TY - JOUR
T1 - Automated Microscopy Identifies Estrogen Receptor Subdomains with Large-Scale Chromatin Structure Unfolding Activity
AU - Carpenter, Anne E.
AU - Ashouri, Anousheh
AU - Belmont, Andrew S.
PY - 2004/4
Y1 - 2004/4
N2 - 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.
AB - 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.
KW - Automated microscopy
KW - Chromatin
KW - Estrogen receptor
KW - High-throughput screening
KW - Steroid
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U2 - 10.1002/cyto.a.10113
DO - 10.1002/cyto.a.10113
M3 - Article
C2 - 15057969
AN - SCOPUS:1942534136
VL - 58
SP - 157
EP - 166
JO - Cytometry. Part A : the journal of the International Society for Analytical Cytology
JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology
SN - 1552-4922
IS - 2
ER -