Supporting Figures

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Fig. 6. Chromatin immunoprecipitation (ChIP) procedures. (A) Sheared genomic DNA by sonication. Agarose gel image of cross-linked DNA after sonication. Note that >90% of DNA fragments (lane 2) fell into a range of 250-500 bp. (for cross-linkage and sonication conditions, see Materials and Methods). Lane 1, nonsheared genomic DNA; Lane 2, sheared genomic DNA; M, M.W. marker. (B) Specificity of MeCP2 ChIP method. Representative PCR amplification of RELN and GAD₆₇ promoters in the MeCP2 ChIP fraction and in the initial nonimmunoprecipitated extract (Input). Controls also include no antibody and immunoglobuline (IgG). Lanes: 1, MeCP2-ChIP for GAD₆₇ promoter; 2, input for glutamic acid decarboxylase (GAD₆₇) promoter; 3, MeCP2-ChIP for RELN promoter; 4, input for RELN promoter; 5, nonantibody; 6, IgG. IgG nonimmune antibody (Santa Cruz Biotechnology) was used at a concentration of 1:180.

Fig. 7. Measurement of RELN and GAD₆₇ promoter fragments by quantitative competitive PCR. (A) Schematic representation of internal standard preparation by using the deletion method. Briefly, two PCR fragments (A and C) were amplified by using a combination of two pair primers: P-5' (external) plus P-3' (internal) and P-5' (internal) plus P-3' (external). Then a second PCR was conducted to overlap A and C by using external primers only. The PCR product AC contains a 100-150 bp deleted sequence corresponding to fragment B. The following deletion primers were used: 5'-atacattctccagcccgcac atatactggcaa-3' (downstream) and 5'-ttgccagtatatgtgcgggctggag aatgtat-3' (upstream) for b-globin gene; 5'-ttggggccactgacggtcctcgcagaacgggc-3' (forward) and 5'-gcccgttctgcg aggaccgtcagtggccccaa-3' (upward) for the RELN promoter region –520 to –225 bp; 5'-ttcgaacgaggtagtgtcagagaaaaccttctgg-3' (sense) and 5'-ccagaaggttttctctgacactacctcgttcgaa-3' (antisense) for the GAD₆₇ promoter region –760 to –339 bp. (B) Representative amplification kinetics of RELN promoter fragment –520 to –225 bp and related internal standards (ISs). (Left) Comparative analysis of independently amplified RELN promoter immunoprecipitated with MeCP2 antibody (RELN) and ISs at consecutive cycles of amplification. IS (10³ amol, closed circle) and the equivalent of 10 ng RELN promoter DNA (open circle) were amplified for 34 successive cycles in separate reactions. Linear regression analysis shows r = 0.99 for both DNAs. (Right) PCR coamplification of RELN promoter ChIP with MeCP2 antibody (RELN) and IS at consecutive cycles of amplification. IS (10³ amol, closed circles) and the equivalent of 10 ng ChIP reelin promoter (open circles) were amplified for 34 successive cycles. Linear regression analysis for the kinetic curve was r = 0.99 for reelin promoter and 0.98 for internal standard DNAs. (C) Representative experiments showing a quantitative PCR analysis for RELN promoter. A constant amount of RELN DNA extracted from frontal cortice (FC) nuclear fraction nonimmunoprecipitated (Input) was incubated with increasing concentrations of RELN IS. (Insert) The higher molecular-size bands correspond to the amplification product arising from the FC extract, whereas the lower molecular-size arises from DNA generated from the internal standard. The data derived from the agarose gel are plotted as follows: the abscissa represents the amount of RELN IS added to the sample, and the ordinate represents the ratio of the OD of a known amount of RELN IS vs. the OD of the RELN DNA amplification product. The OD ratio of RELN IS/RELN DNA equal to 1 gives on the abscissa the concentration of RELN promoter present in the sample. PCR reactions were carried out by using GC melt PCR system (BD Biosciences Clonetch). The thermocycler protocol included an initial denaturation cycle (1 min at 95°C), 28-30 cycles of denaturation (30 sec at 94°C), annealing (1 min at 60°C), and extension (2 min at 68°C), followed by a final extension cycle (3 min at 68°C), terminating at 15°C. DNA signals on agarose gels (1–2%) were visualized and quantified by using Kodak software (EDAS290).

Fig. 8. GAD₆₇ but not GAD₆₅ expression is down-regulated in FC of l-methionine (MET)-treated mice. Representative Western immunoblot of GAD₆₇, GAD₆₅, and b-actin after 10–20% gradient SDS/PAGE. Comparison between a vehicle (VEH) (lanes 1–3, serial dilutions of the same sample) and a MET-treated mouse (lanes 4–6, serial dilution of the same sample). GAD₆₇/₆₅ antibody recognizes bands of 67 and 65 molecular size, respectively. No other major bands were detected with this antibody in our brain extracts.

Fig. 9. MeCP2 expression is not modified by protracted MET treatment. Representative Western immunoblot of crude nuclear extracts from frontal cortices of mice treated with vehicle (VEHa) or methionine (METb) (5.2 mmol/kg twice a day for 15 days) probed with polyclonal antibody against MeCP2. In the insert MeCP2/b-actin OD ratios. Mean ± SE of VEH (a, open bar) and MET (b, closed bar)-treated mice (n = 3). Competitive experiments with the synthetic immunogenic MeCP2 peptide (C-PRPNREEPVDSRTP) completely abolished the immunoreactivity.