Involvement of an octamer-like sequence within a crucial region of the androgen-dependent Slp enhancer.
Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, both in vitro and in vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased Oct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C' delta9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C 'delta2). This underscores that, despite extensive sequence identity of C' delta9 and C' delta2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid responsive.
Scarlett
C O
CO
Department of Human Genetics, University of Michigan Medical School, Medical Science II 4708, Ann Arbor 48109-0618, USA.
Scheller
A
A
Thompson
E
E
Robins
D M
DM
D000728:Androgens / Q000502:physiology*; D000818:Animals; D001798:Blood Proteins / Q000235:genetics*; D051838:COUP Transcription Factor I; D002460:Cell Line; D003181:Complement C4; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051863:Host Cell Factor C1; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D050811:Octamer Transcription Factor-1; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014466:Ultraviolet Rays; D014498:Uracil / Q000378:metabolism
D000728:Androgens; D001798:Blood Proteins; C018386:C4a protein, mouse; D051838:COUP Transcription Factor I; D003181:Complement C4; D004268:DNA-Binding Proteins; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; C497590:Nr2f1 protein, mouse; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D014157:Transcription Factors; D014498:Uracil; D004247:DNA