Supporting Information

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Table 1. The 15 loci that are most frequently aberrantly methylated in different subtypes of grade II gliomas and grade III oligodendrogliomas, as assessed by RLGS

This list includes the 15 most frequently methylated loci, including 1D05 in the order of the most altered at the top of the table. Also listed is their chromosomal position (Chr.), the gene name, whether or not it is a CpG island (Y indicates yes; N indicates no), whether or not the position of the CpG island is 5′ (yes or no), and the no. of methylated tumors out of the total no. of tumors analyzed in each glioma subtype (combined available data from two independent tumor sets): ODII, grade II oligodendrogliomas; OAII, grade II oligoastrocytomas; AstroII, grade II astrocytomas; ODIII, grade III oligodendrogliomas.

Table 2. Bisulfite PCR primer sequences for target locus WNK2CpG185

PCRs were incubated at 95°C for 2 min and then cycled as follows. For touchdown PCR (TD), 40 cycles of 95°C for 30 s, 66°C for 30 s (reducing the annealing temperature by 2°C every 2 cycles until the listed annealing temperature was reached), 72°C for 60 s, with a final extension of 72°C for 10 min. For Nested PCR, 30 cycles of 95°C for 60 s, listed annealing temperature for 60 s, 72°C for 60 s, with a final extension of 72°C for 10 min. A 1-µl aliquot of the first PCR was used for the inner nested PCR using the same parameters as the outer, except that the cycle annealing and extension temperatures were held for 30 s each.

Table 3. Real-time and standard RT-PCR primer and probe sequences

Real-time RT-PCR was conducted using the Applied Biosystems (ABI) Assays-on-Demand. The cycling conditions were according to the instructions of the manufacturer. Standard RT-PCR was conducted with indicated cycle number at 95 °C for 30 s, listed annealing temperature for 30 s, 72 °C for 60 s, and with a final extension of 72 °C for 10 min. FAM, 5-fluorouracil; TAMRA, 5′-6-carboxytetramethylrhodamine.

Table 4. Primer sequences for ChIP assay

Primer sequences for ChIP assay. PCRs were incubated at 95 °C for 2 min and then followed with 30 cycles of 95 °C for 30 s, 66 °C for 30 s, 72 °C for 30 s, with a final extension of 72 °C for 10 min. Q solution was used in PCR.

Table 5. Sequencing results of mutation screen in gliomas

The kinase domain is encoded by exons 1-6, and the initial mutations found in lung cancer, 4856g ® a and 5933g ® t, are within exons 19 and 24, respectively.

Table 6. Primer sequences for mutation screen and truncation

Table 7. Primer sequences for promoter constructs

Primer sequences for cloning constructs of WNK2 promoter region.

SI Experimental Procedures

Restriction landmark genome scanning (RLGS) was conducted for 31 human gliomas with the NotI/EcoRV/HinfI combination as described previously (1). The profiles were analyzed by two independent visual inspections of overlaid autoradiographs. Profiles from three normal brain samples were used as references.

Array comparative genome hybridization (array CGH) (2) was conducted on 42 infiltrative gliomas to identify genetic alterations. DNA from normal and tumor tissues were labeled with Cy5 and Cy3, respectively, and cohybridized to an array consisting of 2,463 mapped BAC clones containing human genomic DNA segments distributed at ≈1 megabase (Mb) intervals across the genome. The log₂ ratios of test-to-reference signal were calculated based on the intensity of fluorescence, and the DNA copy number of each locus in tumor samples was plotted across the genome.

The four human glioma cell lines were treated with 5 mM 5-aza-2′-deoxycytidine (Sigma, St. Louis, MO) for 3 days with drug and media changed every 24 h. All human tissues/cells were lysed with TRIzol (Life Technologies, Rockville, MD) for RNA isolation. Total cellular RNA was treated with Turbo DNase (Ambion, Austin, TX) and then quantified using a spectrophotometer. Following quantification, 800 ng of total cellular RNA was reverse transcribed using MMLV reverse transcriptase (Invitrogen, Carlsbad, CA), oligo-dT, and random^{primers. We amplified human WNK2 with both standard RT-PCR and real-time RT-PCR. For real-time RT-PCR experiments, the gene expression values were derived from the equations: DCt = (Ct_WNK2 − Ct_GusB) followed by (2−DCt) × 100. The conditions and primer sequences are listed in SI Table 3.}

DNA was treated with sodium bisulfite as described (3, 4) with modifications (1). Two micrograms of were was first digested with EcoRV and then phenol-chloroform extracted, ethanol precipitated, and resuspended in water. The digested DNA was treated with sodium bisulfite at 55 °C for 4 h. After purification, DNA was dissolved in 50 ml, and 1 ml was used for PCR. Touchdown or nested PCR was conducted (see SI Table 2). The PCR product was gel purified (Qiagen, Valencia, CA) and cloned into the TOPO TA Cloning/pCR2.1 vector (Invitrogen). Individual transformed bacterial colonies were subject to PCR, and the products were sequenced. Only those clones with greater than 95% conversion of non-CpG cytosines were considered.

P-UTP-labeled WNK2 and actin antisense probes were transcribed by using in vitro transcription (Promega, Madison, WI) and hybridized to paraffin-embedded human glioma tissue specimens by using a previously described protocol (5). The probes correspond to a 300- and 735-bp fragment to actin and WNK2, respectively. For quantitative analysis, a phosphorimaging plate (Fujifilm, Tokyo, Japan) was placed on dried, isotopically hybridized slides for 16 h at room temperature. The phosphorimaging plate was then scanned, and the signal was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

DNA-protein cross-linking was performed as described (6) with^{slight modifications. LN443 Cells were incubated with 0.5 mM 15-deoxyspergualin (Pierce, Rockford, IL)at room temperature for 30 min, then formaldehyde was addedto a final concentration of 1%, and cells were incubated atroom temperature for 10 min. DNA shearing was performed in nucleilysis buffer (1% SDS/10 mM EDTA/50 mM Tris (pH 8.0)/1× proteaseinhibitor mixture; Roche, Indianapolis, IN) with sonication to achieve an averageDNA length of 500-1,000 bp. Samples were diluted to a final concentrationof 0.1% SDS and incubated overnight at 4 °C with 5 µgof rabbit polyclonal anti-histone 3 K9/K18 acetylation (Upstate, Lake Placid, NY) or rabbit IgG (mock). Samples were incubatedwith protein A/G-agarose, and bound complexes were eluted with1% SDS, 0.1 M NaHCO₃. Samples were incubated overnight at 65 °Cto reverse cross-linking, and proteins were digested with proteinaseK. After purification of DNA fragments with phenol/chloroform/isoamyl alcohol extraction, promoter sequences were amplified using PCR. The primer and condition are listed in SI Table 4.}

Forty-eight hours after transfection, cells were washed with PBS and fixed with prechilled methanol/acetone (1:1 vol/vol) for 5 min, washed with PBS, and incubated for 30 min in blocking buffer containing 5% normal goat serum and 2.5% BSA in PBS. After incubation with V5 antibody (Invitrogen) (1:800) at room temperature for 30-45 min, cells were washed twice with 1% BSA in PBS and incubated with secondary goat anti mouse-rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA) (1:50). Cells were counterstained with DAPI, mounted with a coverslip, and viewed under a fluorescence microscope (Zeiss, Thornwood, NY).

The mouse anti-V5 monoclonal antibody was obtained from Invitrogen. Whole-cell extracts were resolved on denaturing 4-15% polyacrylamide Tris×HCL gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad) by using a wet transfer apparatus. Membranes were blocked with Tris-buffered saline with Tween-20 containing 5% nonfat dry milk and incubated with primary antisera (anti-V5; Invitrogen) in Tris-buffered saline with Tween-20 containing 5% BSA. Antigen-antibody complexes were detected by using a proprietary chemiluminescence kit (ECL) according to the instructions of the manufacturer (Pierce).

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