The applicant will test the hypothesis that changes in alpha-fetoprotein (AFP) gene expression are the result of chromatin structure remodeling mediated by 1) transactivator induction 2) repressor depletion and 3) renewed DNA replication as described below:
Specific Aim 1 : In vitro transcription of chromatin assembled AFP and control albumin promoter DNA templates with either adult, regenerating or sham-operated mouse liver nuclear extracts will be used to reconstitute liver regeneration-mediated changes in AFP gene expression. These studies will also allow for determination of the transcription factors mediating AFP gene repression and activation.
Specific Aim 2 : Determine the role that DNA replication plays in genetic reprogramming following partial hepatectomy, which involve proliferation of quiescent adult hepatocytes to repopulate the damaged liver. AFP gene expression states will be determined following assembly of transcription templates into synthetic nuclei, which undergo a single round of semi-conservative DNA replication.
Specific Aim 3 : Determine whether regeneration-mediated changes in gene expression are reflected in chromatin structural changes both in vivo and in vitro. Structure analysis will consists of I) nucleosome positioning and histone modification assays, ii) assays for chromatin structure remodeling/modifying complexes and iii) chromatin structure characterization after passage of a replication fork. The short-term goals are to reconstitute liver regeneration -mediated, genetic reprogramming in vitro and role of increased DNA synthesis and chromatin structural changes in the process. The long-term goal of these studies is to learn how regenerative organs, such as the liver are capable of redirecting their genetic programs in response to traumatic injury.
| Johnson, Amber Buescher; Barton, Michelle Craig (2007) Hypoxia-induced and stress-specific changes in chromatin structure and function. Mutat Res 618:149-62 |
| Wilkinson, Deepti S; Ogden, Stacey K; Stratton, Sabrina A et al. (2005) A direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene. Mol Cell Biol 25:1200-12 |
| Nguyen, Thi T; Cho, Kyucheol; Stratton, Sabrina A et al. (2005) Transcription factor interactions and chromatin modifications associated with p53-mediated, developmental repression of the alpha-fetoprotein gene. Mol Cell Biol 25:2147-57 |
