- Involucrin (hlNV) is a major component of the scaffolding of the keratinocyte cornified envelope. During keratinocyte terminal differentiation, hlNV acts as an amine donor and is crosslinked to various amine acceptors in a trans-glutaminase catalyzed crosslinking reaction that results in scaffolding assembly. Because of the high content of glutamine residues spaced at equal intervals along its length, its elongated rod-like alpha-helical structure and its abundance, hlNV is ideally suited to function as a molecular crossbridge in the cornified envelope. Several interesting abnormalities of hlNV expression are observed in epidermal diseases including squamous cell carcinoma, and studies by the investigators show that hlNV overexpression produces an abnormal epidermal pheonotype in transgenic mice. Expression of involucrin is confined to external surface epithelial cells. In epidermis, it is specifically expressed in the suprabasal, but not basal layers. To ultimately understand the process of normal and abnormal epidermal differentiation, it is essential to understand the molecular mechanisms responsible for regulation of genes specifically expressed in epidermis. The investigators believe that they can now ask fundamental questions about specific sequences that drive tissue-specific and differentiation-appropriate hlNV expression in vivo. In preliminary published and unpublished experiments, the investigators have identified two regions of the hlNV upstream regulatory region, the distal regulatory region (DRR, positions -2473/ 1799) and proximal regulatory region (PRR, positions -241/-7), that are required for optimal promoter activity. In preliminary experiments, they have also identified a 523 bp segment of the hlNV DRR that is required for epidermis-specific expression in transgenic mice. Major goals of this proposal are: (i) To determine whether the 523 bp segment is alone sufficient to target expression to epidermis and, if not, to identify other elements that cooperate with it; (ii), to identify specific transcription factor binding sites within the 523 bp region that are required for expression in epidermis; and (iii) to identify the transcription factors that bind at these sites. Their initial studies identify AP1 and Sp1 as potential regulators of the DRR region and as components of the 523 bp """"""""epidermal targeting"""""""" region, and AP1, ets, and C/EBP factors as regulators of PRR activity. Their experiments are designed (i) to understand the role of AP1 and Sp1 as regulators of the DRR region, (ii) to study the interaction between AP1, ets, and C/EBP as regulators of activity of the PRR, and (iii) to evaluate whether the PRR contributes to epidermis-specific expression. These studies, they expect, will provide new insight regarding mechanisms that regulate hlNV expression during epidermal differentiation. When these studies are completed, the investigators expect to have identified many, perhaps all, of the major transcription factor sites required for hlNV expression in epidermis.
