May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Interaction of the Cone Protein, ZBED4, With an Estrogen Receptor Co-Repressor, SAFB1
Author Affiliations & Notes
  • V. P. Theendakara
    Ophthalmology, Jules Stein Eye Institute - UCLA School of Medicine, Los Angeles, California
  • C. K. Yamashita
    Ophthalmology, Jules Stein Eye Institute - UCLA School of Medicine, Los Angeles, California
  • M. Saghizadeh
    Ophthalmology, Jules Stein Eye Institute - UCLA School of Medicine, Los Angeles, California
  • N. B. Akhmedov
    Ophthalmology, Jules Stein Eye Institute - UCLA School of Medicine, Los Angeles, California
  • D. B. Farber
    Ophthalmology, Jules Stein Eye Institute - UCLA School of Medicine, Los Angeles, California
  • Footnotes
    Commercial Relationships  V.P. Theendakara, None; C.K. Yamashita, None; M. Saghizadeh, None; N.B. Akhmedov, None; D.B. Farber, None.
  • Footnotes
    Support  NIH Grant EY08285
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 149. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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