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 a stably-replicating form, either integrated into the cellular genome or as an episome. Upon binding by 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 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 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. In the past year we have demonstrated that, in addition to functional differences between the two forms of the PR, their localization within the nucleus may be different. Using indirect immunofluorescence methods, we show that both forms of the PR are localized to the nucleus even in the absence of ligand. However, cellular fractionation shows definitively that the two forms of the PR bind to sites in the nucleus with different affinities. One form is loosely bound to the nucleus and fractionates with the cytosol in the unliganded state while the other form is tightly bound to the nucleus and must be extracted with high ionic strength buffers. Its behavior in the unliganded state is very similar to that of the other form of the PR in the liganded state when it is transcriptionally engaged at target genes. Thus our hypothesis is that the two forms of the receptor exist in different nuclear compartments. The form which is bound tightly to the nucleus independent of ligand is probably pre-associated with target genes while the other form is not and must be transported to target sites upon activation by ligand. This difference may provide a basis for some of the distinct functional behaviors we have defined. To elucidate the biochemical basis for the two distinct functional states, we have been developing 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 who has provided us with reagents and expertise invaluble to the project. We have successfully developed this method to the point where we can begin comparative studies of the PR in its two functional states. To further enhance our understanding of PR processing, we have also been developing cell lines in which PR expression is conditional using the tetracycline repressor system. Using these cell lines we can follow the function and processing of the PR over time. This is important biologically because PR expression in the mammary gland is not constant but regulated by estrogen. We would like to use this system to elucidate mechanisms by which mammary cells control PR function. We have recently isolated several conditional cell lines and are currently in the process of characterizing them. - Chromatin, Mouse mammary tumor virus, transcriptional regulation, Progesterone receptor, protein transport, post-translational regulation,
