H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements.
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.
Marks
M S
MS
Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Levi
B Z
BZ
Segars
J H
JH
Driggers
P H
PH
Hirschfeld
S
S
Nagata
T
T
Appella
E
E
Ozato
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004967:Estrogens / Q000235:genetics* / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004967:Estrogens; D011994:Recombinant Proteins; C061606:Rxrb protein, mouse; D004247:DNA