Cloning GADD45a into pCMV-Myc1 or pCMV-nV-His6Gadd45EcoVF ATCCATGACTTTGGAGGAATTCTCGGCTGGAGAGCGadd45XbaR GTAATCTAGATCACCGTTCAGGGAGATTAATCACTGG

Cloning GADD45a into pTXB1G45aNdeF GGCGCGGCATATGACTTTGGAGGAATTCTCGGG45aSapR GGTGGTTGCTCTTCCGCACCGTTCAGGGAGATTAATCAC

nV5-His6 linkerV5HIS6F AGCTGCCACCATGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGA CCGGTCATCATCACCATCACCATV5HIS6R GATCATGGTGATGGTGATGATGACCGGTCGTAGAATCGAGACCGAGGAGAGGGT TAGGGATAGGCTTACCCATGGTGGC

Cloning DNMT1 deletion mutants into pGEX-5X-1D1KOZAKR1F CGGAATTCGCCACCATGCCGGCGCGTACCGCCD1L419R CATGCGGCCGCaggtgaccgtgcttacagtacacD1V412F CTCTGAATTCGCCACCATGGTGTACTGTAAGCACGGTCD1G1114F GGAAGCGGCCGCGTggaaaagggaagggcaagD1V5NOTR GAGCGGCCGCCAGTCCTTAGCAGCTTCCTC

Site directed mutagenesis of GADD45aRT34AA-F CAAAGCCCTGAGTCAGGGCGCGATCACTGTCGGGGGRT34AA-R CCCCCGACAGTGATCGCGCCCTGACTCAGGGCTTTGLC56AA-F CCCGATAACGTGGTGGCGGCCCTGCTGGCGGCGGACGLC56AA-R CGTCCGCCGCCAGCAGGGCCGCCACCACGTTATCGGGED63AA-F TGCCTGCTGGCGGCGGACGCGGCCGACGACAGAGATGTGGCTED63AA-R AGCCACATCTCTGTCGTCGGCCGCGTCCGCCGCCAGCAGGCACE83AA-F CCAGGCGTTTTGCGCCGCGAACGACATCAACATCCTGCGCE83AA-R CGCAGGATGTTGATGTCGTTCGCGGCGCAAAACGCCTGG

Table S2. Primers used for Methyl-DNA Immunoprecipitation (Fig. 2G).Rec2: (2133-2151) 5‘-GAGGGCGAGGGCGATGCC-3‘ (DR-GFP)DRGFP ChIP LOWER R: (2722-2704) 5‘-CGGCGGCGGTCACGAACTC-3‘UBE2B F: 5‘-CTCAGGGGTGGATTGTTGAC-3‘ UBE2B R: 5‘-TGTGGATTCAAAGACCACGA-3‘ H19 ICR F: 5‘-GAGCCGCACCAGATCTTCAG-3‘ H19 ICR R: 5‘-TTGGTGGAACACACTGTGATCA-3‘

Table S1: Primers used in this study. The sequences of forward (F) and reverse (R) primers used for PCR are given in 5’ to 3’ direction. Oligonucleotides used for methyltransferase assays are listed in experimental procedure.

Supplemental Figure S1

Figure S1. Map of the DR-GFP and Methylation Products.A. The DR-GFP Reporter System. The two cassettes are shown (GFP-I with the I-SceI site at stop codons) and GFP-II corresponding BcgI site that serves as donor DNA in the gene conversion process after I-SceI cleavage. Specific primer pairs allow unambiguous detection of HR structures as follows: Parental DR-GFP molecules are amplified by the 5’-UnRec primer and 3’ common primer (the latter does not exist in GFP-II) (Cuozzo et al., 2007). The 5’ REC primer (sequence shown) is indicated with the distinguishing bases in capital letters. An upstream promoter element (CMV enhancer and chicken actin promoter) provides high-level expression of the region. After HR and gene conversion, the proposed hemi-methylated intermediate is shown (primary HR-Product, Hem-methylated DNA). The filled in circles correspond to the location of the strand methylated segment (3’ of the new BcgI or converted I-SceI site, ca. 400 bp region). Subsequent events after DNA replication are depicted in Fig. S2. B. HpaII site in the repaired region between REC primer and 3’ common primer used for methylation sensitive restriction analysis

Transfection with I-SceI

5’-UnRec X

GFPPuromycinGFPGFP

3’

I-SceI BcgI

GFPPuromycinGFPGFP

BcgI

GFP

GFPGFP

Gene conversiton

DSB triggers HR

GFP Rec 5’- GAGGGCGAGGGCGATGCC-3’

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ceI

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GFP+

3’ common

BcgI BcgI

GFP

Primary HR Product

Hemi-methylated DNA:

Rec

3‘commonHpaII

156

437

CCGG

B

A

Supplemental Figure S2

Figure S2. DNA replication results in expression classes based on DNA methylation. Following DNA replication, the primary HR-Product resolves into two populations that differ in methylation status. The low expressor class (HR-L) derives from templating off the methylated strand while the high expressor class (HR-H) derives from the unmethylated parental DNA, yielding hypermethylated and hypomethylated progeny cells, respectively, at the GFP locus. These populations are resolved as indicated in the hypothetical FACS profile (see Fig. 1B for actual FACS data).

5’-Rec 3’

BcgI

GFP+

HR-H = Hypomethylated DNA5’-Rec 3’

BcgI

GFP+

HR-L = Hypermethylated DNA

FL2H

FACS

DNA Replication GivesTwo Expressor

Populations

HR-L HR-H

Puromycin

5’-Rec X

GFP+

3’

BcgI BcgI

GFP

HR Intermediate, Hemi-methylated DNA:

Supplemental Figure S3

Alanine Scanning Mutagenesis of GADD45

Figure S3. Alanine Substitution Locations for mutations in GADD45.

Supplemental Figure S4

M1

M2R2

M1

M2R2

M1

M2R2

24 h

34 h

46 h

1.0

2.0

3.0

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otal

GF

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4 8 12 24 34 46 55

1

2

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mR

NA

G45

a\A

ctin

B

Hrs Post HR

% GFP+mRNA

A

Figure S4. FACS data and time course of GFP.A. HeLa DRGFP cells were transfected with I-SceI and FACS analyses carried out at the times indicated. The regions designated M1 and M2 correspond to the HR-L and HR-H expressing populations, respectively.B. Kinetics of WT GFP Accumulation. HeLa DR-GFP cells were transfected with I-SceI and FACS analysis performed at the indicated times to determine the fraction of GFP expressing cells. The accumulation of stable G45a transcripts (relative to actin) are also shown.

Supplemental Figure S5

Myc

-G45

a +

V5-

G45

a

V5-

G45

aM

yc-R

T34A

A +

V5-

RT34

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RT34

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Myc

-LC

56A

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V5-

LC56

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V5-

LC56

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-ED

63A

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V5-

ED63

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ED63

AA

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-CE83

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+ V

5-C

E83A

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IP : -MycWB: -V5

IP : -MycWB: -Myc

Myc GADD45 + V-5 GADD45

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3

4

5

6

7

% T

ota

l G

FP

B

+ I-SceI

NoG45a

WTG45a

CE83AAG45a

A

Figure S5. Analysis of Dimerization Potential for G45a Mutations. A. Dimerization analysis. HeLa cells were transfected with the indicated Myc or V5 plasmids (3 ug each). Immunoprecipitations were carried with anti-Myc antibody and probed with V5. Co-IP self-association levels (lanes with one protein only) were subtracted in the digitized data analysis (four independent experiments). The digitized data are shown in Fig 5A. For each mutant, recoveries by Co-IP were normalized relative to overall expression levels for each mutant by Western blot analysis using actin as the loading control. Expression levels between mutants varied by as much as 2 fold.B. Influence of ectopic G45a on GFP expression. HeLa DR-GFP cells (a single clone) was transfected with the indicated expression plasmids (I-Sce plus or minus 0.5 ug of wild type G45a or the CE83AA mutant; all plasmids express constitutive levels using the CMV enhancer). Total GFP expression levels were measured 48 hr after transfection (three experimental repeats).

Bound Free KD Value

Unmethylated DNA GADD45a 48M

1600 nM

800 nM

400 nM

200 nM100 nM

A

Bound Free KD Value

Hemimethylated DNA GADD45 0.25 M

Hemimethylated DNA DNMT1 1.07 M

70

55

25

40

35

15

MW GADD45

GADD45

MW Crude Purified

180

115

82

64

DN

MT11600 nM

800 nM

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B

1400

1200

1000

800

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0

Mic

ro R

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10008006004002000Seconds

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onse

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DNMT1

Supplemental Figure S6AB

100 nM200 nM

400 nM

800 nM

Bound Free KD Value

Fully methylated DNA

GADD45 194M

Bound Free KD Value

GADD45 Hemimethylated DNA

1.2M

1600 nM

800 nM

400 nM

200 nM100 nM

C

1600 nM

800 nM

400 nM

200 nM

100 nM

D

Supplemental Figure S6CD

Supplemental Figure S6

Figure S6. SPR Analysis. DNA/protein binding affinities were calculated using Surface Resonance Plasmon (SPR) assays.

Panel A. Biotinylated unmethylated duplex oligonucleotide DNA was immobilized on the gold chip coated with NeutrAvidin as described in “Materials and Methods”. Purified GADD45 (inset gel shows purity of GADD45) was applied to the chip at a concentration of 100, 200, 400, 800 and 1600 nM (flow rate of 13.3 µl/min). The KD value is (inset table) derived from kinetic parameters based on the relationship KD = kd/ka where kd is the dissociation rate constant and ka the association rate constant. The ka and kd rate constants were derived by non-linear curve fitting of sensogram data at the concentrations indicated above. A representative SPR trace is presented that was fit to model data (smoothed red line). Panel B is identical to A except a hemimethylated DNA target was tested (30-mer duplex DNA with a single Me-CpG centrally located; see “Materials and Methods”). The inset data shows a control experiment with DNMT1 (purity of protein is shown along with the sensogram and KD value). Panel C. Analysis of GADD45 binding to fully methylated DNA bound to gold chip. Panel D. Reverse Binding SPR Control: Analysis of hemimethylated DNA binding to gold chip linked GADD45. Purified, biotinylated GADD45 was bound to the gold chip and hemimethylated DNA was at the concentrations indicated was bound and quantified to derive the indicated KD value. The analysis was repeated using unmethylated and hemimethylated DNA; however, binding was sufficiently weak as to preclude accurate KD determination. Curve fitting was based on four experimental determinations.

Supplemental data Fig. S7

con

Rela

tive

Indu

ction

%

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40

80

100

60

#49

#50

Con

G45212 bp

#49

#50

#49

#50

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Actin250 bp

35 cycles

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#50

Con

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ress

ors

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#50- +

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16% 39% 4%

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#50

E

Cycles

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tive

GFP

Exp

ress

ion

%

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#49

#50

Figure S7. Analysis of shRNA G45a knock down cells in three independent shRNAsA. A clonal isolate of HeLa cells, HO1 cells, was selected containing a single integrated copy of the DR-GFP reporter. This isolate converts to GFP+ cells following I-SceI transfection generating two populations of expression classes (low and high) that differ in methylation frequency over the repaired segment. These cells were used in G45a knockdown experiments. The shRNAs used are described in the “Materials and Methods” and represent two different regions, indicated as #49,50, along with a scrambled shRNA control (“con”). Expression of G45a following transient transfection with each construct was determined by RT-PCR along with an actin control. The graphical data correspond to relative G45a mRNA levels based on G45a/actin ratios in UV treated (open boxes) or non-UV treated (shaded) cells. RT-PCR analyses were performed at 48 hr after transfection; however, identical results were seen at 72 hr post transfection (not shown). B. Analysis of HR frequency in shRNA knock down and control cells. Transfected cells were analyzed by Q-PCR using the 5’ REC primer specifically amplifies HR products (graphical data for #49,50 shown in panel E).C. Analysis of GFP expression. HeLa DR-GFP cells were co-transfected with I-SceI plus indicated shRNA expressing plasmid and 48 hr later, cells subjected to FACS to determine total GFP expression (left) or HR-L low expressing population. D. Methylation sensitive restriction enzyme analysis. Cells were transfected with indicated shRNAs or scrambled RNA control and after 48 hr, recovered genomic DNA was subject to HpaII digestion (+) or no digestion (-) followed by PCR amplification using the 5’ REC primers to assess the overall percentage of HpaII sensitivity (see Fig. S1B for map). The values given below each lane correspond to the digestion residuals. Thus, in control cells 4% of the total resisted digestion and were therefore methylated at the HpaII site.E. Graphical data of recombination frequencies for two representative shRNA clones #49,50 (based on Panel B).

Figure 1B, 1C Statistical Analysis of HR-H (High Expressor) and HR-L (Low Expressor) Classes of GFP

Figure 1F. Oneway of G45a siRNA mRNA levels Figure 1F. Oneway Analysis of EzH2 siRNA mRNA levels

Supplemental data Fig. S8

Figure 1D Statistical Analysis of percent of recombination at 5days and 7days after I-SceI transfection

Supplemental data Fig. S9

Figure 1F Statistical Summary of Methyl DIP

Figure 2C Statistical Summary

Figure 2A Statistical Summary

Supplemental data Fig. S10