A RAS recruitment screen identifies ZKSCAN4 as a glucocorticoid receptor-interacting protein.
To search for proteins interacting with the glucocorticoid receptor, we adapted Aronheim's reverse RAS recruitment system relying on the Saccharomyces cerevisiae mutant cdc25-2, which has a temperature-dependent defect in its RAS signaling 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 the 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 (ZKSCAN4) was co-precipitated with the receptor when both proteins were overexpressed in HEK293 cells. Rabbit antisera against ZKSCAN4 were raised, affinity purified, and used to immunoprecipitate endogenous ZKSCAN4 from Hct116 cells, resulting in co-precipitation of endogenous glucocorticoid receptor. Overexpressed ZKSCAN4 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). Overexpressed ZKSCAN4 had no effect on an episomal glucocorticoid receptor-driven reporter plasmid. By contrast, ZKSCAN4 markedly reduced glucocorticoid induction of the mouse mammary tumor virus-promoter-driven reporter gene when this was chromosomally integrated, arguing for a chromatin-dependent inhibition of glucocorticoid receptor-mediated transactivation.
Ecker
Karin
K
Biocenter, Division of Molecular Pathophysiology, Innsbruck Medical University, Fritz Pregl Strasse 3, A 6020 Innsbruck, Austria.
Lorenz
Andreas
A
Wolf
Frank
F
Ploner
Christian
C
Böck
Günther
G
Duncan
Tod
T
Geley
Stephan
S
Helmberg
Arno
A
D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D019913:Nuclear Localization Signals / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015533:Transcriptional Activation / Q000235:genetics; D018631:ras Proteins / Q000378:metabolism*
D002843:Chromatin; D004268:DNA-Binding Proteins; C497713:NR3C1 protein, human; D019913:Nuclear Localization Signals; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C524688:ZKSCAN4 protein, human; D018631:ras Proteins