The broad objective of this proposal is to define the mechanisms mediating spatiotemporal pattern formation during gut development and maturation. The research plan will focus on the molecular mechanisms regulating the spatiotemporal restriction of intestinal lactase gene transcription. Intestinal lactase is the enterocyte disaccharidase responsible for digestion of lactose, the primary carbohydrate in milk. Lactase activity is maximal prior to weaning and then declines significantly during maturation. Decreased lactase activity combined with excessive milk consumption results in symptoms of carbohydrate malabsorption in most mature mammals, including humans. We have characterized several DNA regulatory elements and transcription factors (GATA-4/5/6, Cdx-2, HNF-1 and Pdx-1) involved in regulating lactase gene transcription in intestinal cells. In addition, we have determined that a 2.0-kilobase fragment of the lactase promoter directs appropriate spatial and temporal expression in the small intestine of transgenic mice. We present a global hypothesis that describes the likelihood of deciphering enteric codes for combinatorial interaction between DNA and protein modules mediating spatiotemporal gut gene expression. We will focus on a potential role for Pdx-1 in mediating anterior spatial restriction. Research objectives, therefore, are aimed at characterization of lactase cis regulatory elements and transcription factors.
In Aim 1, we will characterize lactase DNA regulatory elements by analyzing expression of genomic deletions linked to a reporter gene in intestinal cell culture and in transgenic mice. This analysis will be facilitated by methods to allow for in vivo bioluminescent detection of reporter gene expression.
In Aim 2, we will characterize the spatiotemporal expression and DNA binding patterns for known lactase transcription factors during gut development and maturation.
In Aim 3, we will functionally investigate the role of the homeodomain protein Px-1 in mediating spatial restriction of the lactase gene by altering the Pdx-1-lactase promoter interaction in cell culture and in transgenic mice and assaying for transcriptional activity. The combination of ex vivo and in vivo approaches are designed to characterize the mechanisms involved in regulating the spatiotemporal restriction of lactase gene transcription.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK060715-03
Application #
6911730
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
May, Michael K
Project Start
2003-07-01
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$355,218
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Fang, Lin; Ahn, Jong Kun; Wodziak, Dariusz et al. (2012) The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression. Hum Genet 131:1153-9
Chen, Chin; Sibley, Eric (2012) Expression profiling identifies novel gene targets and functions for Pdx1 in the duodenum of mature mice. Am J Physiol Gastrointest Liver Physiol 302:G407-19
Chen, Chin; Fang, Rixun; Chou, Lin-Chiang et al. (2012) PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells. Biochem Biophys Res Commun 423:183-7
Chen, Chin; Leavitt, Tripp; Sibley, Eric (2012) Intestinal Pdx1 mediates nutrient metabolism gene networks and maternal expression is essential for perinatal growth in mice. Biochem Biophys Res Commun 424:549-53
Olds, Lynne C; Ahn, Jong Kun; Sibley, Eric (2011) 13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1. Hum Genet 129:111-3
Sibley, Eric; Ahn, Jong Kun (2011) Theodore E. Woodward Award: lactase persistence SNPs in African populations regulate promoter activity in intestinal cell culture. Trans Am Clin Climatol Assoc 122:155-65
Fu, Ya-Yuan; Lu, Chih-Hsuan; Lin, Chi-Wen et al. (2010) Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution. J Biomed Opt 15:046018
Chen, Chin; Fang, Rixun; Davis, Corrine et al. (2009) Pdx1 inactivation restricted to the intestinal epithelium in mice alters duodenal gene expression in enterocytes and enteroendocrine cells. Am J Physiol Gastrointest Liver Physiol 297:G1126-37
Fu, Ya-Yuan; Lin, Chi-Wen; Enikolopov, Grigori et al. (2009) Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution. Gastroenterology 137:453-65
Fu, Ya-Yuan; Sibley, Eric; Tang, Shiue-Cheng (2008) Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes. J Gen Virol 89:3004-8

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