A critical, unresolved issue in gene regulation is the means by which chromatin that packages developmentally regulated genes is accessed and remodeled to orchestrate cell-type specific gene expression programs informing tissue differentiation and function. This poses a critical dilemma in cells of both the developing embryo, in which much of the chromatin is still compacted, and the adult organism, where alterations in gene expression in response to signaling cascades directing essential physiological functions necessitate rapid alterations in the chromatin landscape. Failure to properly navigate either of these biological challenges can have grave biomedical consequences in the form of congenital malformations, disease, and neoplasia in humans. Based on the ability of the initial "pioneer" chromatin binding transcription factor FoxO1 to remodel histone:DNA contacts within compacted chromatin in vitro, its recruitment of liver-enriched regulatory factors and RNA pol II to regulatory targets in hepatocytes, and regulation of these activities by posttranslational modifications linked to signaling pathways with proven roles in maintenance of glucose, bile acid and cholesterol homeostasis in humans we hypothesize that FoxO1 cooperates with closely juxtaposed liver- enriched transcription factors to carry out chromatin structural alterations necessary for developmental activation of hepatic genes crucial to human hepatocyte function. We argue that posttranslational modification of the FoxO1 DNA binding domain enables hormonal regulation of FoxO1 target genes in the human liver by disrupting this relationship. This proposal addresses two key questions that stem from this hypothesis: 1) How does FoxO1 cooperate with other liver-enriched regulatory factors to carry out chromatin remodeling and modification necessary for developmental activation of hepatic genes? and 2) Is this cooperation necessary for hormonal regulation of transcriptionally active chromatin encoding essential metabolic functions in the human liver? To accomplish this goal, we will use a novel hiPS (human induced pluripotent stem) cell human hepatocyte differentiation system to uncover basic molecular mechanisms informing the function and regulation of FoxO1 under conditions that recapitulate hepatocyte specification, differentiation, and function in humans. We will also use state of the art molecular techniques that enable the assembly and analysis of defined nucleoprotein structures to test mechanisms implicated by our observations. FoxO1 is now established as a key transcriptional regulator integrating hepatic glucose, cholesterol, bile acid, and lipid metabolism in humans;mutant FoxO1 alleles are recently demonstrated in diabetes and hepatic steatosis and fibrosis in human patients. Our hypothesis assumes that the chromatin binding and remodeling capabilities attributed to FoxO1 are essential for the assembly of the nucleoprotein structures tasked with activating FoxO1 target genes and mediating their response to diverse signaling cascades. Subversion of these gene regulatory events is a likely contributor to metabolic derangements and hepatic disease, making it vital that we uncover the key mechanisms and players.

Public Health Relevance

The overall goal of this proposal is to determine how the pioneer initial chromatin binding transcription factor FoxO1 uses its abilities to remodel chromatin and cooperate with closely juxtaposed liver-enriched transcription factors to initiate changes in chromatin structure necessary for activation and hormonal modulation of target genes in the human liver. FoxO1 is now established as a key regulatory protein integrating hepatic glucose, cholesterol, bile acid, and lipid metabolism in humans and FoxO1 mutation/insufficiency is linked to diabetes and hepatic steatosis/fibrosis in humans. If successful, the proposed experiments will reveal novel mechanisms for FoxO1 regulation, enabling targeted intervention in hepatocyte differentiation and function for the treatment of diabetes and liver disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK093763-02
Application #
8486428
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Serrano, Jose
Project Start
2012-06-15
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
2
Fiscal Year
2013
Total Cost
$321,129
Indirect Cost
$111,241
Name
Medical College of Wisconsin
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226