Characterization of the response element and DNA binding properties of the nuclear orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor.
Recently, we have reported the cloning of the germ cell-specific, nuclear orphan receptor germ cell nuclear factor (GCNF)/RTR. In this study, we characterize the RTR response elements by an electrophoretic mobility shift assay/polymerase chain reaction-based, DNA binding site selection strategy. RTR binds with the greatest affinity to response elements containing TCA(AG(G/T)TCA)2 (consensus RTR response element; conRTRE), to which it binds as a homodimer. RTR is also able to bind as a monomer to a single core motif TCAAG(G/T)TCA, albeit with a lower affinity. Mutation analysis supports the specific requirements of the 5'-flanking sequence and the core motif of the RTRE for optimal binding of RTR. An RTR-specific antiserum (RTR-Ab2) was raised that causes supershift of the RTR-conRTRE complex in EMSA. Based on the sequence of the conRTRE, we located a putative RTRE, referred to as P2-RE, in the 5' promoter-flanking region of the mouse protamine 2 gene, which is induced during the same stage of spermatogenesis as RTR. The ability of RTR-Ab2 to cause a supershift of an RTR-RTRE complex with nuclear extracts from different tissues correlated with the tissue- and development-specific expression of RTR. Transfection of RTR in CV-1 cells was unable to cause RTRE-dependent transactivation of a CAT reporter gene; however, an RTR-VP16 fusion protein could induce transactivation through several RTREs, including P2-RE.
Yan
Z H
ZH
Department of Medicine III, Osaka University Medical School, Yamadaoka, Suita, Osaka 565, Japan.
Medvedev
A
A
Hirose
T
T
Gotoh
H
H
Jetten
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017930:Genes, Reporter; D007668:Kidney / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013737:Testis / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; C539954:Nr6a1 protein, mouse; D057136:Nuclear Receptor Subfamily 6, Group A, Member 1; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase