Expression of the estrogen receptor (ER) in human breast cancer is a complex process involving multiple steps subject to hormonal regulation by estrogen and anti-estrogens. The principal goal of the proposed research is to define the mechanisms responsible for this complex regulation. More specifically the aims of this proposal are to define the role of the ER in: 1. translation of receptor MRNA, 2. post-transcriptional processing and stability of ER MRNA, and 3. transcription elongation of the ER gene. The proposed studies on translation are designed to define the molecular basis of this control . The effect of the anti-estrogen ICI-164,384 on receptor half-life will be determined and ribosomal association of ER MRNA will be evaluated. RNase Tl accessible sites on receptor MRNA will also be analyzed. In addition, the role of the 5' open reading frame in translational control will be addressed. Two approaches to the molecular mechanism of post-transcriptional suppression of ER MRNA will be taken. The first approach will measure the stability of ER MRNA and determine the role of the 5' and 3' noncoding regions in stability. The second approach will focus on processing of nascent ER transcripts. The role of antisense RNA in ER regulation will also be addressed. Besides a role in transcription initiation, our data suggest a role for the ER in transcription elongation. To address this question two separate approaches will be taken. To determine whether an attenuation-like model is responsible for the block in elongation, cells will be treated with proflavin. The site of recognition will be mapped. To determine if ICI-164,384 depletes factors involved in elongation, run-on I assays will be complemented with crude nuclear extracts from untreated cells. The factor will be identified.
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