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V. P. Theendakara, C. K. Yamashita, M. Saghizadeh, N. B. Akhmedov, D. B. Farber; Interaction of the Cone Protein, ZBED4, With an Estrogen Receptor Co-Repressor, SAFB1. Invest. Ophthalmol. Vis. Sci. 2008;49(13):149.
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© ARVO (1962-2015); The Authors (2016-present)
ZBED4 is a novel protein expressed in cone photoreceptors that contains four zinc BED finger domains, two nuclear receptor-interacting modules (LXXLL), and a hATC dimerization domain. Nuclear receptor-interacting modules (LXXLL) are known to bind to a hydrophobic cleft in the NHRs as a result of a ligand-dependent conformational change, leading to remodeling of chromatin structure, recruitment of RNA polymerase II, and transcriptional activation. Our purpose is to study whether ZBED4 associates directly with ligand-bound NHRs or if it acts in vivo either as part of a co-activator/co-repressor complex or downstream of a co-activator/co-repressor complex to modulate transcriptional activity.
The recombinant ZBED4 protein from the nuclear extract of HEK 293 cells stably transfected with a pcDNA4/HisMax ZBED4 construct was enriched by using a His-Trap-FF nickel column and eluting with imidazole. The His-tagged ZBED4 and proteins possibly complexed with it were resolved on an inverted gradient PAGE. Western blots were probed with antibodies against the N and/or C termini of ZBED4. Bands corresponding to the recombinant and endogenous ZBED4 proteins were excised and analyzed by mass spectrometry. Co-localization of ZBED4 and SAFB1 was determined by immunofluorescence microscopy.
The presence of both the endogenous and recombinant ZBED4 proteins in the lane of the immunoblot corresponding to the enriched sample, suggests that the His-tagged protein formed a complex with the endogenous ZBED4 protein that was resolved by SDS. Mass spectrometry of excised bands from the region of the gel corresponding to the ZBED4 proteins, also identified SAFB1, a nuclear matrix protein. SAFB1 has been shown to be involved in chromatin organization, transcriptional regulation, RNA metabolism and stress response. SAFB1 also functions as a potent ERα co-repressor. Our immunocytochemistry results revealed nuclear co-localization of ZBED4 and SAFB1 in HEK293 and Y79 retinoblastoma cells.
Our preliminary data suggest that ZBED4 forms a dimer in vivo and that it interacts with SAFB1. We are currently investigating whether the ZBED4/SAFB1 complex modulates transcriptional activity of genes in cone photoreceptors.
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