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|Title:||Insights into ectopic estrogen receptor expression, nucleocytoplasmic distribution and interaction with chromatin obtained with new antibodies to estrogen receptors alpha and beta|
|Author/Creator:||Chantzi, Niki I.|
Meligova, Aggeliki K.
Petrou, Christos C.
Mitsiou, Dimitra J.
Tiniakos, Dina G.
Alexis, Michael N.
|Type:||Journal Article (Scientific Journal article)|
|Abstract:||Recent reports have indicated that in cells ectopically expressing only ER alpha or the full-length hormone-binding isoform of ER beta (ER beta 1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ER alpha or ER beta and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ER alpha or ER beta 1, in order to comparatively study their interaction with reporter ERE. We show that ER alpha was located in the nucleus and ER beta 1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ER alpha, ER beta 1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ER alpha or ER beta 1, ER subtype-specific constraints apply to ER beta 1 nuclear entry: and that in cells displaying cytoplasmic as well as nuclear localization of ER beta 1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.|
|Publication Place:||NEW YORK|
|Subject Category:||Science::Biology (General)|
|Keywords:||Estrogen receptor; Monoclonal antibodies; Chromatin immunoprecipitation; Endocrinology & Metabolism|
|Other Identifiers:||DOI: http://dx.doi.org/10.1016/j.steroids.2011.05.010|
|Journal web Location :||http://www.elsevier.com/locate/steroids|
|Rights holder:||© 2011 Elsevier Inc. All rights reserved.|