H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements

Michael S. Marks, Ben Zion Levi, James H. Segars, Paul H. Driggers, Steven Hirschfeld, Toshi Nagata, Ettore Appella, Keiko Ozato

Research output: Contribution to journalArticlepeer-review

Abstract

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.

Original languageEnglish (US)
Pages (from-to)219-230
Number of pages12
JournalMolecular Endocrinology
Volume6
Issue number2
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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