The objective of this proposal is to define the functional role of the HTRA1 promoter polymorphism that was recently identified as a variant associated with susceptibility to age-related macular degeneration (AMD), and to begin to understand the mechanism regulating HTRA1 expression. AMD is the leading cause of irreversible blindness among elderly people in the developed countries and an etiologically complex disease caused by multiple genetic and environmental factors. Linkage studies revealed two chromosomal regions, 1q31-32 and 10q26, that likely contain susceptibility genes for AMD. Although complement factor H (CFH) appears to be the major susceptibility gene on 1q32, the critical region on 10q26 has been controversial. While several studies identified single nucleotide polymorphism (SNP) rs10490924 in the hypothetical gene LOC387715 as the AMD-associated variant on 10q26, other studies suggested SNP rs11200638 (G>A), a G to A variant located in the putative promoter region of the HTRA1 gene, as the true AMD-associated sequence change on 10q26 and led to the hypothesis that the rs11200638 in the HTRA1 promoter causes higher HTRA1 expression, and thereby increases the risk for wet AMD. Our analyses to explore this hypothesis using in vivo electroporation in mouse RPE showed that the HTRA1 promoter with risk-associated A had a significantly lower activity than the promoter with non-risk-associated G, opposite to the result expected from the hypothesis. Our yeast one-hybrid screen identified two transcription factors that bound to bait with G allele, but not to bait with A allele. More recently, additional SNPs in the HTRA1 locus, including SNP rs2672598 (C>T) in the promoter, have been reported as variants significantly associated with the risk for AMD, supporting the likelihood of HTRA1 as the susceptibility gene for AMD. Based on these findings and our own results, detailed analyses of the HTRA1 promoter and its variants are proposed as below. In Specific Aim 1, HTRA1 promoter activity will be analyzed in bovine RPE primary cells by transient transfection and in mouse RPE by in vivo electroporation. These analyses will involve HTRA1 promoter- luciferase reporter constructs for the two promoter variants, rs11200638 and rs2672598, as well as a series of promoter deletions. In Specific Aim 2, additional transcription factors that bind to the rs11200638 region will be identified by yeast one-hybrid screen, and the two factors already identified, CGGBP1 and SLC2A4RG, will be analyzed. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) will be used to analyze binding of these factors in vitro and in vivo, respectively. Cotransfection with an expression vector and small interfering RNA (siRNA) transfection will be used for gain-of-function analysis by overexpression and loss-of-function analysis by gene knockdown of these factors, respectively. Results of these analyses will help solve the controversy about the HTRA1 promoter variant and gain insights into the pathogenesis of AMD. PUBLIC HEALTH RELEVANCE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly people in the developed countries including the USA. Multiple genes and environmental factors seem to play important roles in developing the disease. A large-scale genetic studies have identified multiple sequence changes in the human genome that are associated with and possibly responsible for the risk of developing AMD, and several of such sequence variants are located in or near the HTRA1 gene. Therefore, the aim of this research is to experimentally analyze the two variants identified in the HTRA1 promoter region and determine their functional consequence on the expression of the HTRA1 gene.