Recently, at least thirteen genes expressed in the context of epidermal differentiation have been mapped to the q21 region of human chromosome 1, including calcyclin, calgranulins A and B, cellular retinoic acid binding protein-II (CRABP-II), and psoriasin. Nonrandom deletions and/or rearrangements of this chromosomal region occur in melanoma and non- melanoma skin cancer, as well as breast cancer. Our hypothesis is that additional novel genes of related function are present in this chromosomal region, and that their genomic co-localization reflects coordinate regulation by locus control region (LCR)-like sequences. The principal objectives of this proposal are (i) to identify novel genes involved in the regulation of epithelial differentiation by virtue of their localization to 1q21, and (ii) to identify the LCR-like regulatory sequences responsible for their coordinate expression in the skin. by improving our understanding of the genetic regulation of epidermal differentiation, these studies will contribute to our understanding the pathogenesis of skin cancer, psoriasis, and disorders of keratinization.
Specific Aim 1 (Year 1): To determine the chromosomal location of other genes which are known to be up-regulated during squamous differentiation (SQ10, SQ37, and C12) or in psoriasis (psoriasis-associated fatty acid binding protein). This will be accomplished by screening somatic cell hybrid panels and yeast artificial chromosome (YAC) clones with cloned cDNAs or PCR products, and by fluorescent in situ hybridization (FISH) using the cognate genomic clones.
Specific Aim 2 (Years 1 and 2): To isolate and map 1q21 YAC clones containing CRABP-II and known members of the calcyclin gene cluster. YAC clones will be identified by PCR screening of a human YAC library, and YAC mapping will be accomplished by a combination of hybridization- and PCR-based strategies.
Specific Aim 3 (Years 1, 2, and 3): To use these 1q21 YAC clones as probes to identify novel genes expressed in skin, and to characterize their expression in skin and other tissues by Northern blotting. cDNA clones will be identified by selection cloning using 1q21 YACs and a psoriatic skin cDNA library, and screened by hybridization against somatic cell hybrid panels and known 1q21 YACs.
Specific Aim 4 (Years 2 and 3): To identify LCR-like regulatory elements in the 1q21 region using enhancer trap assays. 1q21 and globin YACs will be retrofitted with a reporter gene driven by a neutral promoter. YAC sequences which selectively up-regulate the reporter gene in keratinocytes will be further localized in an SV40-based enhancer trap vector.
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