DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

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The central focus of this research is to understand the mechanisms controlling the expression of lactase-phlorizin hydrolase (LPH), an intestinal, microvillus membrane glycoprotein membrane glycoprotein important for the nutrition of mammalian neonates. The genetic expression, enzymatic activity, and intracellular processing of LPH are paradigms for critical processes in the enterocyte, and are all determined by the DNA sequence of the LPH gene. During the last funding period, we have defined the function of 2 kb of the rat LPH promoter in transgenic mice, and identified important cis-acting elements in the proximal promoter, one of which exhibits cell-specific repressor activity. Studies of mRNA localization demonstrate specific patterns, suggesting that the localization of LPH mRNA is important for the distribution of its encoded protein. The hypothesis underlying the proposed experiments are: 1) that hierarchical function of positive and negative factors regulates LPH gene transcription, 2) that cis-acting polymorphism(s) are involved in the genesis of human lactase persistence, and 3) that vectorial movement of LPH mRNA to the apical pole of the enterocyte is a regulated pathway, perhaps involving the cytoskeleton. These concepts form the basis of the specific aims of the present proposal: I) Define the mechanisms of transcriptional control of LPH gene expression: elucidate the individuals and combined effects of specific transcription factors on LPH gene expression, identify the mechanisms of cell-specific negative regulation of the LPH gene, and define candidate elements identified by DNA polymorphisms in the human LPH gene that determine lactase persistence. II) Identify the critical sequence elements in the 3'-UTRs of LPH and other enterocyte mRNAs responsible for their intracellular localization: define the sequences necessary and sufficient for mRNA localization by mapping and mutational analysis of the 3'-UTRs and intracellular translocation studies using adenovirus-mediated gene transfer techniques, characterize mRNA binding proteins required for vectorial mRNA transport, and define the role of the cytoskeleton in localization of mRNA transport, and define the role of the cytoskeleton in localization of mRNAs in enterocytes. Taken together, the studies described in this application should provide an integrated understanding of the role of 5' and 3' regulatory elements and their binding proteins in the transcriptional and post- transcriptional regulation of LPH gene expression.