Identification of Human Epidermal Differentiation Complex (EDC)-Encoded Genes by Subtractive Hybridization of Entire YACs to a Gridded Keratinocyte cDNA Library

The chromosomal band 1q21 has been shown to harbor three gene families involved in terminal differentiation of the human epidermis within 2 Mb of genomic DNA (Volz et al. 1993). They encode precursor proteins of the cornified cell envelope (CE), intermediate filament-associated proteins, and calcium-binding proteins. The clustered organization of these genes and their evolutionarily conserved structural relationship together with functional interdependence of the encoded proteins led to their designation as a gene complex; the epidermal differentiation complex (EDC) (Mischke et al. 1996). Loricrin, involucrin, and small proline-rich proteins (SPRRs) are the major precursors of the CE (Steinert et al. 1998), a highly insoluble and rigid structure that is essential for the barrier function of the skin. In terminally differentiating keratinocytes, the CE is assembled at the intracellular surface of the plasma membrane by transglutaminase-mediated cross-linking of these proteins. The corresponding genes LOR (Hohl et al. 1991), IVL (Eckert and Green 1986) and 10 SPRR genes belonging to 3 subgroups (2 SPRR1, 7 SPRR2, and 1 SPRR3) (Gibbs et al. 1993) are characterized by a similar gene structure, homologies in the terminal protein domains, and a variable number of internal tandem repeats. They constitute a cluster that most likely evolved from a common ancestor (Backendorf and Hohl 1992). Profilaggrin, which is processed to functional filaggrin monomers, and trichohyalin are the main constituents of the keratohyalin granules in the epidermis and the hair follicle, respectively (Steven et al. 1990; Fietz et al. 1993). They serve as keratin filament matrix and are also cross-linked to the CE (Steinert and Marekov 1995; Steinert et al. 1998). Their multifunctional structure combines sequence repeats, similar to the CE precursors, with two calcium-binding EF-hand domains that are typical features of the S100 proteins (see below) (Lee et al. 1993; Markova et al. 1993; Presland et al. 1995). The gene loci FLG and THH are located close together, centromeric to the CE precursor genes (Volz et al. 1993). All of these structural genes of the EDC are flanked by 13 members of the S100 family (S100A1 to S100A13) (Schaefer et al. 1995; Wicki et al. 1996a,b) encoding calcium-binding proteins with two EF-hands (a calcium-binding motif named after the E- and F-helices of parvalbumin). S100 proteins are primarily regulatory proteins involved in different steps of the calcium signal transduction pathway. They mediate effects on cell shape, cell cycle progression, and differentiation (Schaefer and Heizmann 1996). In addition, incorporation of S100A10 and S100A11 into the CE was reported (Robinson et al. 1997), suggesting functional cooperation between calcium-binding and structural proteins in terminal differentiation of human keratinocytes. Several inherited skin diseases have been associated with the EDC. Mutations in the loricrin gene accompanying abnormal CE formation are responsible for Vohwinkel's syndrome, a palmoplantar hyperkeratosis with ainhum-like constrictions of the fingers (Maestrini et al. 1996; Korge et al. 1997), and for progressive symmetric erythrokeratoderma (PSEK), which is characterized by a similar phenotype with expanded erythematous hyperkeratotic plaques (Ishida-Yamamoto et al. 1997). In addition, low levels of profilaggrin have been detected in ichthyosis vulgaris, a mild hyperkeratosis (Nirunsuksiri et al. 1995), and coordinate overexpression of S100A7, S100A8, S100A9, SPRR1, and SPRR2 has been shown in chronic inflammatory and hyperproliferative psoriasis (Hardas et al. 1996), in line with a psoriasis susceptibility locus within the 1q21 region (Capon et al. 1999). Altered expression of certain S100 genes has also been observed in other diseases, such as chronic inflammation (Rammes et al. 1997) and cardiomyopathy (Remppis et al. 1996), as well as in different tumors, such as breast cancer (Lee et al. 1992; Pedrocchi et al. 1994; Moog-Lutz et al. 1995; Albertazzi et al. 1998) and malignant melanoma (Maelandsmo et al. 1997). Furthermore, chromosomal aberrations of the 1q21 region are often implicated in tumorigenesis (Gendler et al. 1990; Hoggard et al. 1995; Weterman et al. 1996; Forus et al. 1998). In summary, identification of further genes located within the EDC should aid (1) to resolve the composition of biological structures in the epidermis, (2) to reveal potential control elements and signaling pathways governing differentiation of keratinocytes, and (3) to uncover genes and processes implicated in skin diseases or tumors associated with this region of chromosome 1. To reach this goal, a gridded keratinocyte cDNA library was constructed and successively hybridized with two entire YAC probes from the 1q21 region. Identified cDNA clones representing potentially new EDC genes were sequenced and their localization was confirmed on the integrated map of the EDC by use of additional 1q21-specific hybridization markers. Furthermore, cDNA sequences were analyzed with regard to their protein-coding regions and their functional domains. Finally, expression of the corresponding new genes during differentiation of cultured human epidermal keratinocytes was investigated.