The epithelial lining of the intestinal tract results from complex developmental patterns of histogenesis and gene expression. Little is known about the molecular mechanisms that direct these developmental programs. Sucrase-isomaltase (SI) will be used as a model gene to examine these mechanisms since it has a pattern of temporal expression that mirrors many important developmental transitions. SI has been utilized for over three decades as a marker for intestinal development and differentiation and recent studies from our laboratory have provided the foundation to examine the molecular mechanisms of its regulation.
SPECIFIC AIM 1 : Question: What regulates the dramatic increase in intestinal gene expression in the small intestine of mice as they progress through the suckling-weaning developmental transition? Hypothesis: The induction of SI gene expression at weaning is transcriptional and occurs via DNA regulatory elements located in the 5'-flanking region of the SI gene.
Aim : To identify the DNA regulatory elements, and their cognate DNA binding proteins, which regulate SI gene expression in the small intestine of mice as they progress through the suckling-weaning developmental transition. Preliminary data are presented which demonstrate the feasibility of using transgenic mice to examine the cis-acting regulatory elements that are required for correct developmental regulation of the SI gene. The precise localization of these regulatory elements will be achieved using a combination of transgenic mice, in vitro analysis of DNase I hypersensitive sites, use of stably transfected intestinal cell lines, and DNA-protein interactions. Finally, the proteins that bind these regulatory elements will be characterized and cloned.
SPECIFIC AIM 2 : Question: What are the mechanisms that restrict the expression of genes to specific cell lineages in the intestinal epithelium? And specifically, what are the mechanisms that restrict SI gene transcription to enterocytes? Hypothesis: SI gene expression is restricted to the enterocyte lineage by the silencing of transcription in other epithelial cell types.
Aim. To identify the DNA regulatory elements that silence SI gene transcription in intestinal epithelial cell lineages other than enterocytes. Preliminary data in both transgenic mice and cell lines suggest that restriction of expression to enterocytes is controlled at the level of repression of transcription. Cell lines stably transfected with minigene constructs will be utilized to identify transcriptionaI silencing elements. Transgenic mice will be used as an in vivo reporter system to determine if the DNA regulatory elements identified in the transfection analysis functions as expected in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047437-05
Application #
2900284
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
May, Michael K
Project Start
1995-04-20
Project End
2000-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz et al. (2008) The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine. Neoplasia 10:8-19
Ezaki, Toshihiko; Guo, Rong-Jun; Li, Hong et al. (2007) The homeodomain transcription factors Cdx1 and Cdx2 induce E-cadherin adhesion activity by reducing beta- and p120-catenin tyrosine phosphorylation. Am J Physiol Gastrointest Liver Physiol 293:G54-65
Keller, Matthew S; Ezaki, Toshihiko; Guo, Rong-Jun et al. (2004) Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells. Am J Physiol Gastrointest Liver Physiol 287:G104-14
Guo, Rong-Jun; Huang, Edward; Ezaki, Toshihiko et al. (2004) Cdx1 inhibits human colon cancer cell proliferation by reducing beta-catenin/T-cell factor transcriptional activity. J Biol Chem 279:36865-75
Boudreau, Francois; Rings, Edmond H H M; Swain, Gary P et al. (2002) A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP. Mol Cell Biol 22:5467-78
Boudreau, Francois; Rings, Edmond H H M; van Wering, Herbert M et al. (2002) Hepatocyte nuclear factor-1 alpha, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene. J Biol Chem 277:31909-17
Tung, J; Markowitz, A J; Silberg, D G et al. (1997) Developmental expression of SI is regulated in transgenic mice by an evolutionarily conserved promoter. Am J Physiol 273:G83-92
Markowitz, A J; Wu, G D; Bader, A et al. (1995) Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines. Am J Physiol 269:G925-39