A RAS recruitment screen identifies ZKSCAN4 as a glucocorticoid receptor-interacting protein

Abstract

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-a) 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-domaincontaining 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.

Association between variants of zinc finger genes and psychiatric disorders: Systematic review and meta-analysis

“…The ZKSCAN4 protein is mainly localized in the nucleus and highly expressed in the brain. Ecker et al found that ZKSCAN4 is a glucocorticoid receptor-interacting protein and co-precipitated with the glucocorticoid receptor when both proteins were overexpressed in HEK293 cells (Ecker et al, 2009). 3.3.2.2. Genetic association.…”

Cellular Genes Derived from Gypsy/Ty3 Retrotransposons in Mammalian Genomes

“…This might be explained by defects in neuronal connectivity, as well as by reduced oxytocin neurons in the hypothalamus, because mutant mothers are deficient in milk ejection. 94 SCAN domain transcription factors are able to interact with proteins as diverse as the von Hippel-Lindau tumor suppressor protein, which is a component of an E3 ubiquitin ligase (ZnF197), 92 the E3 ubiquitin ligase Siah1a (Pw1/Peg3), 95 the neurotrophin receptor p75 (NRIF), 96 peroxisome-proliferator-activated receptors (SDP1 and PGC-2), 97 the glucocorticoid receptor (Znf307), 98 the transcriptional repressor Jumonij/Jarid2 (Zfp496), 99 the NSD1 histone lysine methyltransferase (Nizp1), 100 and the tumour necrosis factor mediator TRAF2 (Pw1/Peg3). 101 Several SCAN domain proteins are involved in the control of cell survival and proliferation.…”

Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity

“…It is GC resistant, due to qualitatively and quantitatively defective GR expression [30], but has been transfected with a functional GR thereby restoring GC sensitivity [26]. Jurkat GR cells [26] were stably transfected with a lentiviral construct (pHR-MMTV-VNP) enabling GC-dependent expression of a fl uorescent reporter gene [27] and subjected to limiting dilution cloning. For this, cells were plated in 96-well plates at densities of 25, 5 or 1 cell per well.…”

“…Th is GBA, based on the measurement of GR-dependent reporter gene activity, can be employed to measure GC bioactivity from small amounts of serum or other biologic fl uids. A human T-cell leukemia derived cell line (Jurkat), stably transfected with a plasmid expressing the GR [26] and a fl uorescent reporter gene under control of the mouse mammary tumor virus (MMTV) LTR promoter [27], was used to establish the assay. Th us, Venus nuclear fl uorescent protein (VNP), with about 10-fold higher fl uorescence intensity than green fl uorescent protein (GFP) [28], has been brought under control of the liganded GR and was used as the assay's reporter.…”