Transcriptional regulation of mouse mu opioid receptor gene by PU.1.
We previously reported that the 34-bp cis-acting element of the mouse micro opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter. Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes. PU.1 is a member of the ets family of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain. PU.1 plays an essential role in the development of both lymphoid and myeloid lineages. Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR. Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development. The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays. We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays. In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site. When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly. This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells. Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells.
Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. hwang025@umn.edu
Cheol Kyu
CK
Hwang
Chun Sung
CS
Kim
Hack Sun
HS
Choi
Scott R
SR
McKercher
Horace H
HH
Loh
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006655:Histone Deacetylases / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099232:Proto-Oncogene Protein Spi-1
D002843:Chromatin; D018076:DNA, Complementary; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D034741:RNA, Small Interfering; D017450:Receptors, Opioid, mu; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C012589:trichostatin A