Reln
mRNA and
protein levels are reduced by approximately 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.