University of Illinois Urbana-Champaign

Analysis of the roles of ligand and DNA binding in estrogen receptor action

Mattick, Sandra Kay

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Description

Title
Analysis of the roles of ligand and DNA binding in estrogen receptor action
Author(s)
Mattick, Sandra Kay
Issue Date
1996
Doctoral Committee Chair(s)
Shapiro, David J.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Biology, Molecular
  • Biology, Cell
  • Chemistry, Biochemistry
Language
eng
Abstract
  • In the first part of this work we provide evidence indicating that under standard cell culture conditions unliganded estrogen receptor (ER) is unable to activate transcription. Studies of Chinese Hamster Ovary cells which were grown under serum-free conditions, suggested that commercial base media may contain traces of estrogenic compounds. Additional studies demonstrated that transcriptional synergy by ER is strongly dependent on both cell-type and ER concentration, and is likely due to the cooperative binding of ER to adjacent estrogen response elements (EREs).
  • In the second portion of this work we employed C and N-terminal deletion mutants to analyze in a more specific way the contributions of the C-terminal HBD, and the N-terminal A/B domain to the ER's ability to both interact with DNA and activate transcription. Deletion of the A/B domain generated a receptor that had an enhanced affinity for the ERE. Deletion of the HBD generated receptors that retained substantial affinity for the ERE. Since stable binding to DNA is normally attributed to the ability of the receptor to form a dimer, mutants lacking the hormone binding domain may be able to dimerize in some fashion which does not depend on the presence of the ER's C-terminal dimerization domain. We conclude from these studies that the A/B domain may have a previously unrecognized roles in receptor dimerization and/or DNA binding.
  • In the final portion of this work, we examined the properties of a truncated receptor containing only the DNA binding domain (DBD). The DBD was unable to activate transcription of either a simple or complex ERE-containing reporter gene. Both in vivo and in vitro assays confirmed that the DBD could still interact with the ERE, albeit with a weaker affinity than full length ER. Numerous cotransfection assays confirmed that the DBD could specifically function as a dominant negative mutant of the ER. The DBD's potency as a dominant negative mutant was dependent on both the number of EREs in the promoter and their degree of saturation with ER. The mechanism by which the DBD exerts its antagonizing effects may involve an interference in the cooperative binding of ER to adjacent EREs.
Type of Resource
text
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http://hdl.handle.net/2142/22308
Copyright and License Information
Copyright 1996 Mattick, Sandra Kay

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Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Biochemistry