The long-term goal of this project is to understand the molecular mechanisms by which progesterone receptor (PR) recognizes and binds to target DNA sites, as this is an essential step for transcriptional signaling by progesterone. We discovered that the nuclear chromatin associated high mobility group proteins-1 and -2, HMGB-1/-2, facilitate binding of PR and all steroid receptors to their cognate target DNA sites, and are limiting factors in the cell required for transcriptional activity for steroid receptors. This interaction is selective within the nuclear receptor super-family, as HMGB-1/-2 does not interact with non-steroidal class II nuclear receptors. HMGB-1/-2 binds linear DNA with little sequence specificity, recognizes specific DNA structures, and can induce sharp bends in DNA. Therefore, it is thought that HMGB-1/-2 functions as a """"""""DNA chaperone"""""""" by facilitating the induction or stabilization of DNA conformations that are most favorable for transcription factor binding. As a non-sequence-specific DNA binding protein, HMGB-1/-2 is recruited to the PR-DNA complexes by a protein interaction with a region of the PR DNA binding domain (DBD) outside of the core zinc finger motif, termed the C-terminal extension (CTE). In addition to simply recruiting HMGB-1 to carry out its DNA chaperone activity in a gene-specific manner, our biochemical and functional data suggest that HMGB-1 interaction with the PR CTE also induces a conformational change in the CTE, enabling it to interact with DNA and stabilize interactions by the core DBD. Further insights into the mechanism of action of HMGB-1 will require structural studies, which are a significant portion of this proposal.
AIM # 1 will explore the biological role and mechanism of interaction of HMGB-1 with receptors on endogenous steroid regulated target genes in wild type and hmgbl-/- cells, and will test the hypothesis that a role of HMGB-1 is to facilitate receptor interaction with genes containing suboptimal half-hormone response elements (HREs).
AIM#2 will define the protein interaction surfaces between HMGB-1 and the CTE of PR, how the CTE interacts with DNA and the interactions that occur in the ternary HMGB/PR/DNA complex.
This aim will use combined approaches of biochemical mutagenesis, chemical cross-linking, DNA footprinting, and structure analysis by X-ray crystallography.
AIM #3 will determine the structure of the full-length PR complexed to DNA as a fundamentally important goal to understand the role of domain-domain interactions in receptor DNA binding and control of total receptor activity.
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