On the epigenetic regulation of the human reelin promoter

Ying Chen

Rajiv Sharma

Robert Costa

Erminio Costa

Dennis Grayson

Psychiatric Institute, Department of Psychiatry, 1601 West Taylor Street, M/C 912 and ^{Department of Molecular Genetics, College of Medicine, University of Illinois, Chicago, IL 60612, USA}

^{To whom correspondence should be addressed. Tel: +1 312 413 4577; Fax: +1 312 413 4569; Email: dgrayson@psych.uic.edu}

Received 2002 Feb 6; Revised 2002 May 7; Accepted 2002 May 7.

Abstract

Reln mRNA and protein levels are reduced by ∼50% in various cortical structures of post-mortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. We show that the reelin promoter directs expression of a reporter construct in multiple human cell types: neuroblastoma cells (SHSY5Y), neuronal precursor cells (NT2), differentiated neurons (hNT) and hepatoma cells (HepG2). Deletion constructs confirmed the presence of multiple elements regulating Reln expression, although the promoter activity is promiscuous, i.e. activity did not correlate with expression of the endogenous gene as reflected in terms of reelin mRNA levels. Co-transfection of the –514 bp human reelin promoter with either Sp1 or Tbr1 demonstrated that these transcription factors activate reporter expression by 6- and 8.5-fold, respectively. Within 400 bp of the RNA start site there are 100 potential CpG targets for DNA methylation. Retinoic acid (RA)-induced differentiation of NT2 cells to hNT neurons was accompanied by increased reelin expression and by the appearance of three DNase I hypersensitive sites 5′ to the RNA start site. RA-induced differentiation was also associated with demethylation of the reelin promoter. To test if methylation silenced reelin expression, we methylated the promoter in vitro prior to transfection. In addition, we treated NT2 cells with the methylation inhibitor aza-2′-deoxycytidine and observed a 60-fold increase in reelin mRNA levels. The histone deacetylase inhibitors trichostatin A (TSA) and valproic acid also induced expression of the endogenous reelin promoter, although TSA was considerably more potent. These findings indicate that one determinant responsible for regulating reelin expression is the methylation status of the promoter. Our data also raise the interesting possibility that the down-regulation of reelin expression documented in psychiatric patients might be the consequence of inappropriate promoter hypermethylation.

Abstract

Values are means ± SEM.

^{Quantitative measurements of mRNA three times using three independent RNA isolates.}

^{Denotes significantly different from NT2 control using one-way ANOVA (}P < 0.01).

NT2 neural progenitor cultures were treated with the indicated concentrations of drugs for 24 h and RNA was isolated. The amount of reelin mRNA was quantified using competitive RT–PCR (4) and is expressed relative to the amount present in the vehicle-treated cultures that were maintained in parallel. The RNA analysis was performed three times per dose on a minimum of three different culture preparations.

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ACKNOWLEDGEMENTS

We would like to thank Mr David Gavin for his excellent technical assistance, Dr Genoveva Davidkova for providing reelin internal standard cRNA and Dr Jai Sung Noh for help with figure preparation. We would like to acknowledge Dr Robert Tjian (UC, Berkeley, CA) for providing the Sp1 expression vector and Dr Morgan Sheng (Massachusetts General Hospital and Harvard University, Cambridge, MA) for the Tbr1 expression vector. Finally, we would like to acknowledge Katwijk Chemie B.V. for the gift of valpromide that was used in these studies. This work was supported by 5R01MH062090-02 to E.C. and in part by 1R01MH062682-01A2 to D.R.G.

ACKNOWLEDGEMENTS

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