Liefke et al. 1

Supplementary Materials and Methods:

ChIP Primers

cDNA Primers Gene 5’ 3’ Deltex-1 GCC ACA TGT ATC ACC TGC TC ATG GCT TTG CAG GTT GGA Hes-1 AGG TGA CCC GCT TCC TGT TCT GGG TCA TGC AGT TGG

preTα CAG CTC TCC TTG CCT TCT GA CCT GGC TGT CGA AGA TTC C

CD25 CAA TGG AGT ATA AGG TAG CAG TGG CAT CTG TGT TGC CAG GTG AG

RBP-J GTC ACC CCT GTG CCT GTC CTG TAA GTT CAA GCA TTG CTA CG KDM5A ATA ACT CAG GGG CCA GCT C CGA CAA AGC CAA GAG TCT CC GUSB TGT GGG CAT TGT GCT ACC T ATT TTT GTC CCG GCG AAC GAPDH CCA AAA CAT CAT CCC ATC GT AAC TGA CAC GTT TGG GGT TG

Gene Position 5‘ 3‘

Deltex-1 -4300 CAG AAA TGA TCT CCC ATG AAC C AGT CAC CCA GGG CAC CTA T Deltex-1 -3000 TCG AAT GTG ACT GAG ATG AGG A ATC TAT TTA AAC CGA TTC TTA TTG

TGG Deltex-1 -2000 ACC AAG GGA TCT GTG AAT GG CGA GGG TCT CTG TAT CGT GAG Deltex-1 -1500 GCC CCA GCT CTA GAT GGA C TGT CAC ACA GAA CAG GAT GGA Deltex-1 -1200 CCT CTC TGA GCT TCC AGA GT TT CCC CTG GAG GTA AGA CTG TG Deltex-1 -900 GAG GAG GGA AGA CCG ATG A AAG AAG AAT CCA GCC AAC TCC Deltex-1 -500 CCC AAA GAG GTC CCT TAA CC AGA TGA GGC TGT GTG GGA CT Deltex-1 TSS GCG TCC TCC TGT CTG TCT GT CCC CCG CTG ACT CTT TCT Deltex-1 300 GAA TTC TGC ATC TCC ACA TCC GAG AAC AGC GGG CAT CA T A Deltex-1 1700 CCT CCC AGA ATC CCT GAA A CCA GGT CAC ACG GTC TCT TT Deltex-1 4700 TCT TTT TAA ACC TGC CTG GTG AGG CGT GCC TAC ATC ACA C Hes-1 -4200 CCA CTC AAA ACA TCC CCA CT CAT ATC TTC CCC TAC TCC CAA A Hes-1 –3300 TGT TTA AAA AGC AAC CGA TTT TC GGG CAC TCC TTT CAST GTC C Hes-1 -2300 TCA TGC AGT CAA GGA AGC AC TCC CAC TGA GAT CAT GTG AAA G Hes-1 -1000 GCG GCA ATA AAA CAT CCT G CCG AGC TGC AGT TTG ACA T Hes-1 -600 AAG ACA CCT TGC TCC GAA AA ACG CTA AAC ATG CCC TTA CC Hes-1 -180 GCC AGA CCT TGT GCC TAG C TCT TTC CCA CAG TAA CTT TCA GC Hes-1 TSS AAG TTT CAC ACG AGC CGT TC CAG CTC CAG ATC CTG TGT GA Hes-1 +300 ACA CCG GAC AAA CCA AAG AC CAC AAC CCA CTT AAT TTC TAG ACG Hes-1 +600 TTT CCC AGG CAC AAA GAA CT TAG TGC TGG CGG GGT AAG Hes-1 +1500 AGG TGA CCC GCT TCC TGT TCT GGG TCA TGC AGT TGG Hes-1 +2400 GAT GCC AAA GAT GTT TGA AAA TG CAG TTC TAA GAC ATA AAA GCC TTC AC Hes-1 +4700 ACTTTC CCC ACA CCC TTT G CCC ATC TGG TTG CAC TTA GAA CD25 Enhancer CCA CCC CAG TTA TGT TTC CTT GAG CCC ACT CCT GAC ACT G CD25 Promoter GAT CAC AGA ACA GAG TAG GCA

CA GCC AGG AGT TGT TCT ATT TAA GCA

preTα Enhancer CTG CAC TGT GGT CGC AGA GGA GGC AGG TGT CCC TAA C

preTα Promoter ATG GGA GTG GGA CCT GAG T CAC TGC TTC CCA GAT CAC AG

TOM22 Promoter AAT TAA GAG GCT GGC GAC AC GGC TAG AGA GAA AGC GTT GG

GAPDH Promoter GGG TTC CTA TAA ATA CGG ACT GC CTG GCA CTG CAC AAG AAG A

    Liefke et al. 2

KDM5A/KDM5C Vectors:

Full length hKDM5A and hKMD5A (H483A) vectors were kindly provided by the Dr. K. Helin (BRIC, Kopenhagen). Full length hKDM5C was kindly provided by Dr. Yang Shi (Harvard Medical School, Boston). pGEX6P1 hKDM5A JmjN-ARID (aa 19-197) Human KDM5A fragments were amplified using the full-length hKDM5A plasmid as template (see above) using primers (forw: CTC GAG GAG TGC CCC GTC TTT GAG, rev: CTC GAG AAC CTC AGG CTC CAC TTT TT). The resulting fragments were cut with XhoI and ligated into pGEX6P1 (GE healthcare). pGEX6P1 hKDM5A PHD1 (aa 246-348) Cloned from full length hKDM5A via ApaI/XhoI cutting sites into pGEX6P1 pGEX6P1 hKDM5A JmjC-ZF (aa 439-861) Cloned from full length hKDM5A by PCR (forw: GAA TTC GCA CTT TCT GGT TGG AAT TTG, rev: CTC GAG TCC TGA GCA CGT TCA TGA AA) via EcoRI/XhoI cutting sites into pGEX6P1. pGEX6P1 hKDM5A PHD2 (aa 1080-1289) Cloned from full length hKDM5A by PCR (forw: GAA TTC ATT GGT GTA TAT GGG AGT GGC, rev: TTG GAG TTC TGC ACT GAT GAT) via EcoRI/XhoI cutting sites into pGEX6P1. pGEX6P1 hKDM5A Inter (aa 1289-1539): 1. Step: Region containing PHD1 and Inter were cloned from full length hKDM5A by PCR (forw: AGC CAT GCA TTC TCT CAG, rev: AAA GGA TCC CTA TTT TAA TTT CTT CTT TCT AGG) via BamHI into pGEX6P1 leading to pGEX6P1 hKDM5A PHD1-Inter. 2. Step: Inter were cloned from pGEX6P1 hKDM5A PHD1-Inter via XhoI/XhoI into the SalI cutting site in pGEX6P1. pGEX6P1 hKDM5A PHD3 (aa 1512-1689) Cloned from full length hKDM5A by PCR (forw: GAA TTC GTA GAG CAA CTT TTT GGA GAA GG, rev: CTC GAG GGT CTC TTT AAG ATC CTC CAT TGG) via EcoRI/XhoI cutting sites into pGEX6P1. RBP-J Vectors: pcDNA 3.1 Flag RBP-J NTD (aa 1-165 from mRBP-J isoform 2) Cloned from full length mRBP-J by PCR (forw: AAC TCG AGA TGG CGC CTG TTG, rev: GGT CTA GAT TAG TCA GCA TTT TTC AAT GAC TGC) via XhoI/XbaI cutting sites into pcDNA3.1 Flag1 (Invitrogen).

    Liefke et al. 3

pcDNA 3.1 Flag RBP-J BTD (aa 166-315 from mRBP-J isoform 2) Cloned from full length mRBP-J by PCR (forw: AAC TCG AGT TAT GCA TTG CCT CAG G, rev: GGT CTA GAT TAC CAG GAA GCG CCA) via XhoI/XbaI cutting sites into pcDNA3.1 Flag1. pcDNA 3.1 Flag RBP-J CTD (aa 316-486 from mRBP-J isoform 2) Cloned from full length mRBP-J by PCR (forw: AAC TCG AGA CAA TCA TTA GCA CAG ATA AGG, rev: GGT CTA GAT TAT ACC ACT GTG GCT GTA G) via XhoI/XbaI cutting sites into pcDNA3.1 Flag1.

Su(H) and Lid Vectors pcDNA 3.1 Flag dmLid 1. Step: N-terminal dmLid was cloned from pOT2dmLID (obtained from the Drosophila Genomics Research Center, Indiana University, [LD40310]) by PCR (forw: ATG AAT TCT CCG CCA AAA CTG AGG CGG, rev: GAT CAA GGA AGT TGA GTT TGA CGC) via EcoRI/XhoI cutting sites into pcDNA3.1 Flag1, leading to pcDNA3.1 Flag1 dmLid-N. 2. Step: C-terminal dmLid was cloned from pOT2dmLID via XhoI cutting sites into pcDNA3.1 Flag1 dmLid-N, resulting in pcDNA 3.1 Flag dmLid. pGEX6P1-dmSu(H) Cloned from pOT2_dmSu(H) (obtained from the Drosophila Genomics Research Center, Indiana University, [GH10914]) by PCR (forw: ATG AAT TCA AGA GCT ACA GCC AAT TTA ATT TAA ACG, rev: ATC TCG AGT CAG GAT AAG CCG CTA CCA TG) via EcoRI/XhoI cuttings sites into pGEX6P1. MIGR dn-MAML-ER IRES GFP and MIGR Notch-IC-ER IRES GFP Plasmid information is available upon request. All constructs were verified by sequencing.  Luciferase Reporter Vectors

The luciferase reporters HES1-Luc and pGA981/6 were described previously (Oswald

et al. 2002). 

Supplementary references in Materials and Methods: 1. Oswald, F. et al. SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway.

Embo J 21, 5417-5426 (2002).

    Liefke et al. 4

Supplementary Figures:

Figure S1: H3K4 trimethylation at Notch target gene Deltex-1 upon induction of

dominant-negative Mastermind: Pre-T-cell line Beko were infected with an

inducible form of dominant-negative Mastermind fused to the estrogen-receptor

ligand binding domain (dn-MAML-ER). Chromatin-immunoprecipitation (ChIP)

experiments using anti-H3K4me3 antibodies (no antibody as mock control) were

performed in the absence (A) or after 24h in presence of tamoxifen (B). H3K4

trimethylation was specifically lost at the RBP-J binding site (-1.2kb upstream, red

asterisk) but not at the transcriptional start site (TSS). H3K4me3 is significantly

enriched with P < 0.05 (Student’s t-test) at the RBP-J -binding site in untreated cells

and at the TSS in both treated and untreated cells.

    Liefke et al. 5

Figure S2: Notch-IC induction in the presence of gamma-secretase inhibitor

(GSI): Pre-T-cell line Beko were infected with an inducible form of Notch

intracellular domain fused to the estrogen-receptor ligand binding domain (Notch-IC-

ER). A) Schematic representation of the experiment: The cells were treated with GSI

for 24 hours. Then the cells were split in two parts and either treated with tamoxifen

for additional 24 hours (without removal of GSI) or kept untouched. Samples for

ChIP were taken before adding GSI (0h), after 24h GSI (24h) and after 48 GSI with or

without treatment with tamoxifen for 24 hours (48h, 48h+Tam) B) In RT-PCR

experiments, expression levels of several Notch target genes were measured at each

timepoint. C) Chromatin-immunoprecipitation (ChIP) experiments using anti-

H3K4me3 antibodies (no antibody as mock control) at the RBP-J binding site (C) at

the transcriptional start site (D) of Deltex-1. The reduced expression and H3K4

trimethylation at the RBP-J binding site after GSI treatment were re-established after

induction of Notch-IC-ER with tamoxifen. (* P < 0.05, Student’s t-test)

    Liefke et al. 6

Figure S3: H3K4 trimethylation at the RBP-J binding site of Hes-1 and CD25 is

Notch-dependent:

A) ChIP experiments in untreated Beko cells using antibodies against H3K4me3 (no

antibody as mock control) were performed to scan the Hes-1 promoter. The sequences

of the RBP-J binding sites information can be found in Fig. S10. There were two

peaks, one at -1kb and the other one downstream of the transcriptional start site.

H3K4me3 was significantly enriched P < 0.05 at the CGIs and downstream of the

RBP-J binding site (P < 0.05, Student’s t-test).

B) The same promoter scan as in A) was performed using Beko cells treated with γ-

secretase inhibitor (GSI) for 24 hours. The H3K4me3 at the RBP-J binding site was

abolished, but retained at the CpG islands indicated by the black bars. After GSI

treatment H3K4me3 was still significantly enriched at the CGIs (P < 0.05, Student’s

t-test).

C-F) H3K4me3 at the conserved RBP-J binding site of CD25 (-26 kb) was abolished

after GSI treatment (C). No H3K4me3 was found at the TSS of CD25 (D). At the

preTα locus H3K4me3 was significantly reduced after GSI treatment at the promoter

(F) but no H3K4 trimethylation detectable at the conserved RBP-J site at the enhancer

(E). CD25 and preTα do not contain a CpG island. (* P < 0.05, Student’s t-test) The

values in A-F are mean ± s.d. for triplicate samples from a representative experiment.

    Liefke et al. 7

    Liefke et al. 8

Figure S4: H3K4 trimethylation at Notch target genes in primary pre-T-cells:

Chromatin immunoprecipitation with anti-H3K4me3 antibodies (no antibody as mock

control) was performed on primary CD4-/CD8- precursor T-cells from RAG1 knock-

out mice (as shown in Figure 2B for Deltex-1). A) The profile for the Hes-1 promoter

region was similar to the one observed in Beko cells (Fig. S3A). B) For preTα

H3K4me3 was detectable at the promoter. No enrichment was found at CD25.

    Liefke et al. 9

Figure S5: DNA affinity purification with mutated RBP-J sites as bait: The same

purification as in Figure 3D-F) was performed; a DNA fragment containing 12

mutated RBP-J binding sites was biotinylated and immobilized on a streptavidin-

Sepharose column. The column was incubated with whole-cell lysates from Beko

cells. After incubation and washing, the DNA-bound RBP-J complexes were eluted

with increasing concentrations of NaCl and analyzed by EMSA (S5A). Western blot

analysis (WB) of the lysate, the washing steps and the eluted fractions (E1-E4) were

performed using antibodies against RBP-J (Fig. S5B), KDM5A (Fig. S5C) and

control NF-κB, p65-RelA (Fig. S5D). RBP-J, KDM5A and p65 were not detected in

the elution fractions.

    Liefke et al. 10

Figure S6: Constructs used for GST-pulldown assays:

A) Schematic representation of constructs used in experiments presented in Figure

3G and H and a representative Coomassie gel (B) of the GST-fusion proteins

immobilized to Glutathione-Sepharose. C) Schematic representation of constructs

used in experiments presented in Figure 3H. Molecular weights are indicated at the

left-hand side.

    Liefke et al. 11

Figure S7: ChIP analysis with an α−p300 antibody and anti-H3K9ac at the

Deltex-1 promoter: A) ChIP analysis with an α−p300 antibody (black bars) or mock

(no antibody, white bars). p300 was significantly enriched around the RBP-J binding

site (-1.2 kb). Analysis was performed before GSI treatment (see also Fig. 3K). B)

ChIP time course experiment with α−p300-specific antibody at 0, 2, 4, 6, 8 and 24

hours after GSI treatment. p300-occupancy at the RBP-J binding site was erased upon

GSI treatment. C) ChIP experiments using anti-H3K9ac antibodies (no antibody as

mock control) showed that the level of this active mark at the Deltex-1 promoter was

reduced after GSI-treatment. (* P < 0.05, Student’s t-test).

    Liefke et al. 12

    Liefke et al. 13

Figure S8: KDM5A alters H3K4me3 on a Hes-1-specific reporter plasmid:

Chromatin-immunoprecipitation assays were performed using anti-H3K4me3

antibodies (no antibody as mock control) at the promoter of the human Hes-1-specific

reporter construct (HES-1-Luc, primers at TSS were used for analysis). Upon

expression of RBP-VP16 H3K4 trimethylation was found enriched. This enrichment

was not observed after coexpression of RBP-VP16 and KDM5A.

    Liefke et al. 14

Figure S9: Su(H) mutations rescue eye growth defect and enhance

tumorigenesis by Notch signaling. (A-C) Representative adult female eyes of wild-

type (a), ey-Gal4>fng/CyO (c) and ey-Gal4>fng/Su(H)2. Animal in B is a sibling of

the cross generating the animal in C. (D) Graph represents the quantification of adult

fly eye tumours and metastasis comparing control tumour, as in Fig. 5H, crossed to

wild type flies and the tumour phenotype in the presence of a mutant copy of

independently generated Su(H) alleles. Bars shown represent the percentage of eyes

with tumor (red), overgrowth (yellow), or undergrowth (blue) in F1 progeny n=597

eyes in tumor [control], n=118 [Su(H)], as in as in Fig. 5H; and n=36 [Su(H)1] and

n=130 in [Su(H)del47]. (E) Depleting lid levels by RNAi (two transgenes were

assayed, lid 9088R-1 and 9088R-2 from NIG-Fly Stock Center, University, Japan) or

by reducing dose of an independently generated P-element mutation lid (lidK06801/+)

similarly increased the frequency of animals with tumor overgrowth and metastases

of the Notch gain-of-function. Control cross of the tumor phenotype (ey-

Gal4>Dl>eyeful, as shown in Fig. 5H) with wt lid activity and effects of lid10424

mutation are shown for comparison. In all cases the transgenes were expressed in the

developing eye imaginal discs using the eye-specific driver ey-Gal4 and UAS-RNAi

lid (lid 9088R-1 and 9088R-2) transgenes.

    Liefke et al. 15

Figure S10: Aligned human and mouse genomic DNA sequences including the

conserved RBP-J sites at Notch target genes Deltex-1, Hes-1, CD25 and preTα:

The RBP-J binding sites are marked in blue.

    Liefke et al. 16

Table S1

Cells Beko Beko + dn-MAML-ER

Name Ratio GSI/DMSO Ratio 4-OHT treated/untreated ACTB 1.18 1.14 Adam10 1.22 1.57 Adam17 1.16 1.53 AES 2.15 2.92 Axin 1.26 0.95 CBL 1.42 1.27 CCND1 1.64 2.16 CCNE1 1.39 1.49 CD44 1.14 1.76 CDC16 0.93 1.44 Cdkn1a 3.65 4.49 CFLAR 1.12 1.54 CHUK 0.87 1.58 CTNNB1 0.88 0.96 DLL1 no signal no signal DTX1 0.10 0.11 EP300 1.47 1.48 ERBB2 no signal no signal FIGF 0.96 2.30 FOS 2.15 1.01 FOSL1 0.74 1.11 FZD1 no signal no signal FZD2 no signal no signal FZD3 no signal no signal FZD4 no signal no signal FZD5 0.60 0.70 FZD6 no signal no signal FZD7 0.83 0.67 Gapdh 0.89 0.81 GBP2 1.70 2.50 GLI1 0.82 1.83 GSK3B 1.29 1.98 Gusb 0.95 1.06 HES1 0.18 0.13 HEY1 no signal no signal HOXB4 1.22 1.55 Hprt1 0.85 1.06 HR 1.07 0.99 HSP90ab1 1.17 1.05 Ifng no signal no signal IL17B no signal no signal IL2ra 0.20 0.74 IL6st 1.53 1.43 JAG1 1.49 2.18 JAG2 1.53 1.07 KRT1 no signal no signal LFNG no signal no signal LMO2 no signal no signal LOR no signal no signal LRP5 1.48 0.97 MAP2K7 1.18 1.22 MFNG 0.79 1.28 MMP7 no signal no signal MTAP1b 0.92 0.69 MYCL1 no signal no signal NCOR2 1.66 1.70 NEURL 0.95 0.71 Nfkb1 1.38 1.39 NFKB2 0.81 1.12 Notch1 0.68 0.62

    Liefke et al. 17

Notch2 1.47 1.00 Notch3 1.01 0.76 Notch4 1.19 1.82 NR4A2 no signal no signal Numb 0.91 1.40 PAX5 no signal no signal PCAF 1.21 1.79 PDPK1 1.15 1.81 POFUT1 1.27 0.98 PPARG no signal no signal PSEN1 1.31 1.04 PSEN2 1.06 2.42 PSENEN 1.25 1.84 PTCRA 0.39 0.74 RFNG 1.21 0.95 RUNX1 1.13 1.01 SEL1L 1.17 1.65 SHH 0.44 0.82 SMO 1.06 1.14 SNW1 0.93 1.31 Stat6 1.10 1.65 Stil 1.26 1.52 SUFU 1.22 0.91 SUPT6H 1.27 1.22 TEAD1 no signal no signal TLE1 no signal no signal WISP1 1.27 1.68 WNT11 no signal no signal ZIC2 no signal no signal

    Liefke et al. 18

Table S2 Su(H)—/+ lid—/lid— animals display phenotypes typical of Notch gain-of-function

bristles wing size wing vein

lidK06801/ lid10424 wt wt wt Su(H)2 lid10424/lidK06801 wt wt 10% showed vein loss Su(H)D47 lid10424/lidK06801 wt wt wt Su(H)D47 lid10424/lid10424 bristle loss and thinner smaller 50% showed vein loss

Animals homozygous for Su(H) mutations do not eclose and reducing one gene dosage of Su(H) has no phenotypical consequences. Homozygous lid flies that eclose (see Table S2) show wild type (wt) wing size and vein pattern and normal bristles. Reducing dose of Su(H) in a lid homozygous background in distinct allelic combinations produces Notch gain-of-function phenotypes such as thinner bristles and loss of notal bristle, wing vein loss and reduced wing size (see also Fig. 5). Table S3 lid and Su(H) genetically interact

Nº of adult progeny a

+ lidK06801/CyO 212 lidK06801/ lid10424 18

Su(H)2 lid10424/CyO 172 Su(H)2 lid10424/lidK06801 20

Su(H)del47 lid10424/CyO 109Su(H)del47 lid10424/lidK06801 13

+ lid10424/lid10424 0 Su(H)del47 lid10424/Cy0 159 Su(H)del47 lid10424/lid10424 5

a Number of adult flies of the genotype shown from the cross of individual lid/CyO-twist>GFP females to males double mutant for Su(H) and lid over CyO-twist>GFP. Previous report showed that control strong hypomorphic allelic combination of lidK06801/lid10424 flies eclose at a 50% frequency of the expected by Mendelian genetics. However, we observed significantly fewer than the expected frequency. We also found that homozygous lid10424 flies did not eclose. In all cases the introduction of a mutant copy of Su(H) increased the viability of homozygous lid mutations from a 17% in lidK06801/ lid10424 to 23,25% in the genotype Su(H)2 lid10424/lidK06801. Expected frequencies were calculated in each cross using as reference the scored frequency of CyO sibblings. We do not rule out that the increased frequency of homozygous lid animals eclosing is not due to the loss of secondary modifiers during the recombination events to generate these double mutant chromosomes.