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 Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)
Carpenter
Anne E
AE
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Ashouri
Anousheh
A
Belmont
Andrew S
AS
D000818:Animals; D001331:Automation; D016466:CHO Cells; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D006224:Cricetinae; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017384:Sequence Deletion / Q000235:genetics; D013997:Time Factors
D002843:Chromatin; D047628:Estrogen Receptor alpha; D011993:Recombinant Fusion Proteins