The objective of this application is to substantially broaden and strengthen the training of the candidate in the field of molecular biology. This will be accomplished under a defined mentoring program by extending the scope of ongoing mouse transgenic studies of long-range gene activation (Aim I) and by acquiring an entirely new set of biochemistry and structural biology tools required to formulate and execute a complementary set of investigations in vitro (Aim II). The experiments that are proposed focus on activation of the human Growth Hormone (hGH) chromatin locus. The GH gene has served as a paradigm for the study of tissue-specific and developmentally-regulated gene expression. The hGH gene cluster is of particular utility as it also serves as an informative model for long-range gene activation. A set of distal regulatory elements, comprising a locus control region (LCR), exerts major regulatory control over expression of the hGH gene cluster in both the pituitary and placenta. HGH LCR determinants activate their target genes over long distances and in a developmentally ordered and tissue-specific manner. A detailed understanding of hGH LCR structure, function, and mechanisms of action is of central importance to understanding of hGH gene regulation and to expanding the available mechanistic models of gene control. A set of specific scientific goals will be addressed within a mentoring environment that will expand the candidate's professional abilities and scientific skills.
Aim I will expand ongoing characterization of hGH LCR activities by developing a novel set of transgenic mouse lines to assess whether determinants at HSI of the LCR are required to maintain the hGH gene in a transcriptionally active state once it is activated, and to determine whether the Pit-1 binding sites at HSI can autonomously target histone acetylation. The in vitro studies of Aim II will test the ability of purified recombinant Pit-1 to bind to Pit-1 elements of the LCR in reconstituted nucleosomes, to subsequently recruit CBP/p300 histone acetylation factors to the Pit-1: nucleosome complexes, and to activate transcription from a reconstituted chromatin template.
These Aims will be carried out under the mentorship of two individuals dedicated to the candidate's training and expert in their respective fields. The ultimate goal of the application is to advance the understanding of gene regulation and to position the candidate to assume an independent research program at an academic institution.
| Shewchuk, Brian M; Ho, Yugong; Liebhaber, Stephen A et al. (2006) A single base difference between Pit-1 binding sites at the hGH promoter and locus control region specifies distinct Pit-1 conformations and functions. Mol Cell Biol 26:6535-46 |
