Translin coactivates steroidogenic factor-1-stimulated transcription.
Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1) (NR5A1), nerve growth factor-inducible protein B (nurr77), COUP-TF, and SET. The -447/-419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, nerve growth factor-inducible protein B, and cAMP. We identified a novel factor, steroidogenic factor-inducer of transcription-2, that binds to this -447/-419 region. We have now purified steroidogenic factor-inducer of transcription-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27-kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promoter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the -447/-419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double- and single-stranded DNA showed that translin binds to single-stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170-225 in SF-1 eliminates translin's ability to augment SF-1-dependent transcription. A translin-like protein, called translin-associated factor X, can substitute for a translin moiety; translin homomers and translin/translin-associated factor X heteromers activated SF-1-stimulated transcription equally. Thus, we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.
Mellon
Synthia H
SH
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143-0556, USA. mellon@cgl.ucsf.edu
Bair
Susanna R
SR
Depoix
Christophe
C
Vigne
Jean-Louis
JL
Hecht
Norman B
NB
Brake
Paul B
PB
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011485:Protein Binding; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011987:Receptors, Steroid / Q000737:chemistry; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C539821:NR4A1 protein, human; C515057:NR5A1 protein, human; C539818:Nr4a1 protein, mouse; C539820:Nr4a1 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C498303:Tsn protein, mouse; C515058:steroidogenic factor 1, mouse; C515060:steroidogenic factor 1, rat; D000242:Cyclic AMP