Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by poly(ADP-ribose) polymerase-1.
The pharmacological actions of morphine and morphine-like drugs such as heroin mediate primarily through the mu opioid receptor (MOR). It represents the target of the most valuable painkiller in contemporary medicine. Here we report that poly(ADP-ribose) polymerase 1 (PARP-1) binds to the double-stranded poly(C) element essential for the MOR promoter and represses promoter activity at the transcriptional level. We identified PARP-1 by affinity column chromatography using the double-stranded poly(C) element, followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. PARP-1 binding to the poly(C) sequence of the MOR gene was sequence-specific as confirmed by the supershift assay. In cotransfection studies, PARP-1 repressed the MOR promoter only when the poly(C) sequence was intact. When PARP-1 was disrupted in NS20Y cells using siRNA, transcription of the endogenous target MOR gene increased significantly. Chromatin immunoprecipitation assays showed specific binding of PARP-1 to the double-stranded poly(C) element essential for the MOR promoter. Inhibition of PARP-1's catalytic domain with 3-aminobenzamide increased endogenous MOR mRNA levels in cultured NS20Y cells, suggesting that automodification of PARP-1 regulates MOR transcription. Our data suggest that PARP-1 can function as a repressor of MOR transcription dependent on the MOR poly(C) sequence. We demonstrate for the first time a role of PARP-1 as a transcriptional repressor in MOR gene regulation.
Choi
Hack Sun
HS
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. choix074@umn.edu
Hwang
Cheol Kyu
CK
Kim
Chun Sung
CS
Song
Kyu Young
KY
Law
Ping-Yee
PY
Loh
Horace H
HH
Wei
Li-Na
LN
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011066:Poly C / Q000235:genetics; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000235:genetics*; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D034741:RNA, Small Interfering; D017450:Receptors, Opioid, mu; D012097:Repressor Proteins; D011066:Poly C; D004247:DNA; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases