P5-03-04: Circadian Modulation of Estrogen Receptor alpha Promoter Activity.

Abstract

The circadian clock is a molecular mechanism that synchronizes environment variations with physiological changes. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, coronary heart disease and cancer). Period 2 (PER2), a circadian protein, is at the center of the clock's function. The complete loss of per2 has been shown to be common in several types of cancer including breast and ovarian. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERa) mRNA oscillates in a 24-hour period and, unlike PER2, ERa peaks during the light phase of the day. Because up regulation of ER relates to tumor development, defining the mechanisms of ERa expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of estrogen receptor alpha (ERa) transcription. Transcriptional activation of ERa was measured using a reporter system in Chinese Hamster Ovary and MCF-7 cell lines. Data show that PER2 influence ERa transcription through a non-canonical mechanism independent of its circadian counterparts. Promoter analysis using the Alibaba 2.0 suite revealed the presence of various putative novel partners. Among them, OCT-1 was found to directly interact with Per2, in vitro, and with corresponding response element within the ERa promoter. The predicted binding site of OCT-1 is 267 bps upstream of the promoter. Pull-down assays were used to map direct interaction of various PER2 recombinant proteins and the DNA-binding domain of OCT-1. Radiolabeled OCT-1 was found to bind to the C-terminal end of PER2. Electrophoretic mobility shift assays were used to determine direct binding of OCT-1 to the promoter. ChIP assay confirmed the recruitment of PER2 to the estrogen promoter by OCT-1. Fluorescence anisotropy and surface plasmon resonance were employed to establish the binding affinity of OCT-1 to the ERa promoter and PER2. OCT-1 was found to have a high affinity for the ERa promoter (∼10 nM). Furthermore, OCT-1 recruits PER2 to the estrogen promoter. Preliminary data suggests that the transcription factor PER2 might play a role in the regulation of the estrogen promoter. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-03-04.