In Vitro Reconstitution of Alpha Fetroprotein Regulation

University Of Tx Md Anderson Can Ctr, Houston TX

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Abstract

DESCRIPTION (provided by applicant): Alpha-fetoprotein (AFP) is abundantly expressed in fetal liver but is repressed after birth as hepatocytes differentiate and exit the cell cycle. AFP is re-activated in adult liver in response to renewed cellular proliferation during liver tumorigenesis and liver regeneration. Our proposed studies will test the hypotheses that (Specific Aim I) aberrant loss of regulated AFP repression is the result of altered chromatin structure, (Specific Aim II) multiple proteins interact to establish specific chromatin structures that preclude AFP transcription and (Specific Aim III) DNA replication contributes to loss of AFP transcription repression by facilitating alterations of chromatin structure and depletion of repressor proteins. Our experiments will take advantage of in vitro chromatin and synthetic nuclei transcription systems used routinely in our laboratory. Together with our proposed analyses of AFP chromatin structure and transcription in vivo, these experiments will define mechanisms of developmentally regulated AFP repression and loss of this repression during tumorigenesis. Specific Aim I: Define a time course of AFP transcription repression at the level of chromatin structure, core promoter occupancy and function in vivo. We will first define the developmental stage at which AFP repression occurs and the associated changes in chromatin structure and promoter occupancy at specific time points. Next, we will test our hypothesis that critical parameters of AFP transcription repression established during development are lost or altered during tumorigenesis when AFP transcription is aberrantly re-activated. Specific Aim II: Determine the mechanisms by which transcription factors repress AFP expression. We will determine the occupancy of candidate transcription regulators at the AFP developmental repressor during specific stages of AFP expression and repression. We found that two tumor suppressor pathways, p53 and TGF-beta, act together to repress aberrant AFP expression in hepatoma cells. We will determine if this cross talk between p53 and TGF-beta signaling also represses AFP during normal development. We will screen for additional proteins that act with p53 and TGF-beta effectors to establish full repression of AFP transcription. Specific aim III: Determine how DNA replication contributes to disruption of transcription repression. Our hypothesis is that DNA replication can facilitate modifications in chromatin structure and gene expression, leading to aberrant activation of AFP gene expression. To test this hypothesis, we will assemble AFP gene templates into developmentally repressed, solid-phase nuclei and analyze chromatin structure and transcription function before and after DNA replication.