Early childhood and old age may simply represent periods of physiological and/or genetic susceptibility in the life of an ocular lens. The lamellar/ Marner cataracts (autosomal dominant) that appear in the early childhood or juvenile years best exemplify this. This developmental aberration has been recently linked to mutations in the DNA binding domain of the heat shock transcription factor 4 (HSF4). Our investigations have revealed that HSF4 appears after the initial development of the lens is complete and increases in expression in the postnatal lens. This pattern of expression suggests a remarkable association with the appearance of the lamellar cataracts in the human lens. In order to understand the molecular mechanism that governs this link between the genotype and the pathological phenotype we propose three specific aims (SA) employing BAC (bacterial artificial chromosome) transgenesis that is based on recent revolution in """"""""Recombineering"""""""" techniques for the manipulation of the BACs. In the first specific aim, we propose to delineate the boundaries of the transcriptional unit of the HSF4 gene, characterize its promoter(s) and elucidate the complexity of the different isoforms produced by alternative splicing. In the second SA, we will engineer a reporter (a LacZ or EGFP) into the HSF4 gene residing in BACs, make transgenic mice and follow the expression of the reporter. Because BACs direct correct expression, spatially and temporally, we will be able to pinpoint essential DMA regions in and around the HSF4 gene that are required for the faithful expression in the ocular lens. Using the BAG identified in specific aim #2, in the SA #3 we will create a mouse paradigm of human cataractogenesis by introduction of the same four mutations (reported in the human HSF4 gene) into transgenic mice. We will investigate these mice to find how the quality of the genotype relates to the temporal presentation of the morphological and the molecular phenotype of the ocular lens, allowing us an insight into cataractogenesis and HSF4 gene activity. ? ? ? ?
Showing the most recent 10 out of 11 publications
