Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERβ (ERβ1), 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 ERα or ERβ 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 ERα or ERβ1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERβ1 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 ERα, ERβ1 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 ERα or ERβ1, ER subtype-specific constraints apply to ERβ1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERβ1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.
- Chromatin immunoprecipitation
- Estrogen receptor
- Monoclonal antibodies