Analyses of Regulatory Activities of h923 in Human Epidermal Differentiation Complex(EDC)

The Epidermal Differentiation Complex (EDC) spans 1.6Mb on human 1q21 and comprises four gene families encoding proteins in the skin epidermis. Genetic linkage studies identified association of the EDC to atopic dermatitis and psoriasis. Conserved non-coding elements (CNEs) were identified in comparative genomic studies as potential cis-regulatory elements in the EDC and may be likely genetic variants in these diseases. The h923 CNE demonstrated high enhancer activity in cell-based reporter and DNase I hypersensitivity (HS) assays and was confirmed in transgenic mice. Four PhastCon (ultra-conserved) blocks within h923 were bioinformatically identified to determine possible enhancer sequences. We hypothesize that PhastCon blocks in h923 are required for enhancer activity. Luciferase assays on four individual PhastCon block deletions in h923 constructs were performed in proliferating and differentiating keratinocytes.

Four h923 deleted constructs were generated by PCR and cloned upstream into luciferase vectors using Gateway Cloning Technology. The luciferase vector clones (Basic, Promoter, and Sprr1a) were co-transfected with the Renilla luciferase into proliferating and differentiating mouse keratinocytes at 0.05mM and 1.3mM Ca²⁺, respectively, and dual luciferase assays were performed.

Under differentiating conditions, Sprr1a 923 del1 and 923 del2 clones showed a two-fold drop in luciferase activity as compared to the h923 positive control. This suggests that PhastCon Block 1 is minimally required for enhancer activity. Experiments on other luciferase vectors and proliferating conditions are underway. By analyzing regulatory 923 activity during epidermal differentiation, we hope to gain better insight into a regulatory element that could likely be a genetic variant in skin diseases.