Nuclear receptors play an important role in the differentiation and development of many types of tissues. It is known that dysfunctional nuclear receptors are involved in diseases such as breast and prostate cancers, however, the central mechanisms of their involvement in the biological responses to such pathologies, remain to be elucidated. Recently, our laboratory has successfully employed a live cell approach that integrates molecular, biochemical and cell biological approaches to investigating nuclear receptor dynamics and the early events following ligand exposure that precede, if not predict, transcription. The proposed work will use this ?molecular cytology? approach to improve our understanding of transcription through direct observations of nuclear dynamics and their effects upon transcription in vivo. Specifically, this work aims to further characterize the dynamic interactions between nuclear receptors and known coregulators through the use of high-resolution fluorescence microscopy. Bulk colocalization studies will be performed in cell lines with transient and stable expression of functional bioluminescent receptors and cofactors (GFP-ER, AR, PR, TR, SRC- 1, SMRT, and a novel indirect means to visualize the non-coding mRNA coactivator, SRA. Cell lines with integrated lac operators (heterochromatic or euchromatic) will be used with lac repressor fusions to investigate protein-protein interactions, effects of ligand, and modification of chromatin structure. Moreover, in 2Od generation cell lines, a natural ER regulated prolactin promoter, flanked by lac operator repeats and driving peroxisome-targeted CFP as a transcriptional reporter will be used to directly link organizational effects of steroid receptors/cofactors with transcriptional activity, chromatin structure and the nuclear dynamics of these proteins. The proposed work will undertake a collective approach, incorporating live, in situ, molecular and cellular biology with the aim to greatly improve our understanding of transcription via simultaneous observations of nuclear dynamics and structure in vivo. Elucidation of these complex mechanisms and interactions will aid the development of novel treatments for diseases related to compromised steroid receptor function.
|Stenoien, David L; Mielke, Marilyn; Mancini, Michael A (2002) Intranuclear ataxin1 inclusions contain both fast- and slow-exchanging components. Nat Cell Biol 4:806-10|