Automated Microscopy Identifies Estrogen Receptor Subdomains with Large-Scale Chromatin Structure Unfolding Activity

Anne E. Carpenter, Anousheh Ashouri, Andrew S. Belmont

Research output: Contribution to journalArticlepeer-review

Abstract

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 α 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.

Original languageEnglish (US)
Pages (from-to)157-166
Number of pages10
JournalCytometry Part A
Volume58
Issue number2
DOIs
StatePublished - Apr 2004

Keywords

  • Automated microscopy
  • Chromatin
  • Estrogen receptor
  • High-throughput screening
  • Steroid

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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