The goal of this project is to understand the mechanism by which the progesterone receptor (PR) interacts with and remodels chromatin at target genes in vivo using the mouse mammary tumor virus (MMTV) promoter as a model system. In the non-activated state, this promoter has a chromatin structure repressive to transcription when it exists in cells as replicating chromatin. Upon binding of the liganded glucocorticoid receptor (GR), the promoter undergoes a chromatin remodeling event which is mechanistically involved in the activation of transcription. Our previous work has shown that the GR and PR have different requirements for chromatin remodeling at the MMTV promoter even though they bind to the same DNA sequences in the promoter. Our observations may form the basis for a mechanism by which the GR and PR control expression of distinct sets of target genes in vivo. This is particularly relevant in the mammary gland where the GR and PR can coexist in the same cell types. We have established that the PR exists in two distinct functional states in cultured mammary adenocarcinoma cells. In one state, it can neither remodel chromatin nor activate transcription at the MMTV promoter; thus its action may be restricted to target genes which do not require remodeling. In addition, this form of the PR can be activated by other signal transduction pathways in a progestin-independent fashion. In the second functional state, the PR is able to remodel and activate the MMTV promoter in chromatin, but is refractory to ligand-independent activation. Thus, this form of the PR responds only to its ligand but would be able to activate target genes even in a repressive chromatin environment. We have also shown that the PR can be converted from the first state to the second by some form of cellular processing. This may represent a mechanism by which cells can restrict or expand the activity of the PR in vivo. To elucidate the biochemical basis for the two distinct functional states, we have developed an immunoprecipitation method to isolate the PR in its native forms and examine associated proteins and post-translational processing. This has been done in collaboration with Dr. David Smith (Mayo Clinic Scottsdale) who has provided us with reagents and expertise invaluble to the project. We have concentrated this year on interactions of the unliganded PR with chaperone proteins and immunophilins. We find that there are two classes of interactions, one of which has been defined previously. The other class of interactions is novel. The PR forms both types of complexes in the two functional states, but the proportion of the two PR complex types is significantly different. We speculate that this altered balance indicates that PR/chaperone interactions can be regulated by means other than the presence of progestins. In addition, the balance of these interactions may have a significant impact on the function of the PR. Future efforts will be directed at defining the role of phosphorylation in determining this balance. In collaboration with Dr. Stoney Simons (NIDDK), we have also initiated a project to examine the activity of the two functional forms of the PR in the presence of anti-progestins. Given recent studies linking progestin exposure to increased risk of breast cancer in postmenopausal women, it is probable that antiprogestins may be used in a chemopreventive role in the future. A better understanding of PR behavior in response to these agents specifically in the context of mammary-derived cells may provide important information to clinicians.
