Reelin promoter hypermethylation in schizophrenia

Abstract

Reelin mRNA and protein levels are reduced by approximately 50% in various cortical structures of postmortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. In addition, the mRNA encoding the methylating enzyme, DNA methyltransferase 1, is up-regulated in the same neurons that coexpress reelin and glutamic acid decarboxylase 67. We have analyzed the extent and pattern of methylation within the CpG island of the reelin promoter in genomic DNA isolated from cortices of schizophrenia patients and nonpsychiatric subjects. Ten (The Stanley Foundation Neuropathology Consortium) and five (Harvard Brain Collection) schizophrenia patients and an equal number of nonpsychiatric subjects were selected from each brain collection. Genomic DNA was isolated, amplified (from base pair -527 to base pair +322) after bisulphite treatment, and sequenced. The results show that within the promoter region there were interesting regional variations. There was increased methylation at positions -134 and 139, which is particularly important for regulation, because this portion of the promoter is functionally competent based on transient transfection assays. This promoter region binds a protein present in neuronal precursor nuclear extracts that express very low levels of reelin mRNA; i.e., an oligonucleotide corresponding to this region and that contains methylated cytosines binds more tightly to extracts from nonexpressing cells than the nonmethylated counterpart. Collectively, the data show that this promoter region has positive and negative properties and that the function of this complex cis element relates to its methylation status.

Fig. 1.

Methylation profile of the reelin promoter in NPS and SZP from The Stanley Foundation brain collection. (A and B) Methylation percentages determined by sequencing bisulphite modified genomic DNA from occipital cortices of NPS (A) and SZP (B) are plotted against the position in the reelin promoter (for numbering, see ref. 17). Six clones from each of 10 patients from each group (NPS vs. SZP) were included in the analysis, yielding a theoretical maximum number of 60 methylated bases at any one position. (C) A linear representation of the reelin promoter (17) aligned with the methylation positions shown in A and B.

Fig. 2.

Methylation profile of the reelin promoter in NPS and SZP from the Harvard Brain Collection. (A and B) Methylation percentages determined by sequencing bisulphite modified genomic DNA from prefrontal cortices of NPS (A) and SZP (B) are plotted against the position in the reelin promoter. Eight clones from each of five patients from each group (NPS vs. SZP) were included in the analysis, yielding a theoretical maximum number of 40 (100%) methylated bases at any one position. (C) A linear representation of the reelin promoter (17) aligned with the methylation positions shown in A and B.

Fig. 3.

Methylation profile summary of The Stanley Foundation (A) and Harvard Brain Collection (B). Positions in which the total number of methylated bases exceeded 12 (or 25%) at any given position are included, with SZP (black bars) alongside NPS (white bars).

Fig. 4.

Transfection of the reelin promoter -514 and deletion/luciferase reporter constructs (-198, -155, -127, -115, and -90) into NT2 cells. (Top) The amount of promoter activity is expressed as a function of 5′ flanking sequence present relative to the simian virus 40 promoter transfected in parallel. Data represent the mean obtained from three measurements made from a minimum of three separate experiments after correcting for transfection efficiency. (Middle) The line below the bar graph indicates the amount of 5′ flanking sequence present in the construct (arrows). (Bottom) A linear representation of the reelin promoter deletions aligned with the promoter.

Fig. 5.

Dissociation rate analysis by electromobility shift competition assays. Labeled oligonucleotides were incubated on ice for 30 min with nuclear extracts prepared from NT2 cells. After a 30-min incubation, a 300-fold excess of unlabeled oligonucleotide was added, and aliquots were analyzed by gel mobility shift assay after the indicated times (0–20 min). (A) NT2 cell nuclear extract incubated with labeled WT (nonmethylated oligonucleotide competed by unlabeled methylated oligonucleotide. (B) NT2 cell nuclear extract incubated with labeled methylated oligonucleotide and competed by unlabeled WT oligonucleotide. (C) Specificity of WT and methyl shifts. (Left) WT oligo. Lanes: 1, control shift (no extract); 2, NT2 cell extract; 3, homologous competition (200-fold); 4, Sp1 oligo (nonspecific) competition (200-fold). (Right) Methyl oligo. Lanes: 5, control shift (no extract); 6, NT2 cell extract; 7, homologous competition (200-fold); 8, Sp1 (nonspecific) oligo competition. (D) The dissociation rate was calculated by scanning the shifted bands with a densitometer and fitting the data to an exponential regression (y = span·e^-kx), where x is time, y is specific binding, and span is the binding at time 0 and k_D is the dissociation rate constant (26). ▪, Methyl probe with nonlabeled WT oligo competition; ▪, WT probe with nonlabeled methyl oligo competition. The affinity for the methylated oligo (k_D = 0.054) was ≈1.7-fold higher than that obtained for the nonmethylated oligo (k_D = 0.094).