Schematic representation of the role of MTHFR in folate and homocysteine metabolism. TS = thymidylate synthase, MTR = methionine synthase, DHF = dihydrofolate, THF = tetrahydrofolate, SHMT = serine hydroxymethyltransferase. Pathway provided by Andrea Lawrance, McGill University.

Effect of VPA on activity of (A) upstream promoter deletion constructs and (B) downstream promoter deletion constructs in HepG2 cells. Promoter activities in VPA-supplemented cells are normalized against the same construct activity in saline-supplemented cells. The promoter activities were corrected for the VPA effect on the normalizing β-gal vector as described in Materials and Methods. Each value represents the mean ± SEM of 3 experiments performed in duplicate.
*, p<0.05 compared to the saline control.

(A) Plasma homocysteine concentrations were determined for Mthfr^+/+ and Mthfr^+/−pregnant females injected with VPA (black bars) or saline (white bars). Values represent the mean ± SEM of 7 mice in each group. #,&, p<0.05 for comparison of genotype and treatment groups, respectively (two-factor ANOVA) (B) MTHFR activity in brain extracts of pregnant Mthfr^+/+ mice injected with either VPA or saline. Values represent the mean ± SEM of 5 mice in each group. *, Treatment effect showed borderline significance (p=0.052) (independent sample t-test).

Schematic representation of upstream and downstream promoter deletion constructs and their activities in HepG2 cells. (A) Location of constructs in the 5 ′ UTR of the human MTHFR gene. The diagram at the top represents a segment of genomic DNA encompassing a portion of the MTHFR gene, upstream of MTHFR exon 2. D1 to D6 are donor splice sites and A1 to A3 are acceptor splice sites. Representative MTHFR cDNAs, that have the potential to encode the short or long MTHFR protein isoforms, are aligned under the complex genomic structure. Promoter deletion constructs hupA through hupF comprise the designated upstream promoter region and deletion constructs hdownA through hdownE comprise the designated downstream promoter region. The human promoter schematic is adapted from that of Tran et al. [2002] and the representation of human promoter deletion constructs is adapted from Pickell et al. [2005]. The relative numbers at the boundaries of the promoter segments (hupA-hupF and hdownA-hdownE) are derived from GenBank Accession no. AF398930, with the base A of the most upstream MTHFR translational start site as the +1 position. (B and C) Activity profile of upstream and downstream promoter deletion constructs in HepG2 cells. Activities for hupA and hdownA show a relative value of 1.0 since upstream promoter deletion constructs are normalized against promoter construct hupA activity whereas downstream promoter deletion constructs are normalized against construct hdownA. Each value represents the mean ± SEM of 3 experiments performed in duplicate.
*, p<0.05 compared to the preceding promoter construct.

Effect of VPA on MTHFR mRNA and protein levels. (A) Quantitative real time RT-PCR measurement of MTHFR transcripts in HepG2 cells treated with VPA or saline. RNA quantity was normalized through GAPDH expression. MTHFR transcript levels in VPA experiments were normalized against MTHFR transcript levels in saline controls. Value represents mean ± SEM for 3 experiments performed in duplicate. *, p<0.01 for comparison of VPA treatment with saline treatment. (B) Representative Western immunoblotting analysis of MTHFR in HepG2 and SK-N-SH cells treated with either VPA or saline. β-actin was used as a loading control. Lanes 1 and 2 are SK-N-SH cell extracts treated with saline and VPA, respectively; lanes 3 and 4 are HepG2 cells treated with saline and VPA, respectively. Lanes 5 and 6 are HepG2 cells untreated and treated with homocysteine, respectively, which are presented as reference bands for the phosphorylated and unphosphorylated MTHFR 70 kDa isoform.