We have identified a novel zinc-finger protein whose mRNA is expressed at high levels in the epidermal layer of the skin and in epithelial cells in the tongue, palate, esophagus, stomach, and colon of newborn mice. Expression in epithelial cells is first detected at the time of their differentiation during embryonic development. In addition, during early embryonic development there is expression in mesenchymal cells of the skeletal primordia and the metanephric kidney which is later downregulated. The expression pattern suggests that the protein could be involved in terminal differentiation of several epithelial cell types and could also be involved in early differentiation of the skeleton and kidney. The carboxyl terminus of the protein contains three zinc fingers with a high degree of homology to erythroid krü ppel-like factor and binds to DNA fragments containing CACCC motifs. The amino-terminal portion of the protein is proline and serine-rich and can function as a transcriptional activator. The chromosomal location of the gene was mapped using mouse interspecific backcrosses and was shown to localize to mouse chromosome 4 and to cosegregate with the thioredoxin gene.During embryogenesis, a single-cell zygote gives rise to a complex organism composed of many cell types which differentiate from their precursors in an intricate process involving the coordinate action of various cytokines, hormones, and growth factors which activate the expression of specific transcription factors in the cell. Many cell-specific transcription factors involved in the differentiation of tissues have been identified: these fall into several broad classes that include helix-loophelix proteins, homeodomain proteins, and zinc-finger proteins. Zinc-finger transcription factors generally contain a cluster of zinc-finger motifs which together bind to specific DNA sequences; they can be divided into several classes based on the sequence and position of the cysteine and histidine residues and other conserved amino acids. The TFIIIA subclass of zincfinger proteins is characterized by an amino acid motif (Cys-X 2-4 -Cys-X 12 -His-X 3-4 -His) that coordinates zinc ions and is involved in DNA binding. Some zinc-finger proteins of the

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Abstract. 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-~l(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 [3-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the [3-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-al(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 proal(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 [3-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific c/s-acting elements that can activate the mouse pro-a(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. D IFFERENT mechanisms can be envisioned to account for the cell-specific activity of a gene that is expressed in a small number of discrete cell types. A first possible mechanism is that a single type of cis-acting element recognized by a unique cell-specific transcription factor be present in the different cell types that are actively expressing this gene. According to another hypothesis, separate cis-acting elements would be present in the regulatory segments of such genes and each of these would be recognized by distinct transcription factors speAddress all correspondence to J6rome Rossert, Dept. of Molecular Genetics, Box 11, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: (713) 792-2590. Fax: (713) 794-4295. cific for e...

Abstract. We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse proa2(I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse proa2(I) collagen gene, cloned upstream of either the Escherichia coli 13-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal proet2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse proal(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro~2(I) collagen gene.T HE genetic programs that control the differentiation of fibroblasts and osteoblasts are still poorly understood. We are using the type I collagen genes as markers for these cell lineages to better understand the mechanisms by which they express their phenotypes. The type I collagen genes are expressed at high levels in osteoblasts, in odontoblasts, and in fibroblasts of tendons and skin, whereas in other tissues, their expression is considerably lower and is due mainly to the presence of fibroblasts and mesenchymal cells in these tissues. Type I collagen belongs to the fibrillar class of collagens and is composed of two al(I) chains and one a2(I) chain (Cole, 1994;Philajaniemi and Rehn, 1995, and references therein for review of collagen types and functions). Excessive amounts of these proteins along with other extracellular matrix proteins are found in fibrotic diseases including cirrhosis, glomerulosclerosis, and scleroderma.

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-␣1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocytespecific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocytespecific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.Acquisition of the chondrocytic phenotype occurs along a major pathway of differentiation of mesenchymal cells (1, 2). With the goal of identifying transcription factors that control chondrocyte-specific gene expression, we used the gene for collagen type II (Col2a1), 1 an early and abundant marker of chondrocytes (3-5), to delineate minimal sequences in this gene that control chondrocyte-specific expression in transgenic mice. Elucidation of the transcriptional mechanisms that control the chondrocyte-specific expression of the Col2a1 gene should provide important insights into the molecular specifications of chondrocytes.We previously identified a 48-bp element in intron 1 of the mouse Col2a1 gene that, when present as four tandem copies, conferred chondrocyte-specific expression both in transgenic mice and in transient expression experiments in tissue culture cells (6). A multimerized 18-bp element located at the 3Ј end of the 48-bp sequence also acted as a powerful chondrocyte-specific enhancer in transient transfection assays of rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not of fibroblasts (6).SOX9 is a member of a family of transcription factors with a DNA-binding domain that shows more than 50% similarity with the high mobility group HMG DNA-binding domain of SRY, the testis-determi...