TY - JOUR
T1 - Ligand-induced large-scale chromatin dynamics as a biosensor for the detection of estrogen receptor subtype selective ligands
AU - García-Becerra, Rocio
AU - Berno, Valeria
AU - Ordaz-Rosado, David
AU - Sharp, Zelton D.
AU - Cooney, Austin J.
AU - Mancini, Michael A.
AU - Larrea, Fernando
N1 - Funding Information:
This study was supported in part by grants from the Contraceptive Research and Development Program, Arlington, Virginia (to A.J.C. and F.L.) and the Consejo Nacional de Ciencia y Tecnología, México (to F.L.). We thank Dr. G.A. García (Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico) for the synthesis and chemical analysis of steroids used in this study. All imaging was performed in the Department of Molecular and Cellular Biology Integrated Microscopy Core (Center for Reproductive Biology, O'Malley; Dan L. Duncan BCM Cancer Center, Osborne; John S. Dunn Gulf Coast Consortium for Chemical Genomics, Davies and Mancini).
PY - 2010/6
Y1 - 2010/6
N2 - Estrogen receptors (ER), members of the nuclear steroid receptor superfamily, act to activate transcription through ligand-dependent recruitment of coregulators and chromatin modifications. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind ERΑ for activated transcription, and to recruit coregulatory factors. In this study, we have analyzed the ability of synthetic 19-nortestosterone derivatives to visibly alter the configuration of ER-target gene chromatin using a novel mammalian promoter transcriptional biosensor (PRL-array) stably transfected into the genome of HeLa cells (PRL-HeLa cells). Results from synthetic steroid-treated cells expressing functional GFP-ERΑ or YFP-ERΒ chimeras were compared to those obtained with estradiol (E2) and the antiestrogen tamoxifen. In the presence of synthetic ligands or E2 a concentration-dependent increase in area of the biosensor array was observed in GFP-ERΑ-expressing PRL-HeLa cells. No significant differences were found between the effects obtained with natural and synthetic steroids. Similarly, E2 or synthetic steroids-treated PRL-HeLa cells also resulted in similar colocalization of SRC-1- and RNAPII-immunofluorescence at the array. YFP-ERΒ-expressing PRL-HeLa cells treated with E2 showed increases in array area that were similar to ERΑ however, treatment of YFP-ERΒ-expressing cells with synthetic ligands was indistinguishable from vehicle controls. These data indicate that A-ring reduced 19-nortestosterone derivatives have an estrogen-like effect on chromatin, including recruitment of transcription factors through selective interactions with ERΑ.
AB - Estrogen receptors (ER), members of the nuclear steroid receptor superfamily, act to activate transcription through ligand-dependent recruitment of coregulators and chromatin modifications. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind ERΑ for activated transcription, and to recruit coregulatory factors. In this study, we have analyzed the ability of synthetic 19-nortestosterone derivatives to visibly alter the configuration of ER-target gene chromatin using a novel mammalian promoter transcriptional biosensor (PRL-array) stably transfected into the genome of HeLa cells (PRL-HeLa cells). Results from synthetic steroid-treated cells expressing functional GFP-ERΑ or YFP-ERΒ chimeras were compared to those obtained with estradiol (E2) and the antiestrogen tamoxifen. In the presence of synthetic ligands or E2 a concentration-dependent increase in area of the biosensor array was observed in GFP-ERΑ-expressing PRL-HeLa cells. No significant differences were found between the effects obtained with natural and synthetic steroids. Similarly, E2 or synthetic steroids-treated PRL-HeLa cells also resulted in similar colocalization of SRC-1- and RNAPII-immunofluorescence at the array. YFP-ERΒ-expressing PRL-HeLa cells treated with E2 showed increases in array area that were similar to ERΑ however, treatment of YFP-ERΒ-expressing cells with synthetic ligands was indistinguishable from vehicle controls. These data indicate that A-ring reduced 19-nortestosterone derivatives have an estrogen-like effect on chromatin, including recruitment of transcription factors through selective interactions with ERΑ.
KW - Chromatin
KW - Estrogen receptor subtypes
KW - Ligand selectivity
KW - Synthetic progestins
KW - Transcription
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U2 - 10.1016/j.gene.2010.03.007
DO - 10.1016/j.gene.2010.03.007
M3 - Article
C2 - 20347019
AN - SCOPUS:77952550322
VL - 458
SP - 37
EP - 44
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1-2
ER -