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Date Submitted
Spring 5-3-2012
Research Mentor and Department
Dr. Cristina Strong
Restricted/Unrestricted
Dissertation/Thesis
Abstract
The skin is an important tissue because it acts as a first line of defense. The human Epidermal Differentiation Complex (EDC) in the genome encodes genes required for the skin barrier; however, the mechanism to coordinate EDC gene expression is unknown. The research focuses on identifying the function of a conserved noncoding element (CNE), 923, in the human EDC. We hypothesize a function for 923 as a locus control region or potential genetic master switch for EDC gene expression during skin cell development and differentiation. The lab’s previous studies showed that 923 has key properties of an enhancer including DnaseI hypersensitivity. Also, in transgenic reporter mice, 923 showed epidermal specificity. Within 923, 4 PhastCons blocks (areas of conserved sequence between multiple species) were uncovered and it was hypothesized that each has a role in 923 enhancer activity. To test this, serial deletions of the blocks were cloned into a vector with a firefly luciferase reporter gene. Each construct was subsequently cotransfected with Renilla luciferase into mouse epidermal cells (SP-1) under proliferating and differentiating conditions before firefly luciferase assays were performed and normalized to Renilla. In both orientations, deletion of Block 4 demonstrated a decrease in enhancer activity. I investigated transcription factor binding sites and hypothesized that AP-1 was a critical transcription factor. Site-directed mutagenesis was employed to alter the AP-1 binding site and test for enhancer activity. A better understanding of the genetic regulation of the EDC could allow progress in treating skin barrier diseases with known genetic mutations.