Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retina • microscopy: light/fluorescence/immunohistochemistry • protein purification and characterization