Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene.
Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N. Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464-46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator.
Kim
Chun Sung
CS
Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. kimxx313@umn.edu
Choi
Hack Sun
HS
Hwang
Cheol Kyu
CK
Song
Kyu Young
KY
Lee
Byung-Kwon
BK
Law
Ping-Yee
PY
Wei
Li-Na
LN
Loh
Horace H
HH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D016716:PC12 Cells; D008926:Plicamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017450:Receptors, Opioid, mu / Q000096:biosynthesis / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional; D012097:Repressor Proteins / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D000099229:RE1-Silencing Transcription Factor
D008926:Plicamycin; D017450:Receptors, Opioid, mu; D012097:Repressor Proteins; D051705:Sp3 Transcription Factor; D014157:Transcription Factors; D000099229:RE1-Silencing Transcription Factor; C404943:Oprm protein, mouse; C066851:mithramycin A