3 gene ( TH012 ) is sufficient for regulating S phase-specific expression of a reporter GUS gene. To define the cis -acting element(s) responsible for S phase-specific expression, GUS fusion genes under the control of wild-type or variously mutated H3 promoters were stably introduced into cultured rice Oc cells and their temporal expression was analyzed during the cell cycle by quantitative S1 analysis. The S phase-specific expression of the full-sized promoter (−1716 to +52) was significantly impaired by short internal deletions disrupting the type I element from −175 to −158 (CCACGTCACCaATCCGCG), composed of the Hex (CCACG-TCA) and reverse-oriented Oct (GATCCGCG) motifs. Moreover, the H3 proximal promoters (−184 to +52) harboring base-substitution mutations in either or both of the Hex and Oct motifs could no longer activate gene expression during the S phase. These results indicate that the type I element is the first cis-acting element identified responsible for the S phase-specific expression of plant histone genes. Results also suggested the presence of a redundant cis -acting element(s) responsible for S phase-specific expression in the H3 far-upstream region (−1716 to −185).     

Differential expression of two exons of the alpha1(XI) collagen gene (Col11a1) in the mouse embryo.

The amino terminal domain of collagen XI has a unique structure, which is believed to participate in the regulation of matrix assembly. Interestingly, several distinct isoforms of the amino terminal domain of alpha1(XI) and alpha2(XI) collagen chains exist as a result of alternative splicing. Here we report the analysis of the alternative splicing pattern of the mouse alpha1(XI) collagen gene (Col11a1). Like other vertebrate species, the mutually exclusive expression of exons 6A and 6B of Col11a1 results in the inclusion in the alpha1 chain of either an acidic peptide (pI 3.14) or a basic peptide (pI 11.66). Expression of these two exons was monitored in several tissues of the 16.5-day mouse embryo by in situ hybridization and immunohistochemistry, with exon-specific cDNA probes and peptide-specific antibodies, respectively. The results documented that isoforms containing the exon 6B-encoded peptide accumulate predominantly in the vertebrae, skeletal muscles and intestinal epithelium. By contrast, exon 6A products were found to be most abundant in the smooth muscle cells of the intestine, aorta and lung. The results using in situ hybridization confirmed those using immunohistochemistry. Albeit correlative, the evidence suggests distinct contributions of the two peptides to the differential assembly of tissue-specific matrices.     

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.