Abstract
The effects of morphine are mediated mainly through the μ opioid receptor (MOR). Expression of the MOR is up-regulated during neuronal differentiation in P19 embryonal carcinoma cells and epigenetic changes play an important role in MOR up-regulation. This study investigates the basis for differentiation-dependent alterations of MOR chromatin by studying the recruitment or dissociation of several factors to the remodeled chromatin locus. Chromatin immunoprecipitation assays were used to demonstrate the recruitment of the transcriptional activator Sp1 and the chromatin remodeling factors Brg1 and BAF155 to this promoter, as well as the dissociation of repressors [histone deacetylases, mSin3A, Brm, and methyl-CpG-binding protein 2 (MeCP2)]. Histone modifications (acetylation, induction of histone H3-lys4 methylation, and reduction of H3-lys9 methylation) were consistently detected on this promoter. Overexpression of Sp1 strongly enhanced MOR promoter activity, and the histone deacetylase inhibitor trichostatin A also increased promoter activity. In vitro DNA CpG-methylation of the promoter partially blocked binding of the Sp1 factor but induced MeCP2 binding. Coimmunoprecipitation studies also found novel evidence of an endogenous MeCP2 interaction with Sp3 but a weaker interaction with Sp1. Overall, the results suggest that during neuronal differentiation, MeCP2 and DNA methylation mediate remodeling of the MOR promoter by chromatin remodeling factors (Brg1 and BAF155) from a compacted state to a conformation allowing access for transcriptional factors. Subsequent recruitment of the activating transcription factor Sp1 to the remodeled promoter results in MOR up-regulation.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA000564, DA001583, DA011806, K05-DA070554, DA011190, DA013926] and by the A and F Stark Fund of the Minnesota Medical Foundation.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064311.
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ABBREVIATIONS:
- MOR
- μ opioid receptor
- MeCP2
- methyl-CpG-binding protein 2
- RA
- all-trans retinoic acid
- TSA
- trichostatin A
- UD
- undifferentiated
- AP2d
- intermediately differentiated
- AP4d
- fully differentiated
- MNase
- micrococcal nuclease
- ChIP
- chromatin immunoprecipitation
- Brm
- Brahma protein
- Brg1
- Brm-related gene 1
- BAF155
- Brg1-association factor 155
- HDAC
- histone deacetylase
- PP
- proximal promoter
- DP
- distal promoter
- AS
- antisense
- S
- sense
- bp
- base pair(s)
- PCR
- polymerase chain reaction
- qPCR
- quantitative polymerase chain reaction
- LM-PCR
- ligand-mediated polymerase chain reaction
- EMSA
- electrophoretic mobility shift assay
- RT-PCR
- reverse transcription-polymerase chain reaction
- qRT-PCR
- quantitative reverse transcription-polymerase chain reaction
- Pol II
- polymerase II.
- Received February 22, 2010.
- Accepted April 12, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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