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Supplemental material

An atomic model of ZFP57 recognition of CpG methylation within a specific DNA sequence

Yiwei Liu1, Hidehiro Toh2, Hiroyuki Sasaki2, Xing Zhang1 and Xiaodong Cheng1

1Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road,

Atlanta, GA 30322, USA

2Division of Epigenomics, Department of Molecular Genetics, Medical Institute of

Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Correspondence should be addressed to:

Xiaodong Cheng (xcheng@emory.edu; phone: 404-727-8491; fax: 404-727-3746)

Email addresses for all authors:

Yiwei Liu (yliu40@emory.edu)

Hidehiro Toh (toh-h@bioreg.kyushu-u.ac.jp)

Hiroyuki Sasaki (hsasaki@bioreg.kyushu-u.ac.jp)

Xing Zhang (xzhan02@emory.edu)

Xiaodong Cheng (xcheng@emory.edu)

VQKTLPWVGEVAATLQEAMK (isoform 3 has additional 20 resides) 1 10 20 30 40 50 60 70 80 | | | | | | | | | hZFP57 1 MFEQLKPIEPRDCWREARVKKKPVTFEDVAVNFTQEEWDCLDASQRVLYQDVMSETFKNLTSVARIFLHKPELITKLEQE mZFP57 1 MAARKQSSQPSRTPVSYEDVAVSFTQEEWEYLTSTQKTLYQKVMSETFKNLTFVGSKKKPQEPSSDLQDKN | | | | | | | | 1 10 20 30 40 50 60 70 90 100 110 120 130 140 150 | | | | | | | hZFP57 81 EEQWREFVHLPNTEGLSEGKKKELREQHPSLRDEGTSDDKVFLACRGAGQCPLSAPAGTMDRTRVLQASQAG mZFP57 72 EEQEKS--------------------------------------------------------SSCTGVFKGG | 80 160 170 180 190 200 210 220 230 | | | | | | | H | h 153 PPFFCYTCDKCFSRRSYLYSHQFVHNPKLTNSCSQCGKLFRSPKSLSYHRRMHLGERPFCCTLCDKTYCDASGLSRHRRVHLG m 88 PFFFCLTCGKCFKKNTFLFNHQFPVRSRRLAV-TN-PQ---SRKGKGY-KAQHRGERPFFCNFCGKTYRDASGLSRHRRAHLG | | | | | | | | 90 100 110 120 130 140 150 160 240 250 260 270 280 290 300 310 |X | N | | | | | | h 236 YRPHSCSVCGKSFRDQSELKRHQKIHQNQEPVDGNQECTLRIPGTQAEFQTPIARSQGSIQGLLDVNHAPVARSQEPIFRTEG m 165 YRPRSCPECGKCFRDQSEVNRHLKVHQNK-PAASNQA------GNQASNQRLKSRVPPTTPRSQAPALKYVKVIQGPVARAKA | | | | | | | | 170 180 190 200 210 220 230 240 320 330 360 350 360 370 380 | | | | | | | h 319 PMAQNQASVLKNQA-PVTRTQAPITGTLCQDARSNSHPVKPSRLNVFCCPHCSLTFSKKSYLSRHQKAHLT m 241 RNSGASTLNVRSNSITVVRS-----------------------REKISCPYCHITFTMRTCLLTHLKIHFR | | | | 250 260 270 280 390 400 410 420 430 440 450 | | | | | D | | h 389 EPPNYCFHCSKSFSSFSRLVRHQQTHWKQKSYLCPICDLSFGEKEGLMDHWRGYKGKDLCQSSHHK m 289 RQPNQHFCCKESAHSSNTLRM-------QKIYTCPVCDSSFRGKESLLDHLCCQRP--IRFS---K | | | | | | 290 300 310 320 330 340 460 470 480 490 500 510 | | | | | | h 454 CRVILGQWLGFSHDVPTMAGEEWKHGGDQSPPRIHTPRRRGLREKACKGDKTKEAVSILKHK 516 m 342 CWEILGHLLGYLHE-PVVLGNIFKVR-DSSGKRMESRRRRRKR--ACTENPETEGLSGKGRVAPWEMEGATSPESPVTEEDSD | | | | | | | | 350 360 370 380 390 400 410 420 Supplementary Figure 1: Sequence alignment of human ZFP57 (CAQ06621.1) (top line) and mouse Zfp57 (NP_001161973.1) (bottom line). Human ZFP57 has at least three isoforms. Isoform 2 used here for alignment is the same one used by Mackay et al (2008) in describing the human mutations (R228H, C241X, H257N and H438D). Isoform 3 (AAI71888.1) has additional 20 residues inserted after amino acid 10. The shorter isoform 1 has 452 residues (Q9NU63.2), missing the 20 residues after amino acid 10 and residues 87-142 (strikethrough). In addition, isoform 1 has a different N-terminal sequence (MAAGEPRSLLFFQ) in replacement of the first 21 residues. White-on-black residues are invariant between the two sequences examined, while gray-highlighted positions are conserved (R and K, E and D, T and S, Q and N, F and Y, V, I, L and M).

ZF1 ZF2 ZF3

ZF4

ZF5

ZF6 ZF7

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Supplementary Figure S2. Comparison of ZN2 and ZF3 of Zfp57 in interaction with DNA

(A) The overhanging A and T of two neighboring DNA molecules form a Hoogsteen base pair.

Also shown is an example of alternative conformations of phosphates.

(B) Superimposition of ZF2 (green) and ZF3 (orange) (left panel). The side chains important for

base specific interactions are shown. All residues, except Arg150 of ZF2 (making a phosphate

contact), are conserved in human ZFP57 (see Supplementary Fig. 1). ZF3 involves three

residues, Arg178 (interacting with C:G pair at position 7), Glu182 (G:C pair at position 8) and

Arg185 (C:G pair at position 9). The corresponding residues in ZF2 are Arg150, Gly154 and

Arg157. Interestingly, both arginines in ZF2 undergo conformational changes from that of ZF3,

resulting in Arg150 interaction with a phosphate group of the A-strand (Fig. 1d) and Arg157

bifurcated interaction with the G:C pair at position 2 (instead of C:G pair at position 3). An

approximate 40º rotation of the helical axis of ZF2 to that of ZF3 is observed by superimposition

of the DNA structure from the 5’ TGC to that of 3’ CGC (right panel). The rotation of the !

helix allowed Asp151 and Ser153 of ZF2 interaction with the T:A base pair. The corresponding

residues in ZF3, Asp179 and Ser181, are conserved and involved in phosphate interaction

(Ser181) and water-mediated inter- and intra-molecular interactions (Asp179).

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Supplementary Figure S3. Zfp57 and MBD proteins share a common mode of 5mC recognition (A) UHRF1 SRA domain uses hydrophobic interactions with a flipped 5mC (adopted from PDB 2ZO1). (B-D) MBD proteins use two pairs of 5mC-Arg-G interactions for recognition of symmetric 5mCpG dinucleotides. Two arginine residues interact with two guanines and the guanidino group of each arginine forms hydrophobic interaction with the methyl group of 5’ neighboring 5mC of the same strand. A tyrosine is involved in bridging water molecules forming a layer of hydration around one methyl group in MeCP2 (panel B). Structural coordinates were used for the MBD domains of human MeCP2 (PDB 3C2I; panel B) (Ho et al., 2008), chicken MBD2 (PDB 2KY8; panel C) (Scarsdale et al., 2011) and human MBD1 (PDB 1IG4; panel D) (Ohki et al., 2001), respectively. Note the conformations of two corresponding arginines of MBD1 (panel d) were not well defined in the early NMR structure (Ohki et al., 2001).

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Supplemental Table 1. Data collection and refinement statistics Data collection (*highest resolution shell is shown in parenthesis)

Space group C2221 Cell dimensions (!="=#=90°) a (Å), b (Å), c (Å) 40.218, 60.641, 96.119 Beamline APS 22-BM (SERCAT) Wavelength (Å) 1.000 Resolution (Å) * 48.6-0.99 (1.07-1.03) (1.03-0.99) Rmerge (%) 0.133 (0.329) (0.404) <I >/$(I) 16.1 (5.8) (2.8) Completeness (%)* 94.4 (85.3) (58.9) Redundancy * 12.9 (7.6) (3.9) Observed reflections 802,194 Unique reflections * 61,985 (5,691) (3,816) Figure of merit (Zn anomalous) 0.68

Refinement Rwork / Rfree (%) 13.14 / 13.96 No. of atoms

Protein 521 DNA 561

Water 288 Others 23 (Zn2+, Ca2+, acetate, MPD)

B-factors (Å2) Protein 9.0

DNA 11.2 Water 20.1 Others 14.9

R.m.s. deviations Bond lengths (Å) 0.009

Bond angle (°) 1.799 The crystals were flash frozen in liquid nitrogen. X-ray diffraction data were collected at the SER-CAT 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. The structure was determined using the phases calculated from single-wavelength anomalous signals from the two bound zinc atoms. Programs HKL2000 (Otwinowski et al., 2003) and CCP4 (Collaborative Computational Project, 1994) were used for the data processing. The PHENIX Autosol option (Adams et al., 2010) located the zinc atoms. COOT (Emsley and Cowtan, 2004) and PHENIX Refinement option performed model building and refinement, respectively. The Department of Biochemistry at the Emory University School of Medicine supported the use of the Southeast Regional Collaborative Access Team synchrotron beamlines at the Advanced Photon Source of Argonne National Laboratory.

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Supplemental Table 2. Local base pair and step parameters (calculated by Web 4DNA (Zheng et al., 2009)) Base pair Shift Slide Rise Tilt Roll Twist

A-T 0 0 0 0 0 0 T-A -0.7 -0.81 3.12 0.07 6.72 28.32 T-A -0.73 -0.24 3.13 2.39 1.99 30.69 G-C -0.69 0.26 3.46 -5.94 4.86 38.93 C-G 0.16 -0.31 3.3 0.17 3.91 32.68 5mC-G 1.45 0.09 3.41 4.04 4.12 34.66 G-5mC -0.09 0.36 3.13 -0.11 11.96 30.58 C-G -1.1 -0.84 3.38 -2.58 3.9 31.72 A-T 1.06 0.2 3.35 4.9 1.19 36.76 G-C -0.12 -0.3 3.05 -3.2 2.64 24.58 B-DNA* 3.4 -6 36 * G. Michael Blackburn and Michael J. Gait (1996). Nucleic Acids in Chemistry and Biology (Second Edition). Oxford University Press. References Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW,

Kapral GJ, Grosse-Kunstleve RW, et al. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213-221.

Collaborative Computational Project, N. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760-763.

Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126-2132.

Ho KL, McNae IW, Schmiedeberg L, Klose RJ, Bird AP, Walkinshaw MD. 2008. MeCP2 Binding to DNA Depends upon Hydration at Methyl-CpG. Mol Cell 29: 525-531.

Ohki I, Shimotake N, Fujita N, Jee J, Ikegami T, Nakao M, Shirakawa M. 2001. Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA. Cell 105: 487-497.

Otwinowski Z, Borek D, Majewski W, Minor W. 2003. Multiparametric scaling of diffraction intensities. Acta Crystallogr A 59: 228-234.

Scarsdale JN, Webb HD, Ginder GD, Williams DC, Jr. 2011. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence. Nucleic Acids Res 39: 6741-6752.

Zheng G, Lu XJ, Olson WK. 2009. Web 3DNA--a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures. Nucleic Acids Res 37: W240-246.

Supplementary Table 3. The distribution of TGCCGC element

Genomic region Size (nt)Number of TGCCGC ACGGCG

Frequency (per 1,000 nt)

Gamete ICR (n = 15) 66,413 104 1.57Blastocyst ICR (n = 5) 12,117 25 2.06Embryo ICR (n = 15) 61,341 101 1.65

CpG island (n = 15,991)* 10,469,353 13,506 1.29Whole genome (mm9)** 2,561,882,518 163,981 0.06

Gamete ICRNespas-Gnasxl 6,702 14

Gnas1A 3,762 4Peg10 2,741 3Mest 5,035 12Peg3 4,471 8Snrpn 4,203 10

Kcnq1ot1 2,075 3Zac1 2,435 6

Grb10 1,974 4U2af1-rs1 2,603 2

Igf2r 1,648 8Impact 3,076 5

H19 7,258 6Rasgrf1 8,039 9

Dlk1-Gtl2 10,391 10Total 66,413 104 1.57

Blastocyst ICRPeg10 2,612 3Snrpn 4,203 10Impact 2,511 5

H19 2,437 6Dlk1-Gtl2 354 1

Total 12,117 25 2.06

Embryo ICRNespas-Gnasxl 8,065 14

Gnas1A 2,293 4Peg10 4,154 3Mest 5,960 13Peg3 5,382 8Snrpn 3,905 10

Kcnq1ot1 3,244 3Zac1 2,046 6

Grb10 1,522 4U2af1-rs1 2,489 2

Igf2r 1,565 8Impact 2,561 5

H19 4,981 6Rasgrf1 9,820 8

Dlk1-Gtl2 3,354 7Total 61,341 101 1.65

*CpG island annotations were downloaded from the UCSC genome database.**The letter N is not included.

Nespas-Gnasxl 8

6

Gnas1A 3

1

Peg10 2

1

Mest 7

5

Peg3 3

5

Snrpn 3

7

Kcnq1ot1 1

2

Zac1 3

3

Grb10 1

3

U2af1-rs1 0

2

Igf2r 4

4

Impact 5

0

H19 2

4

Rasgrf1 7

2

Dlk1-Gtl2 6

4

Gamete ICR

500 nt

TGCCGC

GCGGCA

!"##$%&%'()*+,-"&)',./0,1%2"%'3%1,,!4054,Zeschnigk, M. et al. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet 6, 387-395 (1997) !TAGACATGTCCATTGATCCCAGGTTGCTTATGGTTTCTAGAGGCCCCCTCTCATTGCAACAGTGCTGTGGGGCCCTAGGGGTCCAGTAGCCCCCTCCCCCCAGGTCATTCCGGTGAGGGAGGGAGCTGGGACCCCTGCACTGCGGCAAACAAGCACGCCTGCGCGGCCGCAGAGGCAGGCTGGCGCGCATGCTCAGGCGGGGATGTGTGCGAAGCCTGCCGCTGCTGCAGCGAGTCTGGCGCAGAGTGGAGCGGCCGCCGGAGATGCCTGACGCATCTGTCTGAGGAGCGGTCAGTGACGCGATGGAGCGGGCAAGGTCAGCTGTGCCGGTGGCTTCTCTCAAGAGACAGCCTGGGGAGCGGCCACTTTTATTCATCAGATATTCCAAGTTTTTAGGACTTGGAGTACTGAATAAACGGAATTTGGGCCCTAAAGTCCTTTGTTCTGGAGAACCAGATCCGGAATGTTCAGAGGCTTGCTGTTGTGCCGTTCTGCCCCGATGGTATCCTGTCCGCTCGCATTGGGGCGCGTCCCCCATCCGCCCCCAACTGTGGTGTCGCGACAGGTCCTATTGCGGGTGTCTGCGGTGGGAAGGGCGGT !6/4789:8;<.:8,=6>?@08A,Beatty, L., Weksberg, R. & Sadowski, P. D. Detailed analysis of the methylation patterns of the KvDMR1 imprinting control region of human chromosome 11. Genomics 87, 46-56 (2006) !GGCATTCAGAAGGCGCGCGCCAGCGGCGCGGAGGGGACGGACAAGGGGAGGGGCCGGCAGACGCGGTCAAGTGTCACGCCCGGATGGGGGATGGGAGGTGGGCAGGGCGGGCCCGGCCGGGCGCTGGCAGGCGCGGGGCGCCCTCGGCGCTGCCCTTTGCCAGGTGGGTGGCCTGGCATGTCCCGGCCACCCCTGCCACTCACCCCCTGAGGTCCCAAGTCCTCAGGGGTGAGCGGCAGACACGGGTGTTCCAGGCACAGCAGTGGGGGCTTCAGAACATCCCGATCCCCTGCATGGACCCGCTGGGAGGGGGGAAAACGCCTCCCGCACCCGGTGCCACGGCGGTGGAGACCTTGCCCGGGTTCGAGGGTCGCTGCAGCGGCGCCGGCGGAGGGGCTGGTCTCGGGCGTGGCCTGGACGCCCACAGGCCTCCACACCGAGGGCCCACAGCAGTGTCGGGGTGCGGCCGCTGTCCTCACGCGGTCACCCCGGGGTGGTGAACACATCACGCAGAGAACTGGCCCGTGTCTCCAGCTCACCCCGGCGATGCCAACGCTGAGGCTGGCGCTGGCCCCGGCCCCCGGTGGTGCTCAGGGACGACGGGTGGGGGCAGGGAGGGCGGCGGCTCGCGCCACCTCACACCCAGCCAGTGCCTCATGCGGCGCTGGACCCGCTGGGCCAATCTGAGCCCGGGTGGCATCAAAACGAGACTCTTTCGGCCAATGACAGGACACGGCACATCACTTTCCGCACCCAGCCAATCCGTGCAGCAGCCCGCCGCAAGCCTTCCCCTGCTGCCGCCCAATCAGCAGGTGGGGGGCGGTCGCCACGTCGGCAGCGGCGGGGGCAGTCGGAGCGCTGCCGCAGTCTCCAGGCAGAACGGTCGCCGCGTCGCCTCAGCACGGACCTCCAGGGAGCTCCTCAGCAAGATCCTGCCAGGGCGCCCCTCAGCGCGATTCTGCCGGGGTGCCTCTCAGCGTGGTCCTCCCCGGGGCTCCTCAGCACGATTCTCCCGGTGCGCCCCTCAGCGCGGTCCTCCTCGGTGCGTCAGTCATCGTGGTTCTCCCCGGCGCGCCCCTCGGCGCGGTTCTCCTCGGGGCTCCTCAGCGCGGCGCTCTTCTGGGGGCTCCTCGGCGCAGTTCTCCCCGGGGACTCCTCGGCGCCGTTCTCCTCGGGGCACCCGGGGCTTTTCGGCGCGGTTCTCCCCGGGGGTTCTTCGGCGCGGTTGTCCCCGGGGGTTCTTCGGCGCGGTTCTCTCCGGGGGCCTCTCGCCGCGGTTCTGTTCTCCCCGGGGGCTCCTCAGCATGGTTCTCCTCCGCGCGGTCCTCCCTGGGCCTCCTCAGCGCGGCACTCTCCTGGGGGCTCCTCAGCGCGGCACTCTCCCCGGGGGCTCCTCAGCGCGGCACTCTCCCGGCGGCTCCTCAGTGCGGTTCTCCCAGACTCTCCTCAGCGCGGCCCTCCCCATCTCTCTGGGAGGGTTTGAACACGGTCAGCACGGACCTGGGCGGACGGCGCGGGACGGGTGATCACTGGCGTTGCTGAGGTGAGCTGTGTGCCCCGCGGCCGTCCCAGATCACAGGCGTCAGCAGTGCAGCCTGGCCTGGGCAGTGCGCTCCCATCTGCACCTTATGGACAGCGTGGCCAGGGTCGAGGTCCGAGTTCCTGGCCGCGTCCCAAGGATCGGATTCCGGGTCTATAGTTCTCATGGTGGTTCAGAGTGGGCTGAAT

TGGGATTGGAGTCTGGAATCCGCATCGTGGTTCTGAGTCCGCGCTATTGGGATGGAAGTTGGGAATCCATGTTGTGGTTTTGAGTCAAAGCCCGAATTGGGA ,BC/;5<CD<8!!Valleley, E. M., Cordery, S. F. & Bonthron, D. T. Tissue-specific imprinting of the ZAC/PLAGL1 tumour suppressor gene results from variable utilization of monoallelic and biallelic promoters. Hum Mol Genet 16, 972-981 (2007) !GTGCTTAGGACAGTGCCTGGCTCACGGTCAGTGCTCACGTTTGGGCAGCTGCACTTGGGCGCTGCTGGCACAGGAGGTAAGTTAGTTTGGCCTATTGCAGCGTCCCAGCATCTGTCGCGTTTCTCATGTGTGATTGGGCTCTGGCGGCCCATCCTGGCGGAGACTTCGGCTAGCAGGCCCCGCTGCAGACCCCAGGCCGGCTCGGGTCTACCTGCGCCAGCGCTGTACCTGGGCGACCTTGGCTTTGCCCCCACCGGTGACCCGGCCCGCAGGACGTGTGGGTGCCGCTCAGCTCCCCCCGCCTCGGCCGCCGACCCCCAGCTCCCCGGCGGGGCCTCCTCCTGCCACGTGACGCCCCCGCCAGGGGCCCCAGCGCCCTCCTCGCGGCCGCGCCGTTCCGGCTCCCGAGCCCCGCCTGCGCGCGGCCTCCTCGGCGCAGCCATCCTCTTGGCTGCCGCGGGCGGCAAAGCCCACGGCATCTGCCATTTGTCATTCAGCCCGTCGGTACCGCCCCGAGCCTTGATTTAGACACGGCTGGG D0E8F;@GD8,=/D.H,?@0A,Arnaud, P. et al. Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark. Hum Mol Genet 12, 1005-1019 (2003)!!GGCCGGCCCTGCAGGTGGAAGGTTGCTCGAGCAGGCGGCATCTCTAGCTTGCCGCGGCCGCGATGTCCCCCGCCTGTCTGCGAATGCGGCGTAGCGGGTAGACATGGAGGGCTTTCGGCATCGTCAGAGTGGCCAGTGTGCGCGTCCTTGCCCATCAGGCGGGGGGGCTGTGGGGGAGGAGAGGAGGCAGTGGAGGGAACAAGGGGTTCTTCAGGGTTGCCATGAGAACCAGGGATCCGGGGATTGGGCAGCAGAGGGCCCCCGCCGGGCGTTGGGGCGTGGCCGCGTCACATGGGTTTGGTCCTGGGATTCCTGTTGTGCTCCAGGACCGGGCGCTGCTCCGTCGTCCTCCCGCTCCTCAGGAGCGCCCAGTCCCTCGGAGGCTGAGTATTGCAGCCGGGCGGCAGCCGGCTCCGCGGAGGGGCCCCCGGGCACCTGCGTGGTGATGGCGCTGGGAGCCCCCGGGCACGCTCGGGCGGTGGCGCGGCATCCCACCCTCGCCCGGATGGCGTCCCCAGAGGCGGCGTTGGCCCGCTTTTCGTGCTAGCGCGTTCGCCTGGCGCGCGGTGGCCCCGAGGCCCCGGGTCGGTTTTCTGCGCCGCAGGCCCCTGGCCGGGGCGGAGCCGTGGAGGACCAGCCCGGCCCGGCTCCGAGCGCTGTCCATGCGGAGCGCTGTCCACGCGCCGGGCACTGCGGGGGCCGGGCCCCGAAGCCCTACCCGGGCCGGCGGCGCACACGCAGCGACCCCGTGCGGCCAGTGCTGCCGCCCGCTCTCCAGGTACTCAGGTGGGCTCCGCCGCGGGCGCTGGGCGGTGGGCGGTGGGCGGTGGGCGCGAGCCGGGGCGGCGGGCGGATCGGCCTGCGCAGGCTGCAGAGGCCCCGGCCGCGGCGGACAGGGCCCGGGAGGGAGGCGGGGAGGTCTTGCCGCGCGGCCCCCTGCCCGCCTGCCGCGGGCTCGCTGCAGTCCGAGATCCCGAGCTTCGTTGCCGCCAGCC !.DIH0,Riesewijk, A. M. et al. Maternal-specific methylation of the human IGF2R gene is not accompanied by allele-specific transcription. Genomics 31, 158-166 (1996) !CCGGCGGGCAGCCGCGTGAACCTGGAGCTTGGGGTCTGCAGTGCTGGGAGGCCGAGGCCTGGCATGTTGGGGACAGGTCTTGGGAGCTGACTGGGGGCCTGGCGTGGTGGCGGCAGCCCGCAGTCCCTGCCTGGCCCTTGTCTGGGAGGCCGCGGTTCTCTGCGGCCTCTCGCCGGTGTCGCCCTTGGCCACTGGGCTGCGAGGAAGCCGGCGAGGAGTGGGTGTGCGCCCCGCCCGGCTCTACCGGCGAGGGATGCAGTGGCCGCCAGCGTGGCCGTGCGGCTGGGTCACTCCCTTCCCGCTTCCAGGGCCTTTTTCTG

CCTCCTTTTCAGGACGTGACCCGCACATTATGAGGGACCCGTAGGGGCTCTTGACACAGAGGGGTCACCCTATGTGTGAACAGGTTACGAGGTTGGGGGCTTGGCTTCTGAACTTGCCAGGAAACACCGCATCCCTTTTCCTGCAGCGGCCGCCCCCCATCCCCCACCCGCCCCATTCTCTCCCCTCCCCAACTGCAGCCTTCCGCGCCTCCTTGGCCCCTCGAGCCTCCCTGGGTCCCCTGCACCTCCCCATGTCCCCCACGACTCCTAGGCCTCTCGCGCTTCCCCAGGACCCCGCGCATCCTGGGCCTCTTGCGCCTCCCCGGTCCTCAGCGCCTCCTCGGCCGCCGTGCCTCCCCGCACCTTTTGCGCCTCATGGGGCTCCCGGTGCCTTCTGCCCGCCGCCTCGCCGCGCCCCTCGCCTCCTCATGCCCCTCCGCGCCCCATACCTCCCCACCCCTGTGCCTCTCTGCCTATCCCCTGTACCACCCGGCATCCTCCGTGCCCCATGCGCCTCACCCCTCACGCCTCCCTGTACCCTGCATGCCCCGTGTGCCTGCTGTGCCCCACGCGCCTCCCCCCTCGCGCCTCCCTGTACCCTGCATGCCCTGTGCGCCTGCTGCGCCCCACGCGCCTCCCCCCTCGCGCCTCCCCCCTTCGCGCCTCCCTGTACCCTGCATACCCCGTGAGCCTGCTGCGCCCCATGCGCCTTCCGCGTCTCCAGCCCTCATGCGCCTCCAGCTGCGCATCTCCCTAGGCGGGTACCTCAGCACCGCTGAGCCCACCCCCGCCCCTGCTCAGTGCCTCCCAGACACGCCCACTGCGGGCGGGACAGTGGGAGCTGTCCAGGCGCGGAGCTGTCCAGGCGCGGGAGCTGTCCAGGCGCGGGAGC !-8J,Frevel, M. A., Sowerby, S. J., Petersen, G. B. & Reeve, A. E. Methylation sequencing analysis refines the region of H19 epimutation in Wilms tumor. J Biol Chem 274, 29331-29340 (1999) !AGGCTTCCCATTCAGTCTTGGATGCCAGCCTCACCAAGGGCGGCCCATCTTGCTGACCTCACCAAGGGAGGCCCGTCTCACTGCCCTGATGGCGCAGAATCGGCTGTACGTGTGGAATCAGAAGTGGCCGCGCGGCGGCAGTGCAGGCTCACACATCACAGCCCGAGCACGCCTGGCTGGGGTTCACCCACAGAAACGTCCCAGGTCTCCCAGGCCAGGTGCCGCATTGGTTCCCGAGGGTTGTCAGAGATAGACACTCATGCGACTAACATCGGGCTATGTGTTTGATTCACCCCAGGGTGCATTGTTGAAGGTTGGGGAGATTGGAGGAGATGCTTGGGGGACAATGAGGTGTCCCAGTTCCTTGGATGATAGGGATCTCGGCCTAAGCGTGAGACCCCTCCTACAGGGTCTCTGGCAGGCACAGAGCCTGGGGGCTCTTGCATAGCACATGTGTATTTCTGGAGGCTTCCCCTTCGGTCTCACCGCCCCGATGGTGCAGAATCGGTTGTAGTTGTGGAATCGGAAGTGGCCGCGCGGCGGCAGTGCAGGCTCCCACATCACAGCTCAAGCCCGCCCCAGCTGAGGTTCACCCGCGGAAACGTCCCGGGTCACGCAAGCTAGGTGCCGCAAGGTTCACGGGGGTAGTGAGGGATAGAACACTCATGGGAGCCACATTGGGCTACGTGTCTGATTCACCCCAGGGTGCACTATTGAGGGTTGGGGAGATGAGATACTTTGGTGACAATGAGGTGTCCCCATTCTTTGGATGATGGGGATCTCGGCCTCAGCGTGAGGCCCCTCCCACAGGGTCTCTGGCAGGCACAGAAACTGGGGGCTCTTGCGTAGCACATGGGTATTTGTGGACGCTTCCCCTTCTGTCTCACCACCCGGATGGCACAGAATCGGTTGTAAGTGTGGACTCAAAAGTGGCCGCGCGGCGGCAGTGCAGGCTCACACATCACAGCCCAAGCCCTCCCTGGATGGGGTTCGCCCGCGGAAACGTCCTGGGTCACCCAAGCCAGGTGCCGCAGGGTTCTCGGAGGTCTTCTGGGAATAGGACGCTCATGGGAGCCACACCACGTCTTCGTATCGGGCCATATCCACGGCCGCGTGGCCCCAGGTCACACTCTGAGGGCTTCAGTGTCATGGCCTGGGACTCAAGTCACGCCTACCCGCGTGATGAGCACAGCAAATTCCGCCAAAAGCTTATACTTTCCACATCCATCCCAGAGCACAGATCCGACTAAGGACAGCCCCCAAATCCCGAGCCTTTTTCTGAACTGACAATTGCCTCCCCAGTGAACACTCTGAGCTTGTCAATCTTAAGTGGCCAGACATTAACATTCCCATTCAGTGCAGGTTTGAGATGCTAATTTAGGAGCTTGAGATGCTAAAGAGCTGGGAGTGCCACTGCTGCTTTATTCTGGGGTCTAGGATCCTTGTGTTGGCTGAGATAATCTGCTAATGTGGGTGCAGCAGACATCCCGCGGTTTGTGGAATCGATAAAGGATGGGGATCAATGGTGTTTGTGCACTGTGCGGTCTGTGCCCAATTGCCTGCCTTGTGCTGTGGAATCTGTACACCTGGCCAACATGTGCTTGTGTGAGCCTGACAGTGCATTTTCCAGAGCCTCACCTCGGCTCTGCCCTGGAGGCTCTGTGCTGCTGGAATCAGACTCAAGGACCTCATCAGAGGACCATGGCCCCGTATCACCTGGGTCAGGCACTGAAGCTGGGACAGGAGAGCAGAGACTTCCAAAATGAGGGATCCCTGTGTTCTGAGGTGATCATGACTGGGACCCAAGGACTCAAGCGCATGCTCCAGAGGGAATCGTTTCCCACAAGGCCTTTGGCAGGAACAGGGATCCTGGGAGCCTGCCAAGCAGAGCGCACAGTGTTCCTGGAGTCTCGCTGCCCA

GATGCCACGGAATCAGTTGAAGGTATGGAAACACAGGTGGCCACGTGGTAGCAGGGCAGGCTCAGGCGTCATAGCCCGAGCCCGGCTACCTGTGGTTTGCCTGCAGAAACATCCCGGGTCAACAGGCCAGGCACCGCATTGGTTCGCGAGGGTCATCGGGGGTAGGACCCTTGTACGAGCCACATCGGGCTACGTGCCTGATTCACCCCAGGGTGCACTGTTGAAGGTTGGGGAGATGAGAGGAGATACTTGGGGGACAGTGAAGTGTCCCCATTCTTTGGATGATGGGGATCTCGGCCTCAGCGTGAGACCCCTCCCACAGGGTCTCTGGCAGGCTCAAGAGCCCAGGGGCTCTTGCATAGCACATGAATATTTCTGGAGGCTTCCCCTTCAGTCTCACCACCCGGATGGTGCAGAATTGGTTGTAGCTGTGGAATCGGAAGTGGCCGCGTGGCGGCAGTGCAGGCTCACACATCACAGCCCGAGCCCGCCCCAGCTGGGGTTCGCCCGCGGAAACGTCCCGGGTCCCGCAAGCCAGGCGCCGCAGGGTTCACGGGGGTCATCAGGGATAGGACATTCATGGGAGCCACATCGGGCTATGTGTCTGATTCACCCCAGGGTGCACTATTGAGGGTTGGGGAGATGAGAGGAGATACTTGGGGGACAATGAAGTGTCCCCATTCTTTGGATGATGGGGATCTTGGCCTCAGGGTGAGATCCTTCTTGCAGGGTCTCTGGCAGGCACAGAGCCCGGGGGCTCTTGCATAGCACATGTGTATTTCTGGAGGCTTCCCCTTCAGTCTCACCGCCCGGATGGCACAGAATTGGTTGTAGTTGTGGAATCGGAGGTGGCTGCGCGGCGGCAGTGCAGGCTCACACATCACAGCCTGAGCCCGCCCCAGCTGGGGTTCGCCCGTGGAAACATCCCAGGTCATCCAAGCCGGGCGCCACAGGGTTCACAGGGGTCGTGAGGTATAGGACACTCATGGGAGCCATATCGGGCTACGTGTCTGATTCACCCCAGGGTGCACTGTTGAAGGTTGGGGAGATGGGAGGAGATACTAGGGGAACAATGAGGTGTCCCAGTTCCATGGATGATGGGGATCTCGGCCCTAGTGTGAAACCCTTCTCGCAGGGTCTCTGGCAGGCACAGAGCCCGGGGGCTCTTGCATAGCACATGGGTATTTCTGGAGGCTTCTCCTTCGGTCTCACCGCCTGGATGGCACGGAATTGGTTGTAGTTGTGGAATCGGAAGTGGCCGCGCGGCGGCAGTGCAGGCTCACACATCACAGCCCGAGCCCGCCCCAACTGGGGTTCGCCCGTGGAAACGTCCCGGGTCACCCAAGCCACGCGTCGCAGGGTTCACGGGGGTCATCTGGGAATAGGACACTCATGGGAGCCGCACCAGATCTTCAGGTCGGGCATTATCCACAGCCCCGTGGCCCCGGGTCACACTCCGAGGGCTTCAGTGTCATGGCCTGGGACTCAAG !?<68KD:<H,Kawakami, T. et al. Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma. Hum Mol Genet 15, 821-830 (2006) !TGGGGCTGCAGGGCTGACGCGGGCTGGCACTGTGTCTACGACAGCCTCCCGGGCCCCCGGGTGCGTGGCTGCGGATGCTCTCTGGCGGCCACCACAGTTCCCACGCGCGGCGGGTGAATGCGAGCCCCTTGTCAATGCGTGCCCCTCGCCTCTGCCCCCGAGAGGCTAGCAACCGGGCCTGCCGGCGTCTGATTGGCCGTGGGGGCCTGACGACAGGGGCTCCTGGTTGCTGATTGGCCCCGGCCAGCTCTGGCTGTTTCCTAGCTATTAATACTGTGGCTAATAAACGTTCTCCTGTTGACGCGGGTGCTCGGAGCCATGATTTTTTGAAGCGTTCTTGACGTCTGTGGCTGGTGCGGGCGTTTCTGCTGTGCAAGGCCTGCTTCCGGGGCTCTAGTCCCTGGGGCTCCTGGCAAGCTCCACAGGCTGTAAAGGGGGTGTTTTCTTTCTCCTTCGTGTTACCTGACATATTAAAGCAGCGCCCCATGAGGACCCCAACGTCCCACGTCTCTATCTCCCCAACAGTGCGCCTGTTTATGAAAAAACGAGCCCCCCACACGCCGTCCCAAGGCTCGGCGCCTCTAGTGACCTGACGGTCAATGTTCACCTCCCTAGTCATTAGCTGTGGACGTAGAAATAGCCTTTCTCCCTTCTTTTCAGCCCTGGAATCTCCCGTCTGCTTTACAGTCACCTTGTGGGATCGTTGGGCATGGGGGGCTCCCTCTGACGCCGCTTAAACCCCCCAAAGAAGTGCGGGGGAGCGATTCTGACTTGATGCCCTCGGCGGGGCCTCTCTGCTGTCCTCTTTGGGGCCCCTTGCTCATCCTCACCTGCTTTCTGTCACCGAGCACTCCATCCTTGTCCTCCTGGCTGCCACCCACCTGTCACCCTAAGATAGATCCTCGGTCCCTTTCAACAGCATTTTGACCTTTGCGAGAGGACATTTGATTGACGGCCCTGCACGCTCTGCCACTCAGAGCCGGGCAGAGCTGCCGAGGGCTCCCACCTGTTAGGGATTAACTCCCATGTGCCAGCTCCGGAGCCGAGGCCGCGGCAGGGCTCGGCGCAACATGTGTCGCT