Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity

Background: Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. Methods: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. Results: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. Conclusions: This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementantary (C) 2004 Wiley-liss, Inc.