A distal tyrosinase upstream element stimulates gene expression in neural-crest-derived melanocytes of transgenic mice: position-independent and mosaic expression

Susan D. Porter 0 Cathy J. Meyer 0 0 Department of Pathology and Laboratory Medicine, University of British Columbia , 2211 Wesbrook Mall, Vancouver, British Columbia, V6T 2B5 , Canada crest-derived melanocytes of transgenic mice: position-independent and SUMMARY We have assessed the importance of a melanocyte-specific DNase I hypersensitive site and matrix attachment region situated 15 kb upstream of the mouse tyrosinase gene by analysis in transgenic mice. Transgenes containing all, part, or none of this region linked to the tyrosinase promoter and human tyrosinase cDNA were introduced into genetically albino mice, and pigmentation and transgene message levels were analyzed in the resulting transgenic lines. The effect of the upstream region was to enhance significantly gene expression in melanocytes, and to provide position-independent expression of the transgene. Two exceptions to complete position independence were seen; these lines displayed a mosaic expression pattern in which the transgene was expressed fully in some melanocyte clones but less so in others, resulting in transverse stripes of colours ranging from near white to dark grey. Unexpectedly, pigmentation in the eye of all transgenic lines containing the upstream region was nonuniform, in that the neural-crest-derived melanocytes of the choroid and anterior iris contained significantly more Tyrosinase is the key enzyme of melanin biosynthesis. Its gene is expressed in all pigmented melanocytes, including those of neural crest origin (residing in the skin, choroid and internal organs) and the optic-cup-derived melanocytes of the retinal pigment epithelium (RPE). We are interested in defining the elements required for the establishment of an active tyrosinase chromosomal domain in melanocytes, to understand better the mechanisms involved in this aspect of gene regulation, as well as to help in the identification of trans-acting factors important in melanocyte-specific gene expression and differentiation. The identification of cis-acting sequences important in higher order gene control has been approached primarily by the functional analysis of candidate sequences in transgenes randomly integrated into the genome of mice or cultured cells. High level expression of the genes, which is proportional to the number of copies integrated, is considered to indicate that pigment than those derived from the optic cup (retinal pigment epithelium and posterior iris). Transgenes containing a small part or none of the upstream region were expressed poorly and in a position-dependent manner; of those lines that were visibly pigmented, expression was equal in the neural crest and optic-cup-derived cells of the eye. Mice with transgenes containing DNA sequences encompassing the hypersensitive site but lacking most of the matrix attachment region were, on average, poorer expressors than those containing the entire upstream region; the highest expressing line of this series, however, had a pigmentation level similar to the lines with the full upstream region. Thus, full transcriptional enhancement activity may lie within the segment containing the hypersensitive site, but position-independent expression may require the flanking matrix attachment region containing sequences. sequences required for autonomous establishment of an open chromatin structure and for insulation of the transgene from neighbouring chromatin or regulatory elements, are included in the construct. The first of such elements isolated was the locus control region (LCR) of the b -globin gene cluster (Grosveld et al., 1987), and elements with similar properties from a number of other genes have been identified (Greaves et al., 1989; Abe and Oshima, 1990; Bonifer et al., 1990; Greer et al., 1990; Chamberlain et al., 1991; Aronow et al., 1992). Studies of the tyrosinase gene regulatory elements using transgenic mice have shown that 270 bp of the promoter are sufficient for expression in skin and eye melanocytes (Beermann et al., 1992) and that the expression of the transgene, although lower than that of the endogenous gene, roughly recapitulates its developmental timing. Position independence of expression in this and in other studies using longer tyrosinase promoters (up to 6 kb) with the tyrosinase or heterologous cDNAs (Tanaka et al., 1990; Yokoyama et al., 1990; Bradl et al., 1991a; Klppel et al., 1991) was not specifically addressed; however, visual analysis of coat color suggested that expression levels often did not correspond to copy number. In addition, some transgenic lines (non-founders) displayed a mosaic expression in which melanocyte clones in the coat were pigmented to different degrees (Bradl et al., 1991b; Takeuchi et al., 1993), clearly a variegating type position effect. The three analyzed transgenic lines derived from microinjection of a yeast artificial chromosome (YAC) encompassing 235 kb of the tyrosinase gene (80 kb of the coding region and 155 kb of upstream sequences) showed position-independent transgene expression at levels comparable to that of the endogenous gene (Schedl et al., 1993), suggesting that all elements necessary for higher order gene control are located within these limits. A sequence at 15 kb upstream from the first exon of the tyrosinase gene is hypersensitive to DNase I in melanocytes and has in vitro nuclear matrix attachment activity (Porter et al., 1991). It is located within a region removed by rearrangement from the rest of the tyrosinase gene in the variegating mutation of the tyrosinase locus, chinchilla-mottled (cm) (Porter et al., 1991). This autosomal, somatically stable mutation results in light and dark grey stripes on the coat of homozygous mice, which are accounted for by altered tyrosinase mRNA levels (a roughly 10-fold difference between the light and darkly pigmented cells, and between the dark cm/cm and wild-type melanocytes). The difference in gene expression between the light and dark cells could be attributed to alternate chromatin conformations of the tyrosinase locus. Thus, the rearrangement of the tyrosinase gene causes aberrant chromatin formation in some clones and reduced expression in all clones (even those with apparently normal chromatin structure). The - 15 kb region was hypothesized to encompass a potential regulatory element important in transcriptional enhancement of the tyrosinase gene and/or in the establishment of open chromatin, as it was the only DNase I hypersensitive site within 20 kb of the promoter that was separated from the gene in the cm mutation. That all sequences required for full expression and open chromatin conformation were not present in the immediate vicinity of the coding sequence was concluded independently from the transgenic experiments discussed above. To address the hypothesis that this region is important in tyrosinase gene control in melanocytes, we generated transgenic mice containing the tyrosinase promoter, human tyrosinase cDNA, and part or a (...truncated)