The mammalian intestine continuously renews itself as intestinal stem cells give rise to four epithelial cell types. Enteroendocrine cells represent less than 5% of the total number of epithelial cells but secrete hormones that control numerous physiological processes including appetite and satiety, insulin secretion, and digestive organ function. The transcription factor, Neurogenin 3, initiates the endocrine differentiation program in the intestine and activates expression of NeuroD, another basic helix loop helix protein. NeuroD appears to coordinate terminal differentiation of enteroendocrine cells with cell cycle exit. Activation of constitutive Wnt signaling in neurogenin 3 expressing cells induced intestinal neuroendocrine tumors whereas Wnt activation in NeuroD expressing cells did not, suggesting that NeuroD expression represents a distinct, later stage of differentiation of enteroendocrine cells. The mechanism of transcriptional activation by NeuroD is not well characterized but preliminary results indicate interactions with other DNA binding proteins, CtBP, and the histone modifying enzyme, lysine specific demethylase 1 (LSD1) are involved. The paucity of identified NeuroD targets in enteroendocrine cells, has made it difficult to understand the role of this important transcription factor in their differentiation. The three aims of this proposal will address the function of NeuroD in differentiating enteroendocrine cells.
Aim 1 will examine how NeuroD associates with Sp1, RREB1, and LSD1 at one its known targets, the secretin gene to form a multiprotein coactivator complex. The paradoxical coactivator function of C-terminal binding protein, CtBP, which is generally a corepressor, will be characterized by examining histone modifications and proteins CtBP and NeuroD associate with at the secretin gene enhancer. The goal of Aim 2 will study the transcriptional mechanism of inhibition of Wnt signaling by NeuroD by determining the DNA binding proteins and coactivators/corepressor complexes that NeuroD associates with at promoters regulated by Wnt/?-catenin in vitro. The role of NeuroD in the inhibition of Wnt signaling in vivo will be examined in transgenic mice that either conditionally express NeuroD or a NeuroD knockdown shRNA to determine whether expression of NeuroD at an earlier stage of differentiation prevents development of neuroendocrine tumors following Wnt activation or whether knocking down NeuroD expression removes the block to developing tumors following Wnt activation in NeuroD+ cells.
Aim 3 will identify NeuroD regulated genes in normal enteroendocrine cells by gene expression profiling by high throughput sequencing (RNAseq) of RNA from NeuroD+ cells isolated from mouse small intestine by a new method developed by the principal investigator. Genome-wide chromatin occupancy studies (ChIPseq) will identify a subset of differentially expressed genes as potential direct targets by NeuroD.

Public Health Relevance

A small number of the cells lining the intestine produce hormones in response to meals, hunger, and other stimuli. These hormones have a major role in the control other important body functions such as appetite, food intake, satiety, and insulin secretion. Thus, normal hormone secretion from the intestine may have an important role in preventing or reducing the severity of obesity and diabetes, two major diseases in the U.S. This proposal will study mechanisms controlling the development of these important hormone-producing cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK090000-04
Application #
8481215
Study Section
Special Emphasis Panel (ZRG1-DKUS-C (04))
Program Officer
Serrano, Jose
Project Start
2010-07-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2013
Total Cost
$374,726
Indirect Cost
$146,929
Name
University of Massachusetts Medical School Worcester
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Li, Hui Joyce; Johnston, Brian; Aiello, Daniel et al. (2014) Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus. Gastroenterology 146:754-764.e3
Ray, Subir K; Li, H Joyce; Metzger, Eric et al. (2014) CtBP and associated LSD1 are required for transcriptional activation by NeuroD1 in gastrointestinal endocrine cells. Mol Cell Biol 34:2308-17
Li, H J; Ray, S K; Singh, N K et al. (2011) Basic helix-loop-helix transcription factors and enteroendocrine cell differentiation. Diabetes Obes Metab 13 Suppl 1:5-12