Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Isolation and characterization of the mouse thrombospondin 3 (Thbs3) gene.

The DNA sequence of the coding region of the mouse thrombospondin (TSP) 3 gene has been determined by analysis of both genomic and cDNA clones. Like TSP1 and TSP2, TSP3 has a homologous COOH-terminal domain and seven type III (Ca(2+)-binding) repeats. However, TSP3 contains four, rather than three, type II repeats and lacks the type I (TSP or properdin) repeats and procollagen homology characteristic of TSP1 and TSP2. In addition, the NH2-terminal domain of TSP3 differs markedly, both in sequence and in exon/intron structure, from that in TSP1 or TSP2. The gene is located on mouse chromosome 3, bands E3-F1, immediately upstream from the Muc1 (episialin) gene and is expressed in the developing mouse in a pattern that also differs from that of TSP1 or TSP2. Based on its structure, we suggest that TSP3 may play both a unique role in cell-matrix interactions and perform functions that overlap with those of TSP1 and TSP2.

A highly conserved, 5' untranslated, inverted repeat sequence is ineffective in translational control of the alpha 1(I) collagen gene.

An inverted repeat sequence, extending from the 5' untranslated region of the first exon through the translation initiation codon, is highly conserved in the alpha 1(I), alpha 2(I) and alpha 1(III) collagen genes of mammals and birds. It has been suggested that this sequence functions in translational control of collagen gene expression. When the upstream axis of the dyad of symmetry was deleted, the efficiency of translation of transcripts from a human alpha 1(I) collagen-bovine growth hormone fusion gene was unchanged in either transiently or stably transfected cells. Furthermore, mRNA levels were not affected when the same deletion was transferred to a collagen-human growth hormone fusion gene in which the collagen sequence retained the first intron. Examination of human alpha 1(I) DNA, extending from the start of transcription to the start of translation, by the DNAse I protection procedure revealed evidence for protein binding to a sequence just upstream of the inverted repeat sequence but not to the inverted repeat itself. Our studies therefore indicate that this highly conserved DNA sequence does not function generally in translational or transcriptional control of type I procollagen synthesis.

A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not respond to serum.

A second, expressed thrombospondin (TSP) gene, Thbs2, has been identified in the mouse. The exon/intron organization of Thbs2 is highly conserved in comparison with Thbs1 in that exon size and the pattern of interruption of the reading frame by introns are preserved, but there is a marked divergence in coding sequence, primarily in the first 7 exons. On the other hand, the DNA and translated amino acid sequences of exons coding for the type I, II, and III repeats in the two TSPs are far better conserved. Thbs2 is located on chromosome 17, band A3, whereas Thbs1 was found on chromosome 2, band F. In marked contrast to Thbs1, the Thbs2 gene is not induced by serum in NIH 3T3 cells; promoter sequences in the two genes are also very different. It is therefore likely that the two TSPs perform related but distinct functions.

Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression.

We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.

Regulatory elements in the first intron contribute to transcriptional control of the human alpha 1(I) collagen gene.

Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the alpha 1(I) gene. We therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the alpha 1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an alpha 1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. We propose that normal regulation of alpha 1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.

Thrombospondin 3 (Thbs3), a new member of the thrombospondin gene family.

A third member of the thrombospondin gene family (Thbs3) has been partially characterized in the mouse. In both the mouse and humans, the Thbs3/THBS3 gene is located immediately upstream from the Muc1/MUC1 (episialin) gene; less than 3 kilobases separate the polyadenylation signal of one gene from the start of transcription of the other. The available coding sequence in Thbs3 shows a high degree of amino acid sequence identity to Thbs1 and Thbs2 (58 and 59%, respectively, in exons 15 and 16), but the exon/intron organization of Thbs3 appears to be more disparate than that of the two previously described members of the family. The shorter length of the Thbs3 mRNA (3.5 kilobases) can be attributed largely to a shorter 3'-untranslated region. The Thbs3 gene is expressed in a distinctive pattern in mouse tissues, with the highest level of expression in lung. This pattern suggests a unique function for the translation product of the Thbs3 gene.

Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.

We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease.