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This Article Accepted manuscript (PDF) All Versions of this Article: JME-08-0087v1 42/2/105    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ecker, K. Articles by Helmberg, A. Search for Related Content PubMed PubMed Citation Articles by Ecker, K. Articles by Helmberg, A.

K Ecker, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria
A Lorenz, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria
F Wolf, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria
C Ploner, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria
G Boeck, Biocenter, Developmental Immunology, Innsbruck Medical University, Innsbruck, Austria
T Duncan, Department of Biology, University of Colorado, Denver, Denver, United States
S Geley, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria
A Helmberg, Biocenter, Molecular Pathophysiology, Innsbruck Medical University, Innsbruck, Austria

Correspondence: Arno Helmberg, Email: arno.helmberg{at}i-med.ac.at

Abstract

To search for proteins interacting with the glucocorticoid receptor, we adapted A. Aronheim's reverse Ras recruitment system (rRRS) relying on the S. cerevisiae mutant cdc25-2, which has a temperature-dependent defect in its Ras signalling pathway driving proliferation. The full length human glucocorticoid receptor (NR3C1, isoform alpha) was attached to the yeast plasma membrane in either of two orientations and used as bait to screen a HeLa cell cDNA library. Library proteins were fused to constitutively active, soluble human Ras, complementing the defective yeast pathway in case of bait-prey interaction. Screening of 800.000 clones resulted in isolation of 21 proteins, 8 of which were followed up to evaluate interaction with the receptor in human cell lines. One of these candidates, the SCAN- and KRAB domain-containing zinc finger protein 307 (Znf307; ZKSCAN4) was co-precipitated with the receptor when both proteins were over-expressed in HEK293 cells. Rabbit antisera against Znf307 were raised, affinity purified and used to immunoprecipitate endogenous Znf307 from Hct116 cells, resulting in co-precipitation of endogenous glucocorticoid receptor. Over-expressed Znf307 was found to co-localize in granular nuclear structures with the activated glucocorticoid receptor and partially with chromatin regions characterized by histone H3 mono-methylated on lysine 4 (H3K4me1). Over-expressed Znf307 had no effect on an episomal glucocorticoid receptor-driven reporter plasmid. In contrast, Znf307 markedly reduced glucocorticoid induction of the MMTV promoter-driven reporter gene when this was chromosomally integrated, arguing for a chromatin-dependent inhibition of glucocorticoid receptor-mediated transactivation.