Supplemental Materials Include

(1) Supplemental Methods

(2) References used in Supplemental Materials

(3) Legends to Supplemental Figures

(4) Supplemental Figures 1-7

(5) Supplemental Tables 1-6

Supplemental Methods

In Vitro Methyltransferase Assay. In vitro methyltransferase activity assays were

carried out with the recombinant methyltransferases (full-length of SETD2 or NSD1-3)

or complexes (a trimeric EZH2-EED-SUZ12 complex of PRC2) by monitoring the tritium

incorporation from S-adenosylmethionine (3H-SAM) to the peptide substrates as

previously described(1).

Plasmid. MSCV retroviral expression vectors of MLL-AF9 and MLL-ENL were used as

described before(2). EZH1 construct was a gift from Dr. Vittorio Sartorelli.

Antibodies. Antibodies used in immunoblot include those against H3K27me3 (Millipore

07-449 and Abcam 6002, 1:5,000), H3K27me2 (Millipore 07-452, 1:5,000), H3K27me1

(Active Motif 39377, 1:5000), H3K27ac (Abcam 4729, 1:2,000), H3K4me3 (Abcam 8580

and Active Motif 39159, 1:5,000), H3K36me3 (Abcam 9095, 1:5,000), H3K36me1

(ab9048, 1:1,000); H3K36me2 (Active Motif 39255, 1:1,000), general H3 (Abcam 1791,

1:10,000), EZH2 (BD Biosciences 612666, 1:1,000), SUZ12 (Abcam 12073, 1:1,000),

and Tubulin (Sigma, 1:10,000). The antibodies used for ChIP-Seq are H3K27me3

(Millipore 07-449), H3K27ac (Abcam 4729) and SUZ12 (Abcam 12073).

Flow Cytometry Analysis. Single-cell suspension prepared from tissue culture or the

mouse bone marrow and spleen was stained with the diluted antibodies and analyzed

with a Beckman-Coulter CyAn ADP flow cytometer as previously described(2, 3). The

used antibodies include those against c-Kit (FITC, BD Pharmingen 553354), Gr-1 (PE,

BD Pharmingen 553128), Mac-1/Cd11b (APC, BD Pharmingen 557686), B220 (PE, BD

Pharmingen 553090), Ter119 (Biotin, eBioscience 13-5921), CD3e (Biotin, eBioscience

13-0031), and H3K27me3 (Cell Signaling 9733BF, custom-made and dissolved in PBS

instead of Tris buffer, followed by in-house conjugation to AlexaFlour-488 or AlexaFlour-

647 with AlexaFlour Antibody Labeling Kit [Life Technologies]). For the biotinylated

primary antibodies, we used an anti-Biotin antibody (PE, Miltenyi 130-090-756) for flow

analysis. All antibodies were used at 1:100–1:300 dilutions. Profiles of flow cytometry

were analyzed by the Summit 4.2 software according to manufacturer’s specification.  

Assays of Cell Proliferation, Wright-Giemsa Staining and Apoptosis. All of the

used human cell lines were purchased from the ATCC or DSMZ Cell Bank and cultured

as recommended. Approximately 20,000 of cells were plated in triplets in the presence

of various concentrations of compounds at day 1, and the numbers of viable cells were

counted using a TC20 automated cell counter (BioRad) every 3-4 days. Medium

containing the compounds was refreshed every 2-3 days, and the cell concentration

was kept under 1-2 million per mL as recommended. Wright-Giemsa staining was

performed as previously described(4). Cell apoptotic assay was performed using the

Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling). Images of cell staining

were captured with an EVOS-XL Cell Imaging System (Life Technologies).

Colony Formation Unit (CFU) Assay. Approximately 200-500 leukemia progenitor

cells were initially plated in replicates in the methylcellulose medium (MethoCult™ GF

M3434; StemCell Technologies) pre-added with DMSO or compounds. For serial re-

plating, approximately 5,000-10,000 cells isolated from colonies in the previous plating

were seeded again in the same semi-solid medium. CFUs were scored by direct

quantification every 7-9 days post seeding. Identity of each colony was defined by

morphology according to manufacturer’s specification.

Cell Cycle Profiling. Exponentially dividing cells were seeded in the medium containing

either DMSO or compounds. A million of mock-treated versus compound-treated cells

were washed with ice-cold PBS and fixed in pre-chilled methanol (80%), followed by

staining with PBS added with 20 µg/ml propidium iodide (Sigma), 0.1% Triton-X100, and

200 µg/ml RNase A (Roche). DNA contents of cells were then detected with a CyAn

ADP flow cytometer (Beckman-Coulter), and then analyzed by ModFit Software (Verity

Software House).

Gene Knockdown. miR30-based shRNAs targeting EED (clone 1820) and Renilla

(control) were kindly provided by Dr. Chris Vakoc(5) and then sub-cloned into sites of

XhoI and EcoRI of an inducible shRNA expression system, TRMVP-IR(6) (pSIN-TRE-

dsRed-miR30-PGK-Venus-IRES-rtTA3, Addgene 27994). Cells were infected with

TRMPVIR virus and sorted based on Venus+ signals, following by treatment with

doxycycline (Sigma-Aldrich) at a final concentration of 2 µg/ml to induce shRNA

expression. Gene knockdown efficiency was determined by real time PCR.

Real-Time PCR. Reverse transcription was performed with random primers and the

cDNA Reverse Transcription kit according to the manufacturers protocols (Invitrogen

and Biorad). PCR amplicons (usually 80–150 bp) were designed to span over introns,

and quantitative PCR was performed in triplicate using the SYBR Green master mix

reagent on an ABI 7900 qPCR system (Applied Biosystems). The real-time PCR signals

are typically examined in triplicates and normalized to those of the β-Actin or Gapdh

gene.

ChIP Followed by Quantitative PCR (ChIP-qPCR). ChIP was performed using a

previously described protocol(3). ChIP signals were represented as a percentage of

input chromatin, and fold of enrichment was calculated by normalizing against signals of

nonspecific IgG.

ChIP-sequencing (ChIP-Seq) and Data Analysis. Briefly, all sequencing reads were

mapped to the mouse genome (mm9) using the BWA alignment software(7), and

unique reads mapped to a single best-matching location with no more than two

mismatches were kept, which were then subject to removal of reads generated by PCR-

caused duplicates using the Picard and Samtools. Profiles of ChIP-Seq read densities

were displayed in the Integrative Genomics Browser (IGB, Affymertrix) or Integrative

Genomics Viewer (IGV, Broad Institute). The MACS software(8) was used for peak

identification with data from input as controls and default parameters. In-house scripts

were used to assign peaks to promoter proximal (±2kb of transcription start site, TSS),

promoter distal (-50kb to -2kb of TSS), or gene body (+2kb of TSS to +2kb of

transcription end site), using the mouse RefSeq annotation as reference. In-house

scripts were also used to compute and compare ChIP-Seq reads across samples. The

CpG island annotation from the UCSC browser was used to associate H3K27me3

peaks to CpG islands. In all analyses, 1-bp intersection was considered as peak

overlapping, and unless specified otherwise, the peaks from mock treatment were used

as the base peaks for comparing data between mock and compound treatments. To

generate heatmaps of ChIP-Seq densities, reads from ChIP-Seq and input sequencing

were computed using a 100-bp window across the TSS (-10kb to +10Kb of TSS). After

normalization against the sequencing depths and the subtraction of the input from the

ChIP-Seq signals, the obtained data were used to generate heatmaps.

In Vivo Leukemia Assay and Compound Treatment Studies. The murine model of

MLL-rearranged leukemia was generated by retroviral delivery of MLL-AF9-IRES-GFP

into lineage-negative bone marrow stem/progenitor cells, followed by bone marrow

transplantation as previously described(2, 3). To generate the secondary leukemia, 2

million leukemia cells isolated from bone marrow were suspended in 200µl of sterile

PBS and intravenously introduced to the sub-lethally irradiated (300 Rads) syngeneic

Balb/c mice (12-week-old) by tail-vein injection. Leukemic development was assessed

by complete blood counting (CBC) of peripheral blood (HemaVet ® 950 LV CBC, Drew

Scientific Group) and treatment with UNC1999 was commenced on the day when the

counts in white blood cells (WBC) were found significantly elevated. The powder of

UNC1999 (verified by HPLC and mass spectrometry) was slowly dissolved and

incorporated to vehicle (0.5% of sodium carboxymethylcellulose [NaCMC] and 0.1% of

Tween-80 in sterile water) with continuous trituration by a pestle and mortar. After

transfer to a conical tube, the UNC1999-vehicle mixture was subject to vigorous vortex

and brief sonication to achieve a homogenous suspension at a formulation

concentration of 20 mg/mL. UNC1999 or vehicle control was administered orally twice

daily [BID] at a dose of 50 mg/kg. Mice were inspected daily, and complete blood counts

measured twice per week; mice exhibiting a terminal leukemia phenotype (lethargy,

splenomegaly and limb paralysis) were sacrificed, and cells isolated from leukemic

tissues were subjected to further pathological analysis as described before(2, 3).

All procedures pertaining to animal handling, care, and treatment were performed

according to guidelines approved by the Institutional Animal Care and Use Committee

(IACUC) of the University of North Carolina at Chapel Hill.

Chemistry Synthesis, Purification and Verification. HPLC spectra for all compounds

were acquired using an Agilent 6110 Series system with UV detector set to 254 nm.

Samples were injected (5 µL) onto an Agilent Eclipse Plus 4.6 x 50 mm, 1.8 µM, C18

column at room temperature. Method 1: A linear gradient from 10% to 100% B (MeOH +

0.1% acetic acid) in 5.0 min was followed by pumping 100% B for another 2 minutes

with A being H2O + 0.1% acetic acid. Method 2: A linear gradient from 50% to 100% B

(MeOH + 0.1% acetic acid) in 5.0 min was followed by pumping 100% B for another 2

minutes with A being H2O + 0.1% acetic acid. The flow rate was 1.0 mL/min. Mass

spectra (MS) data were acquired in positive ion mode using an Agilent 6110 single

quadrupole mass spectrometer with an electrospray ionization (ESI) source. Nuclear

Magnetic Resonance (NMR) spectra were recorded at Varian Mercury spectrometer

with 400 MHz for proton (1H NMR) and 100 MHz for carbon (13C NMR); chemical shifts

are reported in ppm (δ). Preparative HPLC was performed on Agilent Prep 1200 series

with UV detector set to 254 nm. Samples were injected onto a Phenomenex Luna 75 x

30 mm, 5 µΜ, C18 column at room temperature. The flow rate was 30 mL/min. A linear

gradient was used with 10% (or 50%) of MeOH (A) in 0.1 % TFA in H2O (B) to 100% of

MeOH (A). HPLC was used to establish the purity of target compounds, all compounds

had > 95% purity using the HPLC methods described above unless noted otherwise.

High-resolution (positive ion) mass spectrum (HRMS) for compound UNC1999 was

acquired using a Thermo LTqFT mass spectrometer under FT control at 100000

resolution.

Chemical Docking Studies. Docking of UNC1999 into the EZH2 apo crystal structure

(PDB: 4MI0) was performed using the Schrodinger Suite, release 2013-1-Maestro,

version 9.4 (Schrödinger, New York, NY). Specifically, glide standard precision docking

was used to obtain an initial conformation before selecting poses for energy

minimization. MacroModel was used to minimize the selected structures using the

OPLS-2005 force field and PRCG method of minimization as described before(9).

Scheme of Chemistry Synthesis used in the study

NN

OHO

Br

NOHNO

NN

Br N

B

NNBoc

OO

NNN

Boc

NN

HNO

NO

NNN

NN

HNO

NO

HNO

H2N

HNO

HN

Boc

HNO

HN

UNC1756 Route

UNC3142 Route

HNO

HNO

NN

Br

HNO

NO

NN

Br

HNO

NO

NN

Br N

B

NNBoc

OO

NNN

Boc

NN

NO

HNO

NNN

NN

NO

HNO

UNC3142

UNC1756

(a) (b) (c), (d)

(e) (f) (g)

(h) (i), (j)

Reagents and Conditions: (a) K2CO3, CH3-I, DMF, rt, 95%; (b) Pd(PPh3)4, K2CO3,

H2O:Dioxane, µ-wave, 150 0C, 60%; (c) TFA:DCM, rt; (d) Acetone, acetic

acid,NaBH3CN, MeOH, 0 0C à rt, 58%; (e) Di-tert-butyl dicarbonate, THF, rt, 97%; (f)

LAH, THF, reflux, 67%; (g) EDCI, HOBt, TEA, DMF, rt, 44%; (h) Pd(dppf)Cl2·DCM,

K2CO3, H2O:Dioxane, reflux, 76%; (i) TFA:DCM, reflux; (j) Acetone, AcOH, NaBH3CN,

MeOH, 0 0C à rt, 53%.

6-bromo-N-((1,6-dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1-

isopropyl-1H-indazole-4-carboxamide. 6-bromo-1-isopropyl-N-((6-methyl-2-oxo-4-

propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide, prepared as

previously reported (10) (.100 g, .225 mmol) was dissolved in DMF (1 mL) and K2CO3

(.080 g, .579 mmol) was added to the reaction. The contents were stirred at RT for ~15

minutes before adding CH3-I (.050 mL, .803 mmol). The contents were then stirred at

RT overnight. After completion of the reaction the mixture was diluted with MeOH and

purified using HPLC to afford 6-bromo-N-((1,6-dimethyl-2-oxo-4-propyl-1,2-

dihydropyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (.096 g, .209 mmol,

93.2%) as a yellow solid. 1H NMR (400 MHz, cd3od) δ 8.33 (s, 1H), 8.02 (s, 1H), 7.62 (s,

1H), 6.23 (s, 1H), 4.95 (dt, J = 10.3, 6.6 Hz, 1H), 4.57 (s, 1H), 3.58 (s, 2H), 2.81 – 2.63

(m, 1H), 2.41 (s, 2H), 1.76 – 1.45 (m, 4H), 1.01 (t, J = 7.4 Hz, 2H). HPLC (method 1):

95%: tR = 5.850 min: MS (ESI): 459 [M+H]+.

NNN

Boc

NN

HNO

NO

tert-butyl 4-(5-(4-(((1,6-dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-

yl)methyl)carbamoyl)-1-isopropyl-1H-indazol-6-yl)pyridin-2-yl)piperazine-1-

carboxylate. 6-bromo-N-((1,6-dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-

1-isopropyl-1H-indazole-4-carboxamide, (.096 g, .209 mmol) was dissolved in 2 mL

H2O:Dioxane (1:5) in a microwave vessel, and then added tert-butyl 4-(5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-carboxylate, prepared as

previously reported[2], (.1 g, .256 mmol), K2CO3 (.088 g, .639 mmol), and

Tetrakis(triphenylphosphine)palladium(0) (.05 eq, ~2 mg). The mixture was then placed

in a microwave reactor for 20 minutes at 150 0C. The crude product was separated

using Brine:EA, washing 3x with EA. The organic layers were combined, dried with

Na2SO4, concentrated in vacuo, and purified using HPLC to yield tert-butyl 4-(5-(4-(((1,6-

dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-1-isopropyl-1H-

indazol-6-yl)pyridin-2-yl)piperazine-1-carboxylate as a white powder (.082 g, .128 mmol,

60.1%). 1H NMR (400 MHz, cd3od) δ 8.46 – 8.30 (m, 1H), 8.09 – 7.98 (m, 1H), 7.84 –

7.69 (m, 1H), 7.45 – 7.30 (m, 1H), 6.32 – 6.18 (m, 1H), 5.19 – 4.97 (m, 1H), 4.61 (s,

1H), 3.85 – 3.63 (m, 4H), 3.57 (s, 2H), 2.81 – 2.67 (m, 1H), 2.41 (s, 2H), 1.70 – 1.54 (m,

4H), 1.51 (s, 4H), 1.02 (t, J = 7.3 Hz, 2H). HPLC (method 1): 95%: tR = 6.176 min: MS

(ESI): 642 [M+H]+.

N-((1,6-dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(6-

(4-isopropylpiperazin-1-yl)pyridin-3-yl)-1H-indazole-4-carboxamide (UNC3142).

tert-butyl 4-(5-(4-(((1,6-dimethyl-2-oxo-4-propyl-1,2-dihydropyridin-3-

yl)methyl)carbamoyl)-1-isopropyl-1H-indazol-6-yl)pyridin-2-yl)piperazine-1-carboxylate

(.040 g, .062 mmol) was dissolved in DCM (0.25 mL) and added TFA (0.25 mL). The

reaction was stirred for ~2 hours until complete. Then, the contents were concentrated

in vacuo, dissolved in MeOH (0.40 mL), and placed in an ice bath. Acetone (.058 mL,

0.79 mmol) and acetic acid (.045 mL, 0.79 mmol) were then added to the reaction. After

cooling to 0 0C, NaBH3CN (.025 g, 0.40 mmol) was added and the reaction was stirred

for 15 minutes. Then the ice bath was removed and the contents were allowed to warm

to RT overnight. After completion, the MeOH was removed in vacuo and the crude

product was isolated by separating with EA:Brine, 3x EA washes. The organic layers

were combined, dried with Na2SO4, and purified using HPLC to give N-((1,6-dimethyl-2-

oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(6-(4-isopropylpiperazin-1-

yl)pyridin-3-yl)-1H-indazole-4-carboxamide as a brown oil (.021 g, .036 mmol, 58.1%).

1H NMR (400 MHz, cdcl3) δ 8.49 (d, J = 2.2 Hz, 1H), 8.39 (s, 1H), 7.80 (td, J = 8.8, 4.2

Hz, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 6.73 (d, J = 8.8 Hz, 1H), 5.97 (s, 1H), 4.88 (s, 1H),

4.65 (d, J = 5.9 Hz, 1H), 3.68 (s, 2H), 3.53 (s, 2H), 3.04 – 2.45 (m, 4H), 2.32 (s, 2H),

1.72 – 1.52 (m, 5H), 1.25 (s, 3H), 1.14 (d, J = 6.0 Hz, 3H), 1.02 (s, 2H). HPLC (method

1): 95%: tR = 4.635 min: MS (ESI): 585 [M+H]+.

tert-butyl ((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)carbamate. 3-

(aminomethyl)-6-methyl-4-propylpyridin-2(1H)-one (1.00 g, 5.60 mmol), prepared as

previously reported(10), was dissolved in THF (20.0 mL) and added di-tert-butyl

dicarbonate (1.22 g, 5.60 mmol). The mixture was then stirred at RT for 4 hours. After

completion, the contents were separated with ethyl acetate (3X) and brine. The organic

layers were combined, dried with Na2SO4, and concentrated in vacuo. The crude

product was then used for the next step (1.50 g, 97%).

6-methyl-3-((methylamino)methyl)-4-propylpyridin-2(1H)-one. tert-butyl ((6-methyl-

2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)carbamate (0.70 g, 2.50 mmol) was

dissolved in THF (50.0 mL) and a solution of LAH (0.95 mg, 25.0 mmol) in THF (50.0

mL) was added to the mixture dropwise; the mixture was heated to reflux for 4 hours.

After completion, the contents were cooled to 0 0C, added 0.5 N NaOH, and extracted

with ethyl acetate 3x. The organic layers were combined, dried with Na2SO4, and

combined to give 6-methyl-3-((methylamino)methyl)-4-propylpyridin-2(1H)-one (.332 g,

67% yield) as a yellow oil. 1H NMR (300 MHz, cdcl3) δ 5.891 (s, 1H), 3.659 (s, 2H),

2.520-2.494 (m, 2H), 2.418 (s, 3H), 2.256(s, 3H), 1.565-1.514 (m, 2H), 0.980-0.931 (t,

3H, J=7.2 Hz).

6-bromo-1-isopropyl-N-methyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-

yl)methyl)-1H-indazole-4-carboxamide. 6-methyl-3-((methylamino)methyl)-4-

propylpyridin-2(1H)-one (.232 g, 1.18 mmol) was dissolved in DMF (10.0 mL) and

added 6-bromo-1-isopropyl-1H-indazole-4-carboxylic acid (.330 g, 1.18 mmol), prepared

as previously reported(10), EDCI (.338 g, 1.77 mmol), HOBt (.239 g, 1.77 mmol) and

TEA (0.49 mL). The reaction was then stirred at room temperature overnight. The

contents were diluted with H2O, stirred for 1 hour, and then filtered to collect a yellow

solid. The product was then washed with H2O and DEE to give 6-bromo-1-isopropyl-N-

methyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-

carboxamide (.230 g, 44%) as a white solid. 1H NMR (300 MHz, cdcl3), (Rotamers), δ

13.137 (m, 1H), (8.072, 7.945) (s, 1H), (7.634, 7.489) (s, 1H), 7.275-7.270 (m, 1H),

5.999(s. 1H), 4.766-4.743(m, 2H), 2.905(s, 3H), 2.712-2.661 (t, 2H, J=7.6 Hz), 2.15 (s,

3H, ), 1.552(m, 8H), 1.042-0.993 (t, 3H J=7.4 Hz).

tert-butyl 4-(5-(1-isopropyl-4-(methyl((6-methyl-2-oxo-4-propyl-1,2-

dihydropyridin-3-yl)methyl)carbamoyl)-1H-indazol-6-yl)pyridin-2-yl)piperazine-1-

carboxylate. 6-bromo-1-isopropyl-N-methyl-N-((6-methyl-2-oxo-4-propyl-1,2-

dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide (.152 g, 0.33 mmol) was

dissolved in a mixture of 5 mL H2O:Dioxane (1:5) and then tert-butyl 4-(5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-carboxylate (.142 g, .364

mmol), prepared as previously reported (1), and KOAc (.098 g, 1.00 mmol) were added

to the flask. The mixture was purged and placed under argon atmosphere and added

1,1'-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex

(.027 g, .033 mmol), and the reaction was heated to reflux for 5 hours. After

completion, the contents were cooled, diluted with EtOAc, washed with H2O, and dried

over Na2SO4. The crude product was then concentrated and prepared for silica

chromatography with (DCM/MeOH = 40/1) to give tert-butyl 4-(5-(1-isopropyl-4-

(methyl((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-1H-

indazol-6-yl)pyridin-2-yl)piperazine-1-carboxylate (.175 g, 76%) as a white solid. 1H

NMR (300 MHz, cdcl3), (Rotamers), δ 8.48 (s, 1H), (8.106, 7.944) (s, 1H), 7.803-

7.775(m, 1H), 7.508-7.389(m, 3H), 6.752-6.723(d, 1H, J=8.7 Hz), (5.990, 5.819) (s,

1H), 4.907(s, 2H), 3.590-3.582(m, 7H), (3.066, 2.935) (s, 3H), 2.712-2.685 (m, 1H),

2.289(s. 4H), 1.601-1.580(m, 8H), 1.489(s, 9H), 1.042-0.995 (t, 3H J=7.0 Hz).

1-isopropyl-6-(6-(4-isopropylpiperazin-1-yl)pyridin-3-yl)-N-methyl-N-((6-methyl-2-

oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide

(UNC1756). tert-butyl 4-(5-(1-isopropyl-4-(methyl((6-methyl-2-oxo-4-propyl-1,2-

dihydropyridin-3-yl)methyl)carbamoyl)-1H-indazol-6-yl)pyridin-2-yl)piperazine-1-

carboxylate (.173 g, 2.96 mmol) was dissolved in DCM (2.0 mL) and added an XS of

TFA. The product (.042 g, .010 mmol) was concentrated in vacuo, dissolved in MeOH

(5.0 mL), and placed in an ice bath. Acetone (1.0 mL, 13.6 mmol) and acetic acid (.065

mL, 1.10 mmol) were then added to the reaction. After cooling to 0 0C, NaBH3CN (.034

g, 0.54 mmol) was added and the reaction was stirred for 15 minutes. Then the ice

bath was removed and the contents were allowed to warm to RT overnight. After

completion, the MeOH was removed in vacuo and the crude product was isolated by

separating with DCM:Brine, 3x DCM washes. The organic layers were combined, dried

with Na2SO4, and purified using HPLC to give 1-isopropyl-6-(6-(4-isopropylpiperazin-1-

yl)pyridin-3-yl)-N-methyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-

1H-indazole-4-carboxamide as a brown oil (.023 g, .393 mmol, 53%). 1H NMR (300

MHz, cdcl3), (Rotamers), δ 8.48 (s, 1H), (8.106, 7.944) (s, 1H), 7.803-7.775(m, 1H),

7.508-7.389(m, 3H), 6.752-6.723(d, 1H, J=8.7 Hz), (5.990, 5.819) (s, 1H), 4.907(s,

2H), 3.590-3.582(m, 7H), (3.066, 2.935) (s, 3H), 2.712-2.685 (m, 1H), 2.289(s. 4H),

1.601-1.580(m, 8H), 1.489(s, 9H), 1.042-0.995 (t, 3H J=7.0 Hz).

Reference: 1.   K.  D.  Konze  et  al.,  An  Orally  Bioavailable  Chemical  Probe  of  the  Lysine  Methyltransferases  EZH2  

and  EZH1.  ACS  Chem  Biol,    (2013).  2.   G.  G.  Wang,  L.  Cai,  M.  P.  Pasillas,  M.  P.  Kamps,  NUP98-­‐NSD1  links  H3K36  methylation  to  Hox-­‐A  

gene  activation  and  leukaemogenesis.  Nature  cell  biology  9,  804-­‐812  (2007).  3.   G.  G.  Wang  et  al.,  Haematopoietic  malignancies  caused  by  dysregulation  of  a  chromatin-­‐binding  

PHD  finger.  Nature  459,  847-­‐851  (2009).  4.   G.  G.  Wang,  M.  P.  Pasillas,  M.  P.  Kamps,  Meis1  programs  transcription  of  FLT3  and  cancer  stem  

cell  character,  using  a  mechanism  that  requires  interaction  with  Pbx  and  a  novel  function  of  the  Meis1  C-­‐terminus.  Blood  106,  254-­‐264  (2005).  

5.   J.  Shi  et  al.,  The  Polycomb  complex  PRC2  supports  aberrant  self-­‐renewal   in  a  mouse  model  of  MLL-­‐AF9;Nras(G12D)  acute  myeloid  leukemia.  Oncogene  32,  930-­‐938  (2013).  

6.   J.   Zuber   et   al.,   Toolkit   for   evaluating   genes   required   for   proliferation   and   survival   using  tetracycline-­‐regulated  RNAi.  Nat  Biotechnol  29,  79-­‐83  (2011).  

7.   H.   Li,   R.   Durbin,   Fast   and   accurate   long-­‐read   alignment   with   Burrows-­‐Wheeler   transform.  Bioinformatics  26,  589-­‐595  (2010).  

8.   Y.  Zhang  et  al.,  Model-­‐based  analysis  of  ChIP-­‐Seq  (MACS).  Genome  Biol  9,  R137  (2008).  9.   E.   R.   Polak,   G,   Note   sur   la   Convergence   de   Méthodes   de   Directions   Conjuguées.   Revenue  

Francaise  Informat.  Recherche  Operationelle,  Serie  Rouge  16,  35  (1969).  10.   S.  K.  Verma,  Tian,  X.,  LaFrance,  L.  V.,  Duquenne,  C.,  Suarez,  D.  P.,  Newlander,  K.  A.,  Romeril,  S.  P.,  

Burgess,  J.  L.,  Grant,  S.  W.,  Brackley,  J.  A.,  Graves,  A.  P.,  Scherzer,  D.  A.,  Shu,  A.,  Thompson,  C.,  Ott,  H.  M.,  Aller,  G.  S.  V.,  Machutta,  C.  A.,  Diaz,  E.,  Jiang,  Y.,  Johnson,  N.  W.,  Knight,  S.  D.,  Kruger,  R.  G.,  McCabe,  M.  T.,  Dhanak,  D.,  Tummino,  P.  J.,  Creasy,  C.  L.,  and  Miller,  W.  H.  ,  Identification  of   Potent,   Selective,   Cell-­‐Active   Inhibitors   of   the   Histone   Lysine  Methyltransferase   EZH2.  ACS  Med  Chem  Lett  3,  1091-­‐1096  (2012).  

11.   H.  R.  Jung,  D.  Pasini,  K.  Helin,  O.  N.  Jensen,  Quantitative  mass  spectrometry  of  histones  H3.2  and  H3.3  in  Suz12-­‐deficient  mouse  embryonic  stem  cells  reveals  distinct,  dynamic  post-­‐translational  modifications  at  Lys-­‐27  and  Lys-­‐36.  Mol  Cell  Proteomics  9,  838-­‐850  (2010).  

 

Supplemental Figure Legends

Supplemental Figure 1. A small-molecule UNC1999, and not its inactive analog

UNC2400, selectively and potently suppresses H3K27me3/2.

(A) A docking model of UNC1999 (green) with the SET domain of EZH2 (blue; PDB:

4MI0) revealing that the backbone carbonyl and backbone amide nitrogen of H688 and

the side chain amide nitrogen of N689 in EZH2 form hydrogen bonds (red dotted lines)

with the acyclic amide and pyridone of UNC1999.

(B) Scheme of chemical synthesis of UNC3142 and UNC1756, with either position of

R1 and R2 (shown in main Figure 1A-B) modified by a single N-methyl group.

(C-D) Summary of relative abundances of the H3(27-40) peptide species bearing dual

methylation of H3K27 and H3K36me1 (panel C) or H3K36me2 (panel D) after the

indicated compound treatments as measured by mass spec (data shown in

Supplemental Table 1). Panels underneath plot show H3K27 ‘demethylation’ effect by

UNC1999, which explain the observed increases in peptide species that contain

H3K36me1 or H3K36me2 alone (shown in blue in bar graph; UNC1999 versus DMSO

or UNC2400).

(E) Relative abundances of peptides with dual methylation of H3K27 and H3K36me2 in

Suz12-/- versus wild-type ES cells as shown by mass spec analysis in a previously

published work(11). Panels underneath plot explain the observed increases in peptide

species that contain H3K36me2 alone (shown in blue in bar graph; Suz12-/- versus wt).

(F) The overall level of H3K36me2, H3K36me1, or un-modified H3K36 after compound

treatments by combining the relative abundances of all the H3(27-40) peptide species

containing each of these modifications (as measured by mass spec, Supplemental

Table 1).

(G) Immunoblots of H3K36me2 and H3K36me1 after treatment with 3 µM of compound

for the indicated duration. hr, hour; d, day.

(H-K) Effect of a wide range of concentrations of UNC1999 on in vitro methylation

mediated by four different H3K36-specific methyltransferases—SETD2 (panel H), NSD1

(panel I), NSD2 (panel J) and NSD3 (panel K). Y-axis represents the relative

percentage of enzyme activities after normalization to mock.

(L-M) Summary of H3K27me3 levels, as quantified by flow cytometry analysis, in EOL-1

human leukemia cells and MLL-ENL-transformed murine leukemia cells after treatment

with DMSO, or 2 µM of UNC2400 or UNC1999 for the indicated duration. Y-axis

represents the mean (white) and medium (black) value of H3K27me3 signals. d, days of

treatment.

(N) Flow cytometry analysis of H3K27me3 in MLL-ENL-transformed murine leukemia

cells after the indicated treatment.

(O-P) Immunoblots show the total cellular level of PRC2 components (EZH2 and

SUZ12) in MLL-AF9- (panel O) or MLL-ENL-transformed (panel P) leukemia cells after

treatment with 2 µM of compounds. Tubulin and total histones serve as control.

Supplemental Figure 2. UNC1999, but not GSK126 and UNC2400, efficiently

suppresses inhibits growth of MLL-rearranged leukemia cells.

(A) Flow cytometry analysis of H3K27me3 in DB lymphoma cells treated with different

concentrations of GSK126 (top) or UNC1999 (bottom) for 4 days.

(B-C) Relative proliferation of an MLL-AF9-transformed murine leukemia progenitor line

(m-MLL-AF9-puro) treated with a range of concentrations of GSK126 (panel B) or

UNC1999 (panel C) for 3, 6, 9 or 12 days. Y-axis represents the percentage of the total

number of cells after normalization to DMSO treatment, and is presented as the mean

of triplicates ± stdev.

(D-E) Relative proliferation of two independent MLL-AF9-transformed leukemia lines (m-

MLL-AF9-puro and m-MLL-AF9-GFP) after a 6-day (panel D) or 12-day (panel E)

treatment with various concentrations of UNC1999 or GSK126. Y-axis represents the

relative proliferation after normalization to DMSO treatment. *, p<0.05; **, p<0.01; ns,

not statistically significant.

(F) Signal intensities extracted from microarray studies showing EZH2, EZH1, EED and

SUZ12 gene expression levels in different MLL-AF9-transformed murine leukemia

progenitors.

(G-I) Relative proliferation of MLL-AF9- (panel G) or MLL-ENL-transformed (panel H)

murine leukemia cells and EOL-1 human leukemia cells (panel I) treated with various

concentrations of UNC1999. Y-axis represents the percentage of the total cell number

normalized to DMSO treatment and presented as the mean of triplicates ± SD.

Supplemental Figure 3. UNC1999, and not UNC2400, promotes differentiation,

suppresses colony formation, and induces apoptosis in MLL-rearranged leukemia

cells.

(A) Light microscopy of Wright-Giemsa staining of MLL-ENL-transformed murine

leukemia progenitors treated with the indicated concentration of UNC1999 for 8 days.

(B) Flow cytometry analysis of Cd11b in MLL-AF9-transformed murine leukemia cells

after treatment with 3 µM of UNC1999 (red) or UNC2400 (blue) for 8 days.

(C) Summary of percentage of live and early or late apoptotic populations of MLL-ENL-

or MLL-AF9-transformed murine leukemic progenitors after the indicated treatment with

DMSO, UNC1999 or UNC2400 for 6 days.

Supplemental Figure 4. UNC1999, and not UNC2400, de-represses the defined

Polycomb target genes.

(A) Summary of the up-regulated (blue) and down-regulated (red) transcripts in two

independent MLL-AF9-transformed leukemia lines following a 5-day treatment with 3

µM of UNC1999 versus control compounds or after knockdown of EED (shEED) or

Renilla (shRen), as identified by microarray analysis with a cut-off of fold-change (FC) of

>1.5 and p value of <0.05.

(B) RT-qPCR detecting the expression of EED six days post induction of an EED-

specific shRNA (shEED) versus control shRNA against Renilla (shREN). ***, p<0.001.

(C) Venn diagram of the up-regulated transcripts shown in panel A.

(D-E) GSEA reveals significant enrichment of H3K27me3+/H3K4me3+ bivalent genes

(panel D) and H3K27me3-positive genes (panel E) in UNC1999-treated versus DMSO-

treated samples.

(F) Gene Ontology (GO) analysis revealing significant enrichment of the indicated gene

pathway in the transcripts up regulated by UNC1999 versus DMSO.

Supplemental Figure 5. Re-activation of the Cdkn2a locus plays a critical role in

UNC1999-mediated growth inhibition.

(A) RT-qPCR showing that UNC1999-mediated de-repression of p16Ink4a and p19Arf

is dose-dependent in MLL-AF9-immortalized leukemia cells. d, days of treatment. *,

p<0.05; **, p<0.01; ns, not significant.

(B) Representative histograms of DNA contents, as measured by propidium iodide (PI)

staining, in MLL-ENL-transformed murine leukemia cells after 8-day treatment with 3 µM

of compound.

(C) Following UNC1999 treatment, RT-qPCR shows lack of p16Ink4a and p19Arf gene

expression in two p16Ink4a-/-;p19Arf-/- progenitor lines transformed by MLL-AF9 in

contrast to their wild-type counterparts.

Supplemental Figure 6. ChIP-Seq reveals UNC1999-induced loss of H3K27me3

and concurrent gain of H3K27ac in MLL-AF9-transformed leukemia progenitors.

(A-C) IGB view of the distribution of ChIP-Seq read densities (normalized by the ChIP-

seq read depths) for input (black), H3K27me3 (red), H3K27ac (blue) or SUZ12 (purple)

at the HoxA gene cluster and Evx1 (panel A), Bcl11a (panel B) and Fzd3 (panel C) in

MLL-AF9-transformed leukemia progenitors after treatment with DMSO or 3µM of

UNC2400 or UNC1999 for 4 days.

(D) Boxplots showing a significant reduction in the H3K27me3 ChIP-Seq signals after

treatment with UNC1999, especially at the H3K27me3 peaks located at non-promoter

regions (distal enhancers and intergenic regions). ChIP-Seq read densities were

normalized against sequencing depths, followed by a comparison between those

obtained from mock-, UNC1999-, or UNC2400-treated samples.

(E) The fractions of SUZ12 peaks showing reduction in ChIP-Seq signals by 1.5-fold or

more in UNC1999-treated (red circle) or UNC2400-treated (cross) samples in

comparison to mock treatment. The SUZ12 peaks and their densities shown on X-axis

were first defined and then grouped by the number of ChIP-Seq reads identified in the

mock-treated sample; Y-axis represents the fraction in each group of SUZ12 peaks that

show reduction in ChIP-Seq reads by 1.5-fold or more in the compound-treated samples

in comparison to mock treatment, after normalization of ChIP-Seq reads to the

sequencing depths and peak sizes.

(F) IGB view of the distribution of SUZ12 (purple) ChIP-Seq read densities (normalized

by the ChIP-seq read depths) across the Cdkn2a/b locus.

(G) Quality control of H3K27ac ChIP showing the high and undetectable H3K27ac

signals at TSS of the active β-actin gene and a silenced intergenic locus on

chromosome 8 (chr8_int), respectively.

Supplemental Figure 7. UNC1999 prolongs survival of MLL-AF9 induced myeloid

leukemia in vivo.

(A) Summary of the counts of pellets as measured by complete blood counting of

peripheral blood cell from the vehicle- and UNC1999-treated leukemic mice at the

indicated date post-transplantation.

(B) Summary of quantifications of the cell cycle status of cells isolated from bone

marrow or spleen of each individual examined leukemic mouse from either the vehicle-

(LV) or UNC1999-treated (LT) cohorts.

(C-D) Representative flow cytometry profiles of leukemia cell population (labeled by the

bicistronic GFP-expression in x-axis) and Mac-1 (y-axis) in the bone marrow (BM) or

spleen (SP) of MLL-AF9-induced leukemic mice after treatment with either vehicle or

50mg/kg UNC1999.

UNC1999 (7d) (4d)

UNC2400 (4d;7d)DMSO (4d;7d)

0

500

1000

1500

DMSO 2d

DMSO 5d

DMSO 7d

UNC24

00 4d

UNC24

00 7d

UNC19

99 2d

UNC19

99 4d

UNC19

99 5d

UNC19

99 7d

H3K

27m

e3 le

vels

(a

rbitr

ary

fluor

esce

nce

units

)

EOL-1 mean median

0

400

800

1200

H3K

27m

e3 le

vels

(a

rbitr

ary

fluor

esce

nce

units

)

C D E F

Supplemental Figure S1

-EZH2

-tubulin

DMSO 3d

UNC2400 3d

UNC1999 3d

DMSO 5d

UNC1999 5d

A B

H I J K

UNC1756

UNC3142

0 10 20 30 40 50 60 70 80

H3K36me2 H3K36me1 H3K36unmodified

DMSO UNC2400 UNC1999

fo segatnecrep llarevO

seditpep gniniatnoc-63K3

H

6hr d1 d2 d4 UNC1999

DM

SO

total H3

α-H3K36me1

α-H3K36me2

0%

20%

40%

60%

80%

100%

K27(me3)SAPATGGVK36(me1)KPHR

K27(me2)SAPATGGVK36(me1)KPHR

K27(me1)SAPATGGVK36(me1)KPHR

KSAPATGGVK36(me1)KPHR

UNC1999

UNC1999

UNC1999

UNC1999(2 replicates) D

MS

O

UN

C24

00

ecnadnubA evitale

R

K27(me3)SAPATGGVK36(me2)KPHR

K27(me2)SAPATGGVK36(me2)KPHR

K27(me1)SAPATGGVK36(me2)KPHR

KSAPATGGVK36(me2)KPHR

UNC1999(2 replicates)

UNC1999

UNC1999

UNC1999

H3(27-40) peptides with K36me2

H3(27-40) peptides with K36me1

G

MLL-ENL (murine)

WT Suz12-/-

K27(me3)SAPATGGVK36(me2)KPHR

K27(me2)SAPATGGVK36(me2)KPHR

KSAPATGGVK36(me2)KPHR

Suz12-/-

Suz12-/-

H3.2(27-40) peptides with K36me2

N692V691

S690

H689

N688

V621

A622

W624

G623

F686

F665L666F667

N668

L669

D725

R727

Y726F724

α-Tubulin

DMSO

UNC1999

α-EZH2

α-SUZ12

Total histone

α-H3K27me3

DM

SO

0042C

NU

0%

20%

40%

60%

80%

100% ecnadnubA evitale

R

0%

20%

40%

60%

80%

100% ecnadnubA evitale

R

DMSO 4d

DMSO 7d

UNC24

00 4d

UNC24

00 7d

UNC19

99 4d

UNC19

99 5d

UNC19

99 7d

L M O

N P

mumixa

m %

FACS signal intensity

mean median

A B C

D E F

G H I

Supplemental Figure S2

m-MLL-ENL

0%

20%

40%

60%

80%

100%

EOL-1

0%

20%

40%

60%

80%

100%

120%

m-MLL-AF9 (puro) : UNC1999

)detaert-O

SM

D . sv( noit ar efil orP

%

m-MLL-AF9 (puro) : GSK126

d4 d8 d12 d16

0%

20%

40%

60%

80%

100%

d4 d8 d12 d16

d3 d6 d9 d12

nsns ns

ns **

**

*

**

**

**

**

2 /1 /0.5 μM DMSO

2 /1 /0.5 μM DMSO

100 mumixa

m %

mumixa

m %

75

50

25

0

100

75

50

25

0

DB(UNC1999)

DB(GSK126)

101 102 10

101 102 10

m-MLL-AF9 (puro)

m-MLL-AF9 (GFP)

GSK126UNC1999 GSK126UNC1999

0%

20%

40%

60%

80%

100%

120%

0.1μM 0.25μM 0.5μM 1μM 2μM 3μM

Day 12

ns nsns ns

***** **

*** *** **

**ns

UNC1999 (μM)0 0.1 1 10

noitarefil orP

% ) det aert-

OS

MD . sv(

100

80

60

40

20

0

m-MLL-AF9

d3 d6 d9 d120%

20%

40%

60%

80%

100%

120%

m-MLL-AF9 (puro)

m-MLL-AF9 (GFP)

GSK126UNC1999 GSK126UNC1999

0%

20%

40%

60%

80%

100%

120%

0.1μM 0.25μM 0.5μM 1μM 2μM 3μM

Day 6

FACS signal intensity

noitarefil orP evit al e

R

noit ar efil orP evit al e

R 0

1000

2000

3000

4000

Ezh2 Ezh1 Eed Suz12 sl eveL noi sser px

E eneG

FACS signal intensity

)detaert-O

SM

D . sv( noit ar efil orP

%

noit ar efil orP

% ) det aert-

OS

MD . sv(

noit ar efil orP

% ) det aert-

OS

MD . sv(

m-MLL-AF9 (six arrays)

3

3

0.10 μM 0.25 μM 0.50 μM 1 μM 2 μM 3 μM

0.10 μM 0.25 μM 0.50 μM 1 μM 2 μM 3 μM

0.10 μM 0.25 μM 0.50 μM 1 μM 2 μM 3 μM

0.10 μM 0.25 μM 0.50 μM 1 μM 2 μM 3 μM

DMSO 3μM UNC1999 2μM UNC1999 0.5μM UNC1999

mumixa

m %

Cd11b

A B

0%

20%

40%

60%

80%

100%

DM

SO

U

NC

2400

3μ

M

0.1μ

M

0.25

μM

1μ

M

2μM

3μ

M

DM

SO

U

NC

2400

3μ

M

UN

C19

99 3μ

M

early apoptotic alive

UNC1999

MLL-ENL MLL-AF9

egatnecreP lle

C

late apoptotic

Supplementary Figure S3

C

DMSOUNC1999

A B C

D E

F

Supplemental Figure S4N

umbe

r of G

enes

Up Down

FC >1.5; p < 0.05

-8 -7 -6 -5 -4 -3 -2 -1 0

regulation of myeloid cell differentiation regulation of erythrocyte differentiation eosinophil fate commitment myeloid cell development tissue development stem cell development regulation of cell differentiation organ development cell morphogenesis cell differentiation regulation of hemostasis negative regulation of cell proliferation developmental process regulation of developmental process negative regulation of cellular process multicellular organismal development regulation of multicellular organismal development cell morphogenesis involved in differentiation cell development nervous system development regulation of multicellular organismal process system development anatomical structure development

Log10 (p value)

MEISSNER_NPC_WITH_H3K4ME3_AND_H3K27ME3

0.20

0.40

0.00

NES = 1.412p = 0.064FDR q = 0.245

erocS tne

mhcirnE

UNC1999 controlenrichment profile hits

shEED vs

shRen

UNC1999 vs

UNC2400 UNC1999

vs DMSO

1

594 668

362

0

136 170

46

0

100

200

300

400

500

600

700

800

UNC2400 vs DMSO

UNC1999 vs DMSO

UNC1999 vs UNC2400

shEED vs shRen

ACEVEDO_LIVER_CANCER_WITH_H3K27ME3_UP

0.20

0.40

0.00

NES = 1.357p = 0.000FDR q = 0.235

erocS tne

mhcirnE

UNC1999 controlenrichment profile hits

0

2

4

6

8

10

shREN shEED

EED

Rel

ativ

e ex

pres

sion

(n

orm

aliz

ed b

y G

AP

DH

)

***

0

100

200

300

400

Arb

itrar

y m

RN

A le

vels

G0-G1 (54.4%) G0-G1 (65.5%)G0-G1 (53.7%)

G2-M (3.5%) G2-M (5.0%) G2-M (1.8%)S (42.1%) S (41.3%) S (32.7%)

DNA content DNA content DNA content

WT

Ink4a-/-;Arf-/-

WT

Ink4a-/-;Arf-/-

A B

C

Supplemental Figure S5

Cel

l cou

nt n

umbe

r

UNC1999UNC2400DMSO

Fold

cha

nge

in g

ene

expr

essi

on

(nor

mal

ized

to D

MS

O)

UNC1999; 7d

0 5

10 15 20 25 30 35 40

DMSO 0.25 M 1.25 M 2 M 3 M

p16Ink4a p19Arf

**

**

**

**

**

ns ns ns

p16Ink4a p19Arf

line#1 line#2

0

2

4

6

8

10

12

-actin_tss chr8_int

H3K

27ac

leve

ls

(rel

ativ

e to

5%

inpu

t) UNC2400 UNC1999

Supplemental Figure S6

10 20 30 40 50 60

020

4060

8010

0

SUZ12 ChIP-seq read density in DMSO

% p

eaks

with

> 1

.5−f

old

redu

ctio

n

UNC2400UNC1999

0

Input (UNC2400)

Input (UNC1999)

H3K27me3 (UNC2400)

H3K27me3 (UNC1999)

H3K27ac (UNC2400)

H3K27ac (UNC1999)

HoxA1 A2 HoxA3 A5/A6 /A7 A9 A10 A11 HoxA13 Evx1

(0, 100)

SUZ12 (UNC2400)

SUZ12 (UNC1999)

SUZ12 (mock)

SUZ12 (UNC2400)

SUZ12 (UNC1999)

SUZ12 (DMSO)

H3K27ac (UNC2400)

H3K27ac (UNC1999)

Bcl11a

Input (UNC2400)

Input (UNC1999)

H3K27me3 (UNC2400)

H3K27me3 (UNC1999)

H3K27ac (UNC2400)

H3K27ac (UNC1999)

Input (UNC2400)

Input (UNC1999)

H3K27me3 (UNC2400)

H3K27me3 (UNC1999)

Input (DMSO)

H3K27me3 (DMSO)

20 Kb 20 Kb

20 KbA

B C

D

E F G

mockUNC2400

UNC1999 mock

UNC2400UNC1999

−20

24

68

10

H3K

27m

e3 re

ad d

ensi

ty, l

og2

promoter non−promoterp < 2.2e-16 p < 2.2e-16

0

10

20

30

40

50

60

70

80

LV2 LV3 LT1 LT3 LT5 LV1 LV2 LV3 LT1 LT3 LT5 LT6

G0/G1 S G2/M

Bone Marrow Spleen

Per

cent

age

of c

ells

104

103

102

101

100

101 102 103 104

Mac

-I

gfp(MLL-AF9)

104

103

102

101

100

101 102 103 104

Mac

-I

gfp(MLL-AF9)

33.7 0.1

4.6 61.5

vehicle (SP)

45.7 0.3

3.3 50.7

UNC1999 (SP)104

103

102

101

100

101 102 103 104

Mac

-Igfp(MLL-AF9)

104

103

102

101

100

101 102 103 104

Mac

-I

gfp(MLL-AF9)

18.5 0.1

4.3 77.3

vehicle (BM)

29.9 0.1

2.3 67.7

UNC1999 (BM)

A B

C D

Supplemental Figure S7

0

250

500

750

1000

Plat

elet

s (x

1,0

00/u

L)

Platelet

10d 16d 18d 25d 27d 30d

Vehicle UNC1999

Peptide  Sequence  (modification*)Peptide  H3(3-­‐8)   DMSO UNC2400 UNC1999

TKQTAR   87.5668 88.889 88.1882TK4(me1)QTAR   10.7014 10.3745 10.7892TK4(me2)QTAR   1.22126 0.527532 0.760891TK4(me3)QTAR   0.510461 0.208985 0.261738

Peptide  H3(9-­‐17)   DMSO UNC2400 UNC1999KSTGGKAPR   19.737 21.5255 16.0454

K9(me1)STGGKAPR   13.0097 15.91 14.3973K9(me2)STGGKAPR   16.1268 15.455 16.6309K9(me3)STGGKAPR   12.788 13.0587 15.8985K9STGGK14(ac)APR   8.77739 11.727 12.0545

K9(me1)STGGK14(ac)APR   10.5788 9.96745 10.458K9(me2)STGGK14(ac)APR   9.10446 7.27957 8.47041K9(me3)STGGK14(ac)APR   4.19161 2.95011 4.18693K9(ac)STGGK14(ac)APR   1.23405 1.0416 1.33674

Peptide  H3(18-­‐26)   DMSO UNC2400 UNC1999KQLATKAAR   70.6333 72.6935 71.8845

KQLATK23(me1)AAR   0.463847 0.746466 0.54664K18(me1)QLATKAAR   0.535328 0.500476 0.324485K18(ac)QLATKAAR   4.75632 4.77685 4.07197KQLATK23(ac)AAR   21.3273 19.0457 20.7263

K18(ac)QLATK23(ac)AAR   2.28389 2.237 2.44619

Peptide  H3(27-­‐40)   replicate  1   replicate  2 average  KSAPATGGVKKPHR   12.5268 15.9538 39.839 39.6905 39.76475

K27(me1)SAPATGGVKKPHR   33.0437 32.9908 18.2758 27.2298 22.7528K27(me2)SAPATGGVKKPHR   21.6247 18.5069 3.14655 3.14913 3.14784K27(me3)SAPATGGVKKPHR   5.19754 4.1425 0.28025 0.141249 0.2107495K27(ac)SAPATGGVKKPHR   0.189522 0.25083 0.442955 0.313998 0.3784765KSAPATGGVK36(me1)KPHR   0.197987 0.0778141 14.2535 6.81673 10.535115

K27(me1)SAPATGGVK36(me1)KPHR 4.27134 4.69443 8.58491 5.94388 7.264395K27(me2)SAPATGGVK36(me1)KPHR 8.86975 9.10557 3.9802 4.55026 4.26523K27(me3)SAPATGGVK36(me1)KPHR 3.20114 2.9349 1.00995 1.07911 1.04453

KSAPATGGVK36(me2)KPHR   2.10592 2.17001 6.31617 6.74054 6.528355K27(me1)SAPATGGVK36(me2)KPHR 5.16873 5.61108 3.07698 3.53976 3.30837K27(me2)SAPATGGVK36(me2)KPHR 0.365458 0.264297 0.0223601 0.0156692 0.01901465K27(me3)SAPATGGVK36(me2)KPHR 0.0997172 0.0731504 undetected undetected 0K27(me1)SAPATGGVK36(me3)KPHR 3.13768 3.22396 0.740181 0.750764 0.7454725

Relative  percentage  (%)**  

UNC1999DMSO UNC2400

Supplemental Table S1. Summary of the relative percentages for each detected histone peptide species from MLL-ENL-transformed murine leukemia progenitor cell lines following 4-day treatment with DMSO, 3uM of UNC2400 or UNC1999, as measured by quantitative mass spectrometry analysis.

Peptide  H3(73-­‐83)   DMSO UNC2400 UNC1999EIAQDFKTDLR   86.1581 88.0067 85.9555

EIAQDFK79(me1)TDLR   10.1282 10.0887 8.96825EIAQDFK79(me2)TDLR   3.71369 1.90458 5.07627

Peptide  H4(4-­‐17)   DMSO UNC2400 UNC1999GKGGKGLGKGGAKR   61.8483 56.24 47.078

GK5(ac)GGKGLGKGGAKR   0.976512 1.69458 1.39404GKGGK8(ac)GLGKGGAKR   0.65592 1.06536 0.631697GKGGKGLGK12(ac)GGAKR   3.2719 4.00504 5.2027GKGGKGLGKGGAK16(ac)R     22.1632 28.7981 36.4307

GK5(ac)GGK8(ac)GLGKGGAKR   0.211515 0.166883 0.164136GK5(ac)GGKGLGK12(ac)GGAKR   0.855339 0.695115 0.707435GK5(ac)GGKGLGKGGAK16(ac)R   1.36173 1.02734 0.794228GKGGK8(ac)GLGK12(ac)GGAKR   0.349738 0.445863 0.369785GKGGK8(ac)GLGKGGAK16(ac)R   2.86879 1.65903 2.16513GKGGKGLGK12(ac)GGAK16(ac)R   3.1588 2.46827 3.22782

GK5(ac)GGK8(ac)GLGK12(ac)GGAKR   0.264648 0.25772 0.237819GK5(ac)GGK8(ac)GLGKGGAK16(ac)R   0.285343 0.206238 0.212775GK5(ac)GGKGLGK12(ac)GGAK16(ac)R   0.630415 0.386243 0.342134GKGGK8(ac)GLGK12(ac)GGAK16(ac)R   0.479671 0.40055 0.600936

GK5(ac)GGK8(ac)GLGK12(ac)GGAK16(ac)R   0.618127 0.483729 0.440624

Peptide  H4(20-­‐23)   DMSO UNC2400 UNC1999KVLR   13.4188 15.6014 13.1106

K20(me1)VLR   34.5502 34.0812 33.1653K20(me2)VLR   48.5287 46.802 49.6076K20(me3)VLR   3.50228 3.51538 4.1165

*ac, acetylation; me1/2/3, mono, di, or tri-methylation; **, relative abundance (in percentage or %) for each listed peptide isoforms following treatment.

Transcripts  Cluster  Id

Gene  Symbol

p  value  (Corrected)  

Regulation

Fold  change  (FC)

Average  Expression  -­‐DMSO  [n=5]

Average  Expression  -­‐UNC1999  [n=6]

Average  Expression  -­‐UNC2400  [n=5] Gene  description

17294458 Rhobtb3 6.834E-­‐06 up 5.80 42.03 243.77 44.49 Rho-­‐related  BTB  domain  containing  3

17354378 Ppic 6.834E-­‐06 up 5.12 107.74 552.05 109.85 peptidylprolyl  isomerase  C

17543691 Phka1 6.834E-­‐06 up 3.26 59.09 192.84 63.68 phosphorylase  kinase  alpha  1

17498847 Lass4 1.126E-­‐05 up 3.11 161.74 503.59 141.19 LAG1  homolog,  ceramide  synthase  4

17249898 Galnt10 1.126E-­‐05 up 2.55 85.36 217.61 85.10 UDP-­‐N-­‐acetyl-­‐alpha-­‐D-­‐galactosamine:polypeptide  N-­‐acetylgalactosaminyltransferase  10

17392937 Sox12 1.126E-­‐05 up 2.46 53.15 130.67 49.34 SRY-­‐box  containing  gene  12

17456710 Tspan33 1.126E-­‐05 up 1.76 38.55 67.72 39.64 tetraspanin  33

17415606 Nfia 1.283E-­‐05 up 2.05 69.91 143.02 72.56 nuclear  factor  I/A

17294694 1.285E-­‐05 up 3.43 87.23 299.16 74.0517233347 Gja1 1.285E-­‐05 up 3.22 82.70 266.32 79.15 gap  junction  protein,  alpha  1

17369368 Ncs1 1.285E-­‐05 up 2.98 133.02 396.46 120.72 neuronal  calcium  sensor  1

17396492 Eif5a2 1.300E-­‐05 up 2.08 142.63 296.10 164.54 eukaryotic  translation  initiation  factor  5A2

17276674 Gphn 1.490E-­‐05 up 1.78 621.64 1106.32 591.38 gephyrin

17220059 Cdc42bpa 1.982E-­‐05 up 4.47 31.56 141.03 30.60 CDC42  binding  protein  kinase  alpha

17458734 Plekha8 1.982E-­‐05 up 2.16 76.76 166.02 83.40 pleckstrin  homology  domain  containing,  family  A  (phosphoinositide  binding  specific)  member  8

17456721 Smo 2.098E-­‐05 up 4.96 282.34 1401.67 261.75 smoothened  homolog  (Drosophila)

17466921 Jazf1 2.221E-­‐05 up 3.60 44.82 161.23 47.91 JAZF  zinc  finger  1

17311179 Fzd6 2.485E-­‐05 up 3.05 32.55 99.38 33.32 frizzled  homolog  6  (Drosophila)

17427033 Mllt3 2.577E-­‐05 up 1.59 203.66 323.73 201.37 myeloid/lymphoid  or  mixed-­‐lineage  leukemia  (trithorax  homolog,  Drosophila);  translocated  to,  3

17368550 Rxra 3.565E-­‐05 up 4.04 70.48 284.62 79.46 retinoid  X  receptor  alpha

17544545 Armcx2 3.565E-­‐05 up 2.64 63.26 166.69 57.21 armadillo  repeat  containing,  X-­‐linked  2

17228588 Ralgps2 3.609E-­‐05 up 3.20 37.94 121.54 36.57 Ral  GEF  with  PH  domain  and  SH3  binding  motif  2

17538096 Rnf128 3.686E-­‐05 up 2.91 48.37 140.52 49.76 ring  finger  protein  128

17223985 Ikzf2 4.491E-­‐05 up 3.09 319.43 988.44 290.79 IKAROS  family  zinc  finger  2

17313811 Fbln1 5.955E-­‐05 up 1.70 39.34 66.75 39.59 fibulin  1

17293103 Gkap1 6.168E-­‐05 up 4.00 124.68 499.09 108.43 G  kinase  anchoring  protein  1

Supplemental  Table  S2.  List  of  the  differentially  expressed  transcripts  in  two  different  MLL-­‐AF9-­‐transformed  leukemia  progenitor  lines  following  treatment  of  3uM  of  DMSO,  UNC2400  or  UNC1999  for  4-­‐5  days  and  then  examined  by  microarray  analysis  with  a  cut-­‐off  of  >1.5-­‐fold  and  p  <0.01.  

17430373 Zbtb8a 6.690E-­‐05 up 3.15 55.52 174.70 57.24 zinc  finger  and  BTB  domain  containing  8a

17235243 Efna2 6.690E-­‐05 up 1.70 59.08 100.46 64.54 ephrin  A2

17468298 Exoc6b 6.813E-­‐05 up 2.54 204.93 521.15 218.87 exocyst  complex  component  6B

17310673 Ank 7.769E-­‐05 up 1.94 317.91 617.90 331.72 progressive  ankylosis

17414212 Fsd1l 7.769E-­‐05 up 1.69 41.47 70.29 42.66 fibronectin  type  III  and  SPRY  domain  containing  1-­‐like

17290865 Epdr1 7.851E-­‐05 up 2.19 47.42 103.71 44.25 ependymin  related  protein  1  (zebrafish)

17324643 Bdh1 7.851E-­‐05 up 1.99 61.00 121.09 65.83 3-­‐hydroxybutyrate  dehydrogenase,  type  1

17417551 Zswim5 8.155E-­‐05 up 4.11 47.41 194.82 51.03 zinc  finger,  SWIM  domain  containing  5

17467171 Vopp1 8.155E-­‐05 up 2.03 131.10 265.96 117.06 vesicular,  overexpressed  in  cancer,  prosurvival  protein  1

17427147 Cdkn2a 8.422E-­‐05 up 4.01 71.34 285.94 68.40 cyclin-­‐dependent  kinase  inhibitor  2A

17428509 Faah 8.635E-­‐05 up 2.51 90.02 225.88 91.09 fatty  acid  amide  hydrolase

17348138 9430020K01Rik8.748E-­‐05 up 2.77 114.20 316.39 116.66 RIKEN  cDNA  9430020K01  gene

17257405 Tanc2 9.132E-­‐05 up 3.08 32.00 98.70 31.84 tetratricopeptide  repeat,  ankyrin  repeat  and  coiled-­‐coil  containing  2

17402193 Arhgap29 9.132E-­‐05 up 2.30 35.63 82.00 35.21 Rho  GTPase  activating  protein  29

17353032 Fam59a 9.132E-­‐05 up 2.27 65.96 149.85 70.22 family  with  sequence  similarity  59,  member  A

17460861 Slc41a3 9.132E-­‐05 up 1.75 124.84 218.02 148.71 solute  carrier  family  41,  member  3

17291275 Lrrc16a 9.132E-­‐05 up 1.74 62.22 108.31 65.47 leucine  rich  repeat  containing  16A

17306477 Slc7a8 9.218E-­‐05 up 1.75 1923.84 3371.07 1912.58 solute  carrier  family  7  (cationic  amino  acid  transporter,  y+  system),  member  8

17456001 Glcci1 9.218E-­‐05 up 1.73 296.42 511.60 277.27 glucocorticoid  induced  transcript  1

17537895 Ngfrap1 9.218E-­‐05 up 1.51 1625.92 2448.33 1532.89 nerve  growth  factor  receptor  (TNFRSF16)  associated  protein  1

17366953 Prkcq 9.796E-­‐05 up 2.87 45.45 130.36 48.95 protein  kinase  C,  theta

17484382 Inpp5a 9.823E-­‐05 up 1.52 186.69 283.33 188.44 inositol  polyphosphate-­‐5-­‐phosphatase  A

17356288 Ctsf 9.944E-­‐05 up 3.41 44.45 151.62 44.52 cathepsin  F

17308361 Bmp1 9.944E-­‐05 up 1.90 41.24 78.46 44.23 bone  morphogenetic  protein  1

17236288 Igf1 1.086E-­‐04 up 5.63 45.29 255.13 51.99 insulin-­‐like  growth  factor  1

17318950 Csf2rb2 1.167E-­‐04 up 2.45 984.22 2413.75 1068.64 colony  stimulating  factor  2  receptor,  beta  2,  low-­‐affinity  (granulocyte-­‐macrophage)

17224771 Serpine2 1.170E-­‐04 up 4.76 55.64 264.94 48.35 serine  (or  cysteine)  peptidase  inhibitor,  clade  E,  member  2

17223138 Pgap1 1.170E-­‐04 up 3.02 69.17 208.56 106.86 post-­‐GPI  attachment  to  proteins  1

17422659 B930041F14Rik1.170E-­‐04 up 2.88 171.45 493.53 170.97 RIKEN  cDNA  B930041F14  gene

17470187 Zfp9 1.170E-­‐04 up 1.98 48.85 96.85 42.36 zinc  finger  protein  9

17277170 Acot6 1.170E-­‐04 up 1.95 40.62 79.36 36.79 acyl-­‐CoA  thioesterase  6

17539241 Map3k15 1.170E-­‐04 up 1.75 149.25 261.55 129.70 mitogen-­‐activated  protein  kinase  kinase  kinase  15

17430645 Nkain1 1.370E-­‐04 up 3.78 84.77 320.45 85.83 Na+/K+  transporting  ATPase  interacting  1

17312054 Khdrbs3 1.392E-­‐04 up 4.06 68.17 276.59 56.75 KH  domain  containing,  RNA  binding,  signal  transduction  associated  3

17341975 Npw 1.420E-­‐04 up 2.20 59.06 129.82 53.90 neuropeptide  W

17323589 Klhl22 1.429E-­‐04 up 2.24 175.57 392.95 160.07 kelch-­‐like  22  (Drosophila)

17271168 Prkca 1.453E-­‐04 up 1.98 159.68 316.02 168.56 protein  kinase  C,  alpha

17321263 Adcy6 1.719E-­‐04 up 2.82 37.43 105.61 37.26 adenylate  cyclase  6

17268120 Pdk2 1.719E-­‐04 up 2.13 77.98 166.05 78.18 pyruvate  dehydrogenase  kinase,  isoenzyme  2

17262600 Tcf7 1.719E-­‐04 up 1.67 50.09 83.85 44.44 transcription  factor  7,  T  cell  specific

17411103 Lphn2 1.843E-­‐04 up 4.28 39.03 167.06 40.67 latrophilin  2

17215576 Agap1 1.921E-­‐04 up 2.61 216.68 565.87 176.17 ArfGAP  with  GTPase  domain,  ankyrin  repeat  and  PH  domain  1

17462202 Rasgef1a 2.034E-­‐04 up 1.81 34.02 61.59 39.65 RasGEF  domain  family,  member  1A

17433055 Kif1b 2.146E-­‐04 up 2.07 186.83 386.41 199.09 kinesin  family  member  1B

17240226 Marcks 2.152E-­‐04 up 2.76 53.62 147.92 58.79 myristoylated  alanine  rich  protein  kinase  C  substrate

17374098 Lgr4 2.152E-­‐04 up 2.30 67.86 155.74 65.97 leucine-­‐rich  repeat-­‐containing  G  protein-­‐coupled  receptor  4

17276934 Ttc9 2.329E-­‐04 up 1.57 33.80 52.99 34.17 tetratricopeptide  repeat  domain  9

17233273 Dcbld1 2.605E-­‐04 up 3.31 40.76 135.11 42.22 discoidin,  CUB  and  LCCL  domain  containing  1

17228164 Lamc1 2.613E-­‐04 up 2.05 225.54 461.52 313.22 laminin,  gamma  1

17218349 Glul 2.827E-­‐04 up 2.01 352.16 706.13 411.88 glutamate-­‐ammonia  ligase  (glutamine  synthetase)

17310912 Laptm4b 2.838E-­‐04 up 3.11 291.99 906.90 287.42 lysosomal-­‐associated  protein  transmembrane  4B

17400622 Lix1l 3.061E-­‐04 up 1.65 357.67 590.76 348.50 Lix1-­‐like

17312191 4930572J05Rik3.141E-­‐04 up 1.73 459.72 796.75 444.46 RIKEN  cDNA  4930572J05  gene

17498251 Phlda2 3.332E-­‐04 up 2.07 34.90 72.11 40.90 pleckstrin  homology-­‐like  domain,  family  A,  member  2

17226203 Epb4.1l5 3.332E-­‐04 up 2.03 106.35 215.78 104.10 erythrocyte  protein  band  4.1-­‐like  5

17479183 Fam174b 3.356E-­‐04 up 2.33 229.88 535.30 238.91 family  with  sequence  similarity  174,  member  B

17415391 3.485E-­‐04 up 2.98 40.15 119.78 42.2317313705 Parvb 3.485E-­‐04 up 2.44 99.58 243.43 105.87 parvin,  beta

17358375 Smarca2 3.485E-­‐04 up 1.81 437.42 789.54 404.82 SWI/SNF  related,  matrix  associated,  actin  dependent  regulator  of  chromatin,  subfamily  a,  member  2

17475580 Blvrb 3.485E-­‐04 up 1.63 146.33 238.73 151.58 biliverdin  reductase  B  (flavin  reductase  (NADPH))

17237170 Pawr 3.510E-­‐04 up 1.97 65.11 128.41 63.44 PRKC,  apoptosis,  WT1,  regulator

17433977 Agrn 3.846E-­‐04 up 1.85 115.32 213.14 107.60 agrin

17351473 Slmo1 3.925E-­‐04 up 1.93 44.26 85.47 45.93 slowmo  homolog  1  (Drosophila)

17520886 Ryk 4.039E-­‐04 up 5.11 74.78 382.02 55.77 receptor-­‐like  tyrosine  kinase

17296084 Zswim6 4.434E-­‐04 up 1.69 404.45 681.89 411.56 zinc  finger,  SWIM  domain  containing  6

17248304 Ubtd2 4.531E-­‐04 up 1.67 520.31 870.95 503.02 ubiquitin  domain  containing  2

17510735 Smad1 4.653E-­‐04 up 2.81 46.55 130.87 45.35 SMAD  family  member  1

17460831 Aldh1l1 4.655E-­‐04 up 2.46 48.58 119.62 51.65 aldehyde  dehydrogenase  1  family,  member  L1

17246284 Suox 4.852E-­‐04 up 1.74 198.21 344.45 201.89 sulfite  oxidase

17287536 Tspan17 4.883E-­‐04 up 2.03 254.56 517.77 288.21 tetraspanin  17

17217500 Kdm5b 4.883E-­‐04 up 1.84 363.63 668.45 303.45 lysine  (K)-­‐specific  demethylase  5B

17397497 Foxo1 5.573E-­‐04 up 1.68 105.14 176.66 107.87 forkhead  box  O1

17409284 Celsr2 5.670E-­‐04 up 1.70 47.00 80.10 41.78 cadherin,  EGF  LAG  seven-­‐pass  G-­‐type  receptor  2  (flamingo  homolog,  Drosophila)

17468828 Plxna1 5.696E-­‐04 up 3.59 119.58 429.32 137.28 plexin  A1

17325159 Mylk 5.696E-­‐04 up 1.77 61.64 109.29 52.89 myosin,  light  polypeptide  kinase

17247852 Bcl11a 5.754E-­‐04 up 2.35 255.78 601.84 260.00 B  cell  CLL/lymphoma  11A  (zinc  finger  protein)

17334638 Prss34 5.905E-­‐04 up 7.03 70.06 492.26 69.82 protease,  serine,  34

17470930 Leprel2 5.905E-­‐04 up 2.11 106.51 225.19 107.35 leprecan-­‐like  2

17516450 Pvrl1 5.905E-­‐04 up 1.88 49.87 93.71 55.67 poliovirus  receptor-­‐related  1

17285097 Dip2c 5.937E-­‐04 up 4.77 43.76 208.85 40.44 DIP2  disco-­‐interacting  protein  2  homolog  C  (Drosophila)

17305520 Ear1 6.123E-­‐04 up 5.74 25.24 144.82 24.77 eosinophil-­‐associated,  ribonuclease  A  family,  member  1

17538628 Fgd1 6.123E-­‐04 up 1.87 48.42 90.42 46.40 FYVE,  RhoGEF  and  PH  domain  containing  1

17320720 Prickle1 6.123E-­‐04 up 1.55 45.02 69.88 46.77 prickle  homolog  1  (Drosophila)

17307354 Atp8a2 6.215E-­‐04 up 1.72 47.11 80.96 50.92 ATPase,  aminophospholipid  transporter-­‐like,  class  I,  type  8A,  member  2

17519699 Ddx43 6.400E-­‐04 up 1.92 31.53 60.48 30.18 DEAD  (Asp-­‐Glu-­‐Ala-­‐Asp)  box  polypeptide  43

17431607 C1qb 6.402E-­‐04 up 5.58 57.67 321.61 65.57 complement  component  1,  q  subcomponent,  beta  polypeptide

17292058 Elovl2 6.412E-­‐04 up 1.52 41.73 63.56 44.22 elongation  of  very  long  chain  fatty  acids  (FEN1/Elo2,  SUR4/Elo3,  yeast)-­‐like  2

17305042 Bmpr1a 6.434E-­‐04 up 4.06 35.29 143.12 32.21 bone  morphogenetic  protein  receptor,  type  1A

17317858 Ptk2 6.434E-­‐04 up 1.91 37.59 71.83 37.22 PTK2  protein  tyrosine  kinase  2

17428094 Zfyve9|3110021N24Rik6.434E-­‐04 up 1.68 49.82 83.90 46.49 zinc  finger,  FYVE  domain  containing  9  |  RIKEN  cDNA  3110021N24  gene

17326510 Pros1 6.500E-­‐04 up 2.16 41.97 90.82 53.26 protein  S  (alpha)

17470468 Mical3 6.670E-­‐04 up 1.55 108.80 168.11 112.21 microtubule  associated  monoxygenase,  calponin  and  LIM  domain  containing  3

17522958 Ctdspl 6.839E-­‐04 up 1.63 303.13 493.90 288.16 CTD  (carboxy-­‐terminal  domain,  RNA  polymerase  II,  polypeptide  A)  small  phosphatase-­‐like

17308772 9030625A04Rik6.913E-­‐04 up 1.83 73.35 134.59 60.12 RIKEN  cDNA  9030625A04  gene

17405746 Rarres1 7.001E-­‐04 up 2.36 56.23 132.81 55.13 retinoic  acid  receptor  responder  (tazarotene  induced)  1

17307738 Fzd3 7.106E-­‐04 up 3.07 44.97 137.91 37.14 frizzled  homolog  3  (Drosophila)

17221792 Fam135a 7.189E-­‐04 up 4.16 45.96 191.22 51.08 family  with  sequence  similarity  135,  member  A

17286641 Dsp 7.793E-­‐04 up 2.72 390.66 1064.20 238.96 desmoplakin

17511377 Neto2 7.975E-­‐04 up 1.70 373.26 633.20 391.88 neuropilin  (NRP)  and  tolloid  (TLL)-­‐like  2

17427494 Dock7 8.046E-­‐04 up 3.03 158.80 480.94 182.46 dedicator  of  cytokinesis  7

17302092 Tsc22d1|Gm195978.046E-­‐04 up 1.91 56.73 108.10 65.93 TSC22  domain  family,  member  1  |  predicted  gene,  19597

17239539 Hebp2 8.046E-­‐04 up 1.88 41.43 77.86 40.68 heme  binding  protein  2

17392690 Acss1 8.046E-­‐04 up 1.63 287.99 469.32 286.43 acyl-­‐CoA  synthetase  short-­‐chain  family  member  1

17248276 Hba-­‐a2|Hba-­‐a18.116E-­‐04 up 5.17 45.27 234.11 45.02 hemoglobin  alpha,  adult  chain  2  |  hemoglobin  alpha,  adult  chain  1

17320614 Kif21a 8.116E-­‐04 up 1.98 39.53 78.42 38.58 kinesin  family  member  21A

17438389 C530008M17Rik8.459E-­‐04 up 1.94 67.03 129.73 65.24 RIKEN  cDNA  C530008M17  gene

17223283 Satb2 8.502E-­‐04 up 2.11 183.52 386.99 195.86 special  AT-­‐rich  sequence  binding  protein  2

17291935 8.502E-­‐04 up 1.66 63.27 104.89 54.6717465373 Impdh1 8.502E-­‐04 up 1.62 369.34 597.06 290.13 inosine  5'-­‐phosphate  dehydrogenase  1

17313000 Kdelr3 8.708E-­‐04 up 2.77 74.02 204.68 76.66 KDEL  (Lys-­‐Asp-­‐Glu-­‐Leu)  endoplasmic  reticulum  protein  retention  receptor  3

17404585 Zmat3 8.845E-­‐04 up 4.16 101.81 423.10 120.91 zinc  finger  matrin  type  3

17357640 Ms4a4a 8.845E-­‐04 up 3.15 58.56 184.40 89.68 membrane-­‐spanning  4-­‐domains,  subfamily  A,  member  4A

17290420 Zmynd11 9.228E-­‐04 up 1.58 553.16 876.26 567.77 zinc  finger,  MYND  domain  containing  11

17358905 Hectd2 9.555E-­‐04 up 2.09 35.67 74.45 33.54 HECT  domain  containing  2

17390823 Slc30a4 9.576E-­‐04 up 2.73 44.23 120.74 43.71 solute  carrier  family  30  (zinc  transporter),  member  4

17510823 Gab1 9.646E-­‐04 up 1.68 87.08 146.13 87.11 growth  factor  receptor  bound  protein  2-­‐associated  protein  1

17312746 Kctd17 9.715E-­‐04 up 1.90 105.76 201.30 118.83 potassium  channel  tetramerisation  domain  containing  17

17370807 Fmnl2 9.783E-­‐04 up 2.92 29.87 87.20 29.44 formin-­‐like  2

17453242 Auts2 1.038E-­‐03 up 1.87 131.20 245.81 122.75 autism  susceptibility  candidate  2

17249036 Gfpt2 1.038E-­‐03 up 1.81 36.83 66.50 35.96 glutamine  fructose-­‐6-­‐phosphate  transaminase  2

17251728 Zbtb4 1.038E-­‐03 up 1.52 80.39 122.00 75.67 zinc  finger  and  BTB  domain  containing  4

17498673 Camsap3 1.038E-­‐03 up 1.50 42.76 64.33 43.90 calmodulin  regulated  spectrin-­‐associated  protein  family,  member  3

17363903 Prkg1 1.041E-­‐03 up 2.52 59.56 150.32 57.40 protein  kinase,  cGMP-­‐dependent,  type  I

17371019 Tanc1 1.041E-­‐03 up 2.00 73.85 147.38 57.01 tetratricopeptide  repeat,  ankyrin  repeat  and  coiled-­‐coil  containing  1

17464803 Ica1 1.076E-­‐03 up 2.35 99.97 235.42 83.85 islet  cell  autoantigen  1

17333418 Mllt4 1.076E-­‐03 up 1.74 70.67 122.68 69.70 myeloid/lymphoid  or  mixed-­‐lineage  leukemia  (trithorax  homolog,  Drosophila);  translocated  to,  4

17318750 Rbfox2 1.076E-­‐03 up 1.57 50.16 78.93 54.83 RNA  binding  protein,  fox-­‐1  homolog  (C.  elegans)  2

17261575 Ranbp17 1.121E-­‐03 up 2.43 60.32 146.86 61.44 RAN  binding  protein  17

17284065 Cdc42bpb 1.121E-­‐03 up 1.89 172.70 326.92 182.35 CDC42  binding  protein  kinase  beta

17362953 Ms4a7 1.147E-­‐03 up 5.06 62.32 315.46 100.89 membrane-­‐spanning  4-­‐domains,  subfamily  A,  member  7

17404835 Bbs7 1.167E-­‐03 up 1.84 64.76 118.84 71.31 Bardet-­‐Biedl  syndrome  7  (human)

17241512 Tet1 1.195E-­‐03 up 1.93 103.45 199.65 88.20 tet  methylcytosine  dioxygenase  1

17534746 Cxx1c 1.201E-­‐03 up 2.18 36.82 80.18 39.61 CAAX  box  1  homolog  C  (human)

17306507 Slc22a17 1.237E-­‐03 up 1.84 71.34 131.56 71.88 solute  carrier  family  22  (organic  cation  transporter),  member  17

17355026 Atp8b1 1.282E-­‐03 up 2.12 40.86 86.80 41.28 ATPase,  class  I,  type  8B,  member  1

17403866 Ptger3 1.296E-­‐03 up 2.41 73.88 177.73 71.71 prostaglandin  E  receptor  3  (subtype  EP3)

17363025 Stx3 1.303E-­‐03 up 3.32 85.47 283.78 96.93 syntaxin  3

17459520 Reep1 1.304E-­‐03 up 1.91 60.00 114.65 59.95 receptor  accessory  protein  1

17450618 Tgfbr3 1.315E-­‐03 up 2.33 47.03 109.49 43.57 transforming  growth  factor,  beta  receptor  III

17540059 Porcn 1.333E-­‐03 up 1.65 68.90 113.73 71.92 porcupine  homolog  (Drosophila)

17221197 Tcf24 1.361E-­‐03 up 2.85 51.93 147.85 49.81 transcription  factor  24

17369613 Aif1l 1.361E-­‐03 up 1.59 76.38 121.34 74.90 allograft  inflammatory  factor  1-­‐like

17266698 Myo1d 1.362E-­‐03 up 3.72 51.84 193.03 53.37 myosin  ID

17273714 Adcy3|Cenpo1.364E-­‐03 up 1.74 84.45 146.73 81.73 adenylate  cyclase  3  |  centromere  protein  O

17498365 Mrgpre 1.395E-­‐03 up 1.67 71.20 119.12 79.47 MAS-­‐related  GPR,  member  E

17367163 Cacnb2 1.441E-­‐03 up 2.16 78.30 168.79 65.65 calcium  channel,  voltage-­‐dependent,  beta  2  subunit

17401610 Gstm5|4933431E20Rik1.448E-­‐03 up 2.72 76.16 206.93 77.55 glutathione  S-­‐transferase,  mu  5  |  RIKEN  cDNA  4933431E20  gene

17418035 Cited4 1.462E-­‐03 up 4.66 88.85 414.18 67.66 Cbp/p300-­‐interacting  transactivator,  with  Glu/Asp-­‐rich  carboxy-­‐terminal  domain,  4

17384021 Stxbp1 1.505E-­‐03 up 1.65 237.10 392.09 228.82 syntaxin  binding  protein  1

17218186 Fam129a 1.506E-­‐03 up 2.65 126.61 335.37 116.24 family  with  sequence  similarity  129,  member  A

17342581 Decr2 1.557E-­‐03 up 1.66 325.70 542.18 349.42 2-­‐4-­‐dienoyl-­‐Coenzyme  A  reductase  2,  peroxisomal

17543286 Spin4 1.603E-­‐03 up 1.64 35.20 57.86 36.45 spindlin  family,  member  4

17460634 Gata2 1.604E-­‐03 up 1.87 1746.79 3271.60 1852.96 GATA  binding  protein  2

17346618 Nudt12 1.604E-­‐03 up 1.62 68.40 110.64 63.68 nudix  (nucleoside  diphosphate  linked  moiety  X)-­‐type  motif  12

17436545 Nat8l 1.607E-­‐03 up 2.09 133.84 280.16 129.79 N-­‐acetyltransferase  8-­‐like

17408414 Fam46c 1.610E-­‐03 up 2.50 66.78 167.26 69.93 family  with  sequence  similarity  46,  member  C

17517831 Cyp11a1 1.638E-­‐03 up 9.90 104.52 1034.79 143.07 cytochrome  P450,  family  11,  subfamily  a,  polypeptide  1

17276386 Rhoj 1.747E-­‐03 up 3.29 51.85 170.86 48.57 ras  homolog  gene  family,  member  J

17497076 Chst15|Gm105841.747E-­‐03 up 2.63 82.69 217.88 76.45 carbohydrate  (N-­‐acetylgalactosamine  4-­‐sulfate  6-­‐O)  sulfotransferase  15  |  predicted  gene  10584

17544816 Esx1 1.751E-­‐03 up 1.63 43.80 71.49 45.23 extraembryonic,  spermatogenesis,  homeobox  1

17366359 Fam171a1 1.808E-­‐03 up 1.67 52.08 87.03 54.13 family  with  sequence  similarity  171,  member  A1

17492826 Homer2 1.812E-­‐03 up 2.44 93.44 227.93 89.09 homer  homolog  2  (Drosophila)

17300030 1.817E-­‐03 up 1.66 52.17 86.77 52.0517251816 Ybx2 1.838E-­‐03 up 1.56 47.29 73.99 47.11 Y  box  protein  2

17270222 Itga2b 1.864E-­‐03 up 1.67 268.04 447.50 274.01 integrin  alpha  2b

17340177 Prkce 1.894E-­‐03 up 1.78 133.61 237.52 134.76 protein  kinase  C,  epsilon

17272877 Tbc1d16 1.922E-­‐03 up 1.84 104.27 192.32 98.97 TBC1  domain  family,  member  16

17548987 2810025M15Rik1.945E-­‐03 up 3.93 119.74 471.15 100.57 RIKEN  cDNA  2810025M15  gene

17535607 Slc6a8 1.945E-­‐03 up 3.12 133.00 415.01 150.27 solute  carrier  family  6  (neurotransmitter  transporter,  creatine),  member  8

17272770 Timp2 1.945E-­‐03 up 2.79 232.51 649.17 232.94 tissue  inhibitor  of  metalloproteinase  2

17363572 Tjp2 1.945E-­‐03 up 1.70 134.11 227.51 117.64 tight  junction  protein  2

17502994 Lphn1 2.025E-­‐03 up 2.82 134.84 379.90 129.88 latrophilin  1

17301697 Tnfrsf10b 2.049E-­‐03 up 1.84 111.45 204.84 107.04 tumor  necrosis  factor  receptor  superfamily,  member  10b

17431612 C1qc 2.049E-­‐03 up 1.71 41.92 71.81 44.91 complement  component  1,  q  subcomponent,  C  chain

17229974 Fcer1a 2.101E-­‐03 up 4.39 29.17 127.94 30.59 Fc  receptor,  IgE,  high  affinity  I,  alpha  polypeptide

17378359 Acss2 2.116E-­‐03 up 1.82 520.35 946.94 489.27 acyl-­‐CoA  synthetase  short-­‐chain  family  member  2

17514853 Slc36a4 2.119E-­‐03 up 5.04 51.69 260.60 49.50 solute  carrier  family  36  (proton/amino  acid  symporter),  member  4

17291787 Slc22a23 2.127E-­‐03 up 2.64 45.12 118.96 55.13 solute  carrier  family  22,  member  23

17229988 Cadm3 2.127E-­‐03 up 1.63 41.67 67.90 40.27 cell  adhesion  molecule  3

17276020 Frmd6 2.127E-­‐03 up 1.61 87.54 140.74 91.23 FERM  domain  containing  6

17470616 C3ar1 2.222E-­‐03 up 3.61 69.00 249.08 74.85 complement  component  3a  receptor  1

17459014 Abcg2 2.222E-­‐03 up 2.11 25.57 53.98 25.71 ATP-­‐binding  cassette,  sub-­‐family  G  (WHITE),  member  2

17458911 Fkbp9 2.233E-­‐03 up 1.76 31.07 54.64 29.83 FK506  binding  protein  9

17544734 Bex1 2.233E-­‐03 up 1.72 29.70 51.01 29.13 brain  expressed  gene  1

17349304 Tslp 2.273E-­‐03 up 1.59 39.74 63.24 40.55 thymic  stromal  lymphopoietin

17249542 septin  8 2.289E-­‐03 up 1.73 105.91 183.29 110.34 septin  8

17500453 Tex15 2.289E-­‐03 up 1.70 35.72 60.75 36.80 testis  expressed  gene  15

17316826 Tmem74 2.289E-­‐03 up 1.63 41.10 67.14 45.75 transmembrane  protein  74

17373283 Lrp4 2.348E-­‐03 up 1.92 55.34 106.11 49.72 low  density  lipoprotein  receptor-­‐related  protein  4

17403463 Syde2 2.348E-­‐03 up 1.88 63.64 119.44 59.30 synapse  defective  1,  Rho  GTPase,  homolog  2  (C.  elegans)

17519533 Lysmd2 2.348E-­‐03 up 1.83 45.79 83.83 42.03 LysM,  putative  peptidoglycan-­‐binding,  domain  containing  2

17413221 Unc13b 2.348E-­‐03 up 1.62 40.77 66.04 39.30 unc-­‐13  homolog  B  (C.  elegans)

17257903 2.411E-­‐03 up 2.02 42.77 86.57 40.5317282216 Zfp36l1 2.446E-­‐03 up 2.83 96.15 271.85 99.14 zinc  finger  protein  36,  C3H  type-­‐like  1

17444372 Cyth3 2.447E-­‐03 up 1.59 215.35 343.34 235.46 cytohesin  3

17221193 2.453E-­‐03 up 1.89 45.00 85.07 41.3017357971 Gna14 2.490E-­‐03 up 2.21 48.93 107.99 46.22 guanine  nucleotide  binding  protein,  alpha  14

17361304 Lrfn4 2.529E-­‐03 up 1.61 94.95 152.75 96.29 leucine  rich  repeat  and  fibronectin  type  III  domain  containing  4

17441396 Vsig10 2.529E-­‐03 up 1.53 44.39 67.89 46.09 V-­‐set  and  immunoglobulin  domain  containing  10

17265229 Alox15 2.531E-­‐03 up 2.15 37.09 79.85 40.21 arachidonate  15-­‐lipoxygenase

17472517 Ldhb 2.547E-­‐03 up 2.70 63.04 170.15 50.95 lactate  dehydrogenase  B

17545839 Reps2 2.641E-­‐03 up 1.73 76.66 132.27 92.52 RALBP1  associated  Eps  domain  containing  protein  2

17312716 Csf2rb 2.692E-­‐03 up 1.61 166.96 269.28 181.40 colony  stimulating  factor  2  receptor,  beta,  low-­‐affinity  (granulocyte-­‐macrophage)

17415396 2.705E-­‐03 up 1.87 50.38 94.08 55.7917461942 Pparg 2.774E-­‐03 up 2.28 53.24 121.52 52.37 peroxisome  proliferator  activated  receptor  gamma

17426497 Megf9 2.774E-­‐03 up 1.83 442.27 809.10 392.56 multiple  EGF-­‐like-­‐domains  9

17238848 Syne1 2.785E-­‐03 up 2.56 63.81 163.05 43.55 synaptic  nuclear  envelope  1

17482110 Xylt1 2.785E-­‐03 up 1.60 1457.54 2326.80 1303.98 xylosyltransferase  1

17222777 Slc40a1 2.842E-­‐03 up 3.03 43.30 131.10 49.10 solute  carrier  family  40  (iron-­‐regulated  transporter),  member  1

17422996 Plag1 2.922E-­‐03 up 2.20 66.87 147.12 62.69 pleiomorphic  adenoma  gene  1

17382203 Hnmt 2.940E-­‐03 up 2.80 51.41 143.73 45.17 histamine  N-­‐methyltransferase

17529398 Me1 2.969E-­‐03 up 2.02 40.54 81.93 44.28 malic  enzyme  1,  NADP(+)-­‐dependent,  cytosolic

17335017 2.999E-­‐03 up 2.46 45.17 111.27 53.6917429057 Ptprf 3.015E-­‐03 up 1.78 56.88 101.16 54.76 protein  tyrosine  phosphatase,  receptor  type,  F

17404180 Car1 3.039E-­‐03 up 3.44 75.58 259.84 101.94 carbonic  anhydrase  1

17404601 Gnb4 3.039E-­‐03 up 2.69 61.64 165.96 73.70 guanine  nucleotide  binding  protein  (G  protein),  beta  4

17541856 Mtap7d3 3.039E-­‐03 up 2.11 31.07 65.59 32.19 MAP7  domain  containing  3

17227089 Btg2 3.039E-­‐03 up 1.99 167.58 334.21 179.36 B  cell  translocation  gene  2,  anti-­‐proliferative

17519738 Cd109 3.039E-­‐03 up 1.87 25.00 46.86 25.71 CD109  antigen

17305221 5730469M10Rik3.057E-­‐03 up 1.98 192.14 381.33 177.33 RIKEN  cDNA  5730469M10  gene

17267471 Ppm1e 3.082E-­‐03 up 3.11 83.49 259.68 78.54 protein  phosphatase  1E  (PP2C  domain  containing)

17212211 Il1rl1 3.086E-­‐03 up 3.67 66.59 244.06 67.54 interleukin  1  receptor-­‐like  1

17228751 Rabgap1l 3.097E-­‐03 up 1.89 113.38 214.58 110.12 RAB  GTPase  activating  protein  1-­‐like

17334666 Tpsb2 3.141E-­‐03 up 1.91 25.43 48.54 27.04 tryptase  beta  2

17462705 Foxj2 3.146E-­‐03 up 1.62 377.64 612.78 420.50 forkhead  box  J2

17416616 Echdc2 3.157E-­‐03 up 1.98 143.72 284.13 137.08 enoyl  Coenzyme  A  hydratase  domain  containing  2

17394297 Pltp 3.318E-­‐03 up 4.17 85.21 354.99 84.86 phospholipid  transfer  protein

17362986 Ms4a2 3.332E-­‐03 up 5.41 78.92 426.79 90.23 membrane-­‐spanning  4-­‐domains,  subfamily  A,  member  2

17241013 Ccdc109a 3.338E-­‐03 up 1.55 698.33 1081.39 641.85 coiled-­‐coil  domain  containing  109A

17292333 Ptpdc1 3.381E-­‐03 up 1.86 41.72 77.66 36.04 protein  tyrosine  phosphatase  domain  containing  1

17248288 Sh3pxd2b 3.427E-­‐03 up 3.64 92.19 335.10 104.73 SH3  and  PX  domains  2B

17255663 Copz2 3.427E-­‐03 up 2.34 79.11 184.92 78.52 coatomer  protein  complex,  subunit  zeta  2

17512221 Cmtm4 3.427E-­‐03 up 1.83 127.33 233.44 124.82 CKLF-­‐like  MARVEL  transmembrane  domain  containing  4

17457504 Rab19 3.465E-­‐03 up 1.65 76.61 126.34 89.32 RAB19,  member  RAS  oncogene  family

17376541 Prnp|Prnd 3.539E-­‐03 up 1.87 53.27 99.80 65.67 prion  protein  |  prion  protein  dublet

17374880 Tyro3 3.539E-­‐03 up 1.51 58.85 88.70 54.13 TYRO3  protein  tyrosine  kinase  3

17548315 LOC1008621323.642E-­‐03 up 2.37 52.25 123.70 52.7817318083 Ly6a 3.642E-­‐03 up 1.53 89.30 136.87 105.43 lymphocyte  antigen  6  complex,  locus  A

17231477 Myct1 3.642E-­‐03 up 1.81 68.71 124.48 58.80 myc  target  1

17229259 Mpzl1 3.709E-­‐03 up 1.62 304.41 492.44 348.10 myelin  protein  zero-­‐like  1

17326405 Dcbld2 3.717E-­‐03 up 1.63 44.24 72.19 45.52 discoidin,  CUB  and  LCCL  domain  containing  2

17526707 Zbtb16 3.717E-­‐03 up 1.54 587.87 903.65 539.91 zinc  finger  and  BTB  domain  containing  16

17348840 Rnf125 3.804E-­‐03 up 1.51 1176.03 1774.94 1007.32 ring  finger  protein  125

17300998 Shisa2 3.863E-­‐03 up 1.85 112.98 209.29 111.77 shisa  homolog  2  (Xenopus  laevis)

17470988 Ptms 3.863E-­‐03 up 1.53 106.32 162.75 117.73 parathymosin

17539966 Gata1 3.868E-­‐03 up 2.42 108.71 262.59 111.21 GATA  binding  protein  1

17295130 F2r 3.922E-­‐03 up 1.62 125.18 203.03 118.35 coagulation  factor  II  (thrombin)  receptor

17260668 Grb10 4.026E-­‐03 up 2.71 116.48 315.66 77.02 growth  factor  receptor  bound  protein  10

17500662 Zdhhc2 4.034E-­‐03 up 1.68 206.75 347.10 213.93 zinc  finger,  DHHC  domain  containing  2

17339013 Emr1 4.291E-­‐03 up 2.00 261.16 521.07 333.90 EGF-­‐like  module  containing,  mucin-­‐like,  hormone  receptor-­‐like  sequence  1

17503841 Lpcat2 4.296E-­‐03 up 1.57 43.82 68.90 44.46 lysophosphatidylcholine  acyltransferase  2

17504572 Ces2g 4.298E-­‐03 up 3.97 63.17 250.96 66.77 carboxylesterase  2G

17239142 Samd5 4.392E-­‐03 up 1.51 42.67 64.57 40.84 sterile  alpha  motif  domain  containing  5

17264792 Atp1b2 4.406E-­‐03 up 2.68 62.06 166.62 67.75 ATPase,  Na+/K+  transporting,  beta  2  polypeptide

17459870 Loxl3 4.406E-­‐03 up 1.59 64.35 102.50 76.20 lysyl  oxidase-­‐like  3

17350824 Slc12a2 4.429E-­‐03 up 2.55 96.87 246.64 97.94 solute  carrier  family  12,  member  2

17372670 Prg3 4.553E-­‐03 up 6.44 108.03 695.87 82.84 proteoglycan  3

17411751 Trp53inp1 4.553E-­‐03 up 1.74 239.32 416.22 291.99 transformation  related  protein  53  inducible  nuclear  protein  1

17388353 Gyltl1b 4.751E-­‐03 up 1.65 50.60 83.68 52.24 glycosyltransferase-­‐like  1B

17380915 Col20a1 4.795E-­‐03 up 1.55 84.47 131.19 88.00 collagen,  type  XX,  alpha  1

17355189 Spire1 4.822E-­‐03 up 1.93 148.23 285.46 146.29 spire  homolog  1  (Drosophila)

17409963 Fnbp1l 4.847E-­‐03 up 1.72 30.18 51.76 26.97 formin  binding  protein  1-­‐like

17217247 Pik3c2b 4.847E-­‐03 up 1.94 76.46 148.46 74.30 phosphoinositide-­‐3-­‐kinase,  class  2,  beta  polypeptide

17457876 Gstk1 4.847E-­‐03 up 1.66 742.10 1228.70 677.29 glutathione  S-­‐transferase  kappa  1

17423577 Atp6v0d2 4.893E-­‐03 up 3.63 42.24 153.40 41.43 ATPase,  H+  transporting,  lysosomal  V0  subunit  D2

17228624 Rasal2 4.904E-­‐03 up 1.95 42.83 83.52 42.48 RAS  protein  activator  like  2

17238878 Syne1 4.942E-­‐03 up 2.95 56.23 166.13 35.07 synaptic  nuclear  envelope  1

17514678 Maml2 4.942E-­‐03 up 1.86 158.32 294.62 176.82 mastermind  like  2  (Drosophila)

17337706 Rhag 4.942E-­‐03 up 1.80 48.91 88.02 54.60 Rhesus  blood  group-­‐associated  A  glycoprotein

17238844 Syne1 4.976E-­‐03 up 1.92 40.28 77.38 34.42 synaptic  nuclear  envelope  1

17299101 Ear2|Ear12|Ear34.985E-­‐03 up 3.57 37.29 133.10 36.61 eosinophil-­‐associated,  ribonuclease  A  family,  member  2  |  eosinophil-­‐associated,  ribonuclease  A  family,  member  12  |  eosinophil-­‐associated,  ribonuclease  A  family,  member  3

17360823 Slc18a2 5.046E-­‐03 up 5.53 136.18 752.71 158.44 solute  carrier  family  18  (vesicular  monoamine),  member  2

17525263 Aplp2 5.109E-­‐03 up 1.68 940.97 1580.64 909.63 amyloid  beta  (A4)  precursor-­‐like  protein  2

17413852 Galnt12 5.157E-­‐03 up 2.19 64.71 141.55 70.26 UDP-­‐N-­‐acetyl-­‐alpha-­‐D-­‐galactosamine:polypeptide  N-­‐acetylgalactosaminyltransferase  12

17515819 Ets1 5.232E-­‐03 up 2.63 68.21 179.42 58.05 E26  avian  leukemia  oncogene  1,  5'  domain

17375327 Casc4 5.232E-­‐03 up 2.35 98.14 231.03 88.08 cancer  susceptibility  candidate  4

17411262 St6galnac3 5.232E-­‐03 up 2.06 53.34 109.69 59.47 ST6  (alpha-­‐N-­‐acetyl-­‐neuraminyl-­‐2,3-­‐beta-­‐galactosyl-­‐1,3)-­‐N-­‐acetylgalactosaminide  alpha-­‐2,6-­‐sialyltransferase  3

17337796 Pla2g7 5.232E-­‐03 up 1.75 47.15 82.34 44.80 phospholipase  A2,  group  VII  (platelet-­‐activating  factor  acetylhydrolase,  plasma)

17300961 Rcbtb1 5.232E-­‐03 up 1.69 144.02 243.39 178.27 regulator  of  chromosome  condensation  (RCC1)  and  BTB  (POZ)  domain  containing  protein  1

17355722 Sall3 5.232E-­‐03 up 1.67 34.52 57.67 35.35 sal-­‐like  3  (Drosophila)

17217035 Ctse 5.232E-­‐03 up 1.57 2899.40 4550.77 2466.20 cathepsin  E

17466360 Kel 5.232E-­‐03 up 1.56 33.43 52.18 33.5717238834 Syne1 5.233E-­‐03 up 2.49 55.00 136.69 43.66 synaptic  nuclear  envelope  1

17239546 D10Bwg1379e5.366E-­‐03 up 2.11 114.72 242.34 133.18 DNA  segment,  Chr  10,  Brigham  &  Women's  Genetics  1379  expressed

17524847 Epor 5.474E-­‐03 up 1.80 74.99 134.82 72.49 erythropoietin  receptor

17533269 Tspan7 5.480E-­‐03 up 1.99 32.55 64.92 39.93 tetraspanin  7

17283155 Eml5 5.526E-­‐03 up 3.78 92.84 350.49 93.97 echinoderm  microtubule  associated  protein  like  5

17466060 Parp12 5.526E-­‐03 up 1.87 319.33 597.42 397.02 poly  (ADP-­‐ribose)  polymerase  family,  member  12

17413403 Npr2 5.537E-­‐03 up 2.62 42.64 111.65 41.36 natriuretic  peptide  receptor  2

17356526 Ltbp3 5.539E-­‐03 up 1.56 49.27 76.81 51.11 latent  transforming  growth  factor  beta  binding  protein  3

17415570 Hook1 5.605E-­‐03 up 1.61 120.14 193.82 118.45 hook  homolog  1  (Drosophila)

17373930 0610012H03Rik5.737E-­‐03 up 1.53 79.16 121.41 76.08 RIKEN  cDNA  0610012H03  gene

17403514 Ssx2ip 6.169E-­‐03 up 1.52 208.94 318.01 223.29 synovial  sarcoma,  X  breakpoint  2  interacting  protein

17417138 Tal1 6.257E-­‐03 up 1.54 121.90 187.50 119.64 T  cell  acute  lymphocytic  leukemia  1

17252081 Pld2 6.369E-­‐03 up 1.58 87.06 137.48 102.85 phospholipase  D2

17500832 Fat1 6.457E-­‐03 up 1.52 33.18 50.52 32.52 FAT  tumor  suppressor  homolog  1  (Drosophila)

17315245 Krt18 6.552E-­‐03 up 1.83 33.45 61.08 32.14 keratin  18

17502816 Inpp4b 6.569E-­‐03 up 1.90 51.80 98.47 48.61 inositol  polyphosphate-­‐4-­‐phosphatase,  type  II

17329636 Gp5 6.574E-­‐03 up 1.93 33.02 63.89 33.27 glycoprotein  5  (platelet)

17328742 Slc7a4 6.574E-­‐03 up 1.78 39.03 69.42 36.11 solute  carrier  family  7  (cationic  amino  acid  transporter,  y+  system),  member  4

17264984 Nlgn2 6.584E-­‐03 up 1.81 257.07 464.47 249.03 neuroligin  2

17512732 Nqo1 6.655E-­‐03 up 1.80 62.01 111.35 68.30 NAD(P)H  dehydrogenase,  quinone  1

17320486 Odf3b 6.686E-­‐03 up 1.78 53.86 95.60 53.03 outer  dense  fiber  of  sperm  tails  3B

17414009 5730528L13Rik6.813E-­‐03 up 3.72 58.82 218.94 54.01 RIKEN  cDNA  5730528L13  gene

17530406 Acpp 6.813E-­‐03 up 2.93 62.35 182.90 57.16 acid  phosphatase,  prostate

17371969 Sp9 6.894E-­‐03 up 2.60 51.92 134.81 55.71 trans-­‐acting  transcription  factor  9

17304523 Stab1 6.971E-­‐03 up 1.57 56.62 88.65 72.95 stabilin  1

17239113 Sash1 7.021E-­‐03 up 1.75 58.30 102.00 57.44 SAM  and  SH3  domain  containing  1

17266038 Fam101b 7.522E-­‐03 up 2.27 235.24 533.44 180.21 family  with  sequence  similarity  101,  member  B

17279509 Crip1 7.522E-­‐03 up 1.91 630.46 1207.17 628.37 cysteine-­‐rich  protein  1  (intestinal)

17501633 Lpl 7.604E-­‐03 up 2.61 36.10 94.18 33.37 lipoprotein  lipase

17479099 Igf1r 7.612E-­‐03 up 1.89 883.85 1666.16 738.72 insulin-­‐like  growth  factor  I  receptor

17383467 Ntng2 7.630E-­‐03 up 1.66 236.41 393.22 206.79 netrin  G2

17340050 Plekhh2 7.746E-­‐03 up 1.79 205.95 368.00 170.94 pleckstrin  homology  domain  containing,  family  H  (with  MyTH4  domain)  member  2

17417407 Pik3r3 7.864E-­‐03 up 2.00 56.32 112.84 75.21 phosphatidylinositol  3  kinase,  regulatory  subunit,  polypeptide  3  (p55)

17545785 Cdkl5 7.864E-­‐03 up 1.81 33.86 61.15 32.46 cyclin-­‐dependent  kinase-­‐like  5

17498188 Ascl2 7.864E-­‐03 up 1.67 53.36 89.28 45.58 achaete-­‐scute  complex  homolog  2  (Drosophila)

17238916 Syne1 7.954E-­‐03 up 3.32 91.09 302.31 55.24 synaptic  nuclear  envelope  1

17238932 Syne1 8.004E-­‐03 up 2.85 126.86 361.60 85.36 synaptic  nuclear  envelope  1

17238912 Syne1 8.004E-­‐03 up 2.81 81.48 229.27 50.99 synaptic  nuclear  envelope  1

17238938 Syne1 8.004E-­‐03 up 2.75 67.99 187.16 46.33 synaptic  nuclear  envelope  1

17238908 Syne1 8.004E-­‐03 up 1.70 41.23 69.99 37.04 synaptic  nuclear  envelope  1

17399266 Pklr 8.004E-­‐03 up 1.52 57.63 87.50 60.52 pyruvate  kinase  liver  and  red  blood  cell

17217666 Tmem9 8.042E-­‐03 up 1.90 220.57 419.51 209.93 transmembrane  protein  9

17238850 Syne1 8.054E-­‐03 up 2.44 46.15 112.74 35.69 synaptic  nuclear  envelope  1

17543870 Zdhhc15 8.054E-­‐03 up 1.54 38.42 59.23 34.49 zinc  finger,  DHHC  domain  containing  15

17398406 Fam198b 8.072E-­‐03 up 1.53 40.77 62.50 45.91 family  with  sequence  similarity  198,  member  B

17502330 Zfp709 8.085E-­‐03 up 1.87 57.94 108.37 59.13 zinc  finger  protein  709

17496150 Lat 8.141E-­‐03 up 1.82 100.82 183.06 107.47 linker  for  activation  of  T  cells

17464588 Sgce 8.155E-­‐03 up 1.65 26.39 43.56 26.22 sarcoglycan,  epsilon

17372662 Prg2 8.158E-­‐03 up 9.77 221.52 2164.20 111.85 proteoglycan  2,  bone  marrow

17257235 Itgb3 8.158E-­‐03 up 1.55 291.31 452.14 310.76 integrin  beta  3

17480030 Rab38 8.158E-­‐03 up 1.52 500.51 762.46 469.20 RAB38,  member  of  RAS  oncogene  family

17485389 Shank2 8.190E-­‐03 up 1.64 55.17 90.50 53.78 SH3/ankyrin  domain  gene  2

17228234 Npl 8.237E-­‐03 up 3.26 465.07 1515.84 492.87 N-­‐acetylneuraminate  pyruvate  lyase

17392748 Ninl 8.237E-­‐03 up 1.69 58.30 98.66 63.06 ninein-­‐like

17343088 Mdga1 8.468E-­‐03 up 2.14 66.27 141.77 67.48 MAM  domain  containing  glycosylphosphatidylinositol  anchor  1

17325892 Cd200r3 8.540E-­‐03 up 1.75 62.89 110.21 58.96 CD200  receptor  3

17445715 Cd36 8.603E-­‐03 up 3.55 24.24 85.97 24.55 CD36  antigen

17215820 Gpc1 8.603E-­‐03 up 2.15 698.54 1503.77 701.43 glypican  1

17375997 Mertk 8.736E-­‐03 up 1.83 58.28 106.91 74.14 c-­‐mer  proto-­‐oncogene  tyrosine  kinase

17502729 Zfp827 8.876E-­‐03 up 2.61 27.55 71.93 27.14 zinc  finger  protein  827

17238934 Syne1 9.067E-­‐03 up 4.79 94.94 455.13 50.44 synaptic  nuclear  envelope  1

17238862 Syne1 9.097E-­‐03 up 2.24 33.54 75.19 25.20 synaptic  nuclear  envelope  1

17391915 Erv3 9.108E-­‐03 up 2.07 32.77 67.85 33.64 endogenous  retroviral  sequence  3

17310872 Sdc2 9.190E-­‐03 up 2.18 36.25 78.89 38.61 syndecan  2

17356216 Pcx 9.244E-­‐03 up 1.55 165.56 257.06 143.42 pyruvate  carboxylase

17238928 Syne1 9.413E-­‐03 up 2.82 112.46 317.49 82.53 synaptic  nuclear  envelope  1

17436907 Afap1 9.452E-­‐03 up 2.64 115.03 303.37 97.78 actin  filament  associated  protein  1

17340197 Epas1 9.608E-­‐03 up 1.61 62.34 100.24 61.39 endothelial  PAS  domain  protein  1

17404337 Cpa3 9.874E-­‐03 up 5.90 1037.10 6117.65 1049.43 carboxypeptidase  A3,  mast  cell

17238868 Syne1 9.874E-­‐03 up 2.97 55.62 164.97 39.42 synaptic  nuclear  envelope  1

17268714 Stac2 9.874E-­‐03 up 2.17 53.01 114.82 58.42 SH3  and  cysteine  rich  domain  2

17401710 Psrc1 9.874E-­‐03 up 1.88 123.17 231.80 112.74 proline/serine-­‐rich  coiled-­‐coil  1

17287984 Dapk1 9.874E-­‐03 up 1.66 46.22 76.55 44.70 death  associated  protein  kinase  1

17518109 Uaca 9.874E-­‐03 up 1.63 29.41 48.00 31.32 uveal  autoantigen  with  coiled-­‐coil  domains  and  ankyrin  repeats

17466075 Jhdm1d 9.874E-­‐03 up 1.55 177.81 274.86 227.39 jumonji  C  domain-­‐containing  histone  demethylase  1  homolog  D  (S.  cerevisiae)

17238826 Syne1 9.993E-­‐03 up 1.71 45.50 77.80 39.07 synaptic  nuclear  envelope  1

Transcripts  Cluster  Id

Gene  Symbol

p  value  (Corrected)  

Regulation

Fold  change  (FC)

Average  Expression  -­‐shEED  [n=3]

Average  Expression  -­‐shRenilla  [n=3] Gene  description

17260625 3.380E-­‐07 up 13.10 462.49 35.3217325324 Stfa2l1 7.390E-­‐07 up 11.48 378.10 32.94 stefin  A2  like  117372662 Prg2 5.000E-­‐05 up 10.68 823.31 77.05 proteoglycan  2,  bone  marrow17267520 Epx 7.970E-­‐05 up 8.96 466.73 52.11 eosinophil  peroxidase17404337 Cpa3 8.080E-­‐05 up 8.13 3352.91 412.58 carboxypeptidase  A3,  mast  cell17504572 Ces2g 3.130E-­‐05 up 6.92 488.88 70.65 carboxylesterase  2G17404180 Car1 3.340E-­‐04 up 6.88 320.97 46.62 carbonic  anhydrase  117248276 Hba-­‐a2|Hba-­‐a16.809E-­‐03 up 6.41 358.34 55.87 hemoglobin  alpha,  adult  chain  2  |  hemoglobin  alpha,  adult  chain  117231477 Myct1 2.740E-­‐05 up 5.35 271.70 50.82 myc  target  117365098 Scd1 1.130E-­‐04 up 5.14 931.20 181.29 stearoyl-­‐Coenzyme  A  desaturase  117294458 Rhobtb3 1.732E-­‐03 up 4.80 144.58 30.14 Rho-­‐related  BTB  domain  containing  317456721 Smo 2.380E-­‐04 up 4.63 951.85 205.69 smoothened  homolog  (Drosophila)17360823 Slc18a2 1.569E-­‐03 up 4.57 345.02 75.44 solute  carrier  family  18  (vesicular  monoamine),  member  217453874 A630081J09Rik8.756E-­‐03 up 4.32 266.89 61.76 RIKEN  cDNA  A630081J09  gene17294694 4.560E-­‐05 up 3.99 228.91 57.3617492431 Anpep 3.850E-­‐04 up 3.97 279.73 70.38 alanyl  (membrane)  aminopeptidase17231844 Perp 7.080E-­‐05 up 3.82 452.76 118.46 PERP,  TP53  apoptosis  effector17485430 Fgf3 6.570E-­‐04 up 3.80 219.24 57.69 fibroblast  growth  factor  317233347 Gja1 3.510E-­‐04 up 3.54 208.84 59.01 gap  junction  protein,  alpha  117375327 Casc4 1.200E-­‐04 up 3.42 148.94 43.59 cancer  susceptibility  candidate  417539966 Gata1 5.550E-­‐04 up 3.40 205.97 60.65 GATA  binding  protein  117325347 Stfa1 1.390E-­‐04 up 3.29 87.05 26.46 stefin  A117427494 Dock7 2.640E-­‐04 up 3.29 287.31 87.44 dedicator  of  cytokinesis  717305520 Ear1 1.832E-­‐03 up 3.26 75.19 23.03 eosinophil-­‐associated,  ribonuclease  A  family,  member  117517831 Cyp11a1 4.913E-­‐03 up 3.25 120.85 37.24 cytochrome  P450,  family  11,  subfamily  a,  polypeptide  117337706 Rhag 6.520E-­‐04 up 3.23 116.38 36.04 Rhesus  blood  group-­‐associated  A  glycoprotein

Supplemental  Table  S3.  List  of  the  differentially  expressed  transcripts  in  an  MLL-­‐AF9-­‐transformed  leukemia  progenitor  line  (MLL-­‐AF9-­‐puro)  following    induction  of  shRNA  against  EED  or  Renilla  for  6  days,  as  detected  by  microarray  analysis  with  a  cut-­‐off  of  >1.5-­‐fold  and  p<0.01.  

17283155 Eml5 1.314E-­‐03 up 3.22 162.89 50.55 echinoderm  microtubule  associated  protein  like  517276386 Rhoj 7.080E-­‐05 up 3.20 183.18 57.29 ras  homolog  gene  family,  member  J17372670 Prg3 9.870E-­‐05 up 3.18 220.57 69.37 proteoglycan  317217136 Slc45a3 4.990E-­‐03 up 3.12 126.22 40.42 solute  carrier  family  45,  member  317354378 Ppic 1.190E-­‐04 up 3.11 215.85 69.49 peptidylprolyl  isomerase  C17372621 Slc43a1 5.370E-­‐06 up 3.10 270.43 87.15 solute  carrier  family  43,  member  117228164 Lamc1 1.200E-­‐04 up 3.07 253.56 82.48 laminin,  gamma  117362986 Ms4a2 1.506E-­‐03 up 3.06 136.20 44.51 membrane-­‐spanning  4-­‐domains,  subfamily  A,  member  217368550 Rxra 4.460E-­‐05 up 3.04 232.62 76.56 retinoid  X  receptor  alpha17286641 Dsp 1.091E-­‐03 up 2.89 835.74 289.14 desmoplakin17422996 Plag1 2.970E-­‐04 up 2.89 197.35 68.31 pleiomorphic  adenoma  gene  117293103 Gkap1 5.120E-­‐05 up 2.87 282.42 98.57 G  kinase  anchoring  protein  117299101 Ear2|Ear12|Ear37.080E-­‐05 up 2.86 74.72 26.10 eosinophil-­‐associated,  ribonuclease  A  family,  member  2  |  eosinophil-­‐associated,  ribonuclease  A  family,  member  12  |  eosinophil-­‐associated,  ribonuclease  A  family,  member  3

17418035 Cited4 5.048E-­‐03 up 2.86 114.31 40.02 Cbp/p300-­‐interacting  transactivator,  with  Glu/Asp-­‐rich  carboxy-­‐terminal  domain,  417458734 Plekha8 5.550E-­‐04 up 2.82 153.19 54.37 pleckstrin  homology  domain  containing,  family  A  (phosphoinositide  binding  specific)  member  817340817 Slc22a3 6.250E-­‐03 up 2.74 402.27 147.03 solute  carrier  family  22  (organic  cation  transporter),  member  317396143 Car13 1.150E-­‐04 up 2.72 130.30 47.92 carbonic  anhydrase  1317270222 Itga2b 2.717E-­‐03 up 2.67 463.65 173.63 integrin  alpha  2b17306147 Ndrg2 7.970E-­‐05 up 2.67 118.64 44.44 N-­‐myc  downstream  regulated  gene  217430645 Nkain1 7.596E-­‐03 up 2.67 203.25 76.20 Na+/K+  transporting  ATPase  interacting  117220059 Cdc42bpa 1.482E-­‐03 up 2.63 71.30 27.07 CDC42  binding  protein  kinase  alpha17524847 Epor 6.660E-­‐04 up 2.63 116.32 44.22 erythropoietin  receptor17248621 Ccnjl 8.080E-­‐05 up 2.60 148.62 57.06 cyclin  J-­‐like17307738 Fzd3 5.630E-­‐05 up 2.60 132.59 51.09 frizzled  homolog  3  (Drosophila)17449084 Igfbp7 1.514E-­‐03 up 2.57 1908.19 742.51 insulin-­‐like  growth  factor  binding  protein  717352549 Mpp7 1.986E-­‐03 up 2.53 131.00 51.73 membrane  protein,  palmitoylated  7  (MAGUK  p55  subfamily  member  7)17256975 Fzd2 1.482E-­‐03 up 2.52 104.71 41.48 frizzled  homolog  2  (Drosophila)17276674 Gphn 3.520E-­‐05 up 2.51 1084.49 432.58 gephyrin17428509 Faah 1.130E-­‐04 up 2.46 141.20 57.32 fatty  acid  amide  hydrolase17232358 Ptprk 3.320E-­‐04 up 2.46 294.74 119.89 protein  tyrosine  phosphatase,  receptor  type,  K17312054 Khdrbs3 7.957E-­‐03 up 2.46 127.35 51.83 KH  domain  containing,  RNA  binding,  signal  transduction  associated  317314190 Shank3 1.920E-­‐04 up 2.43 163.72 67.31 SH3/ankyrin  domain  gene  317226203 Epb4.1l5 8.080E-­‐05 up 2.43 130.99 53.86 erythrocyte  protein  band  4.1-­‐like  5

17502994 Lphn1 3.320E-­‐04 up 2.43 402.96 166.02 latrophilin  117540059 Porcn 2.950E-­‐04 up 2.41 87.64 36.40 porcupine  homolog  (Drosophila)17301926 Htr2a 6.640E-­‐05 up 2.40 114.30 47.56 5-­‐hydroxytryptamine  (serotonin)  receptor  2A17328958 Gp1bb|Sept52.591E-­‐03 up 2.40 116.86 48.79 glycoprotein  Ib,  beta  polypeptide  |  septin  517224771 Serpine2 3.804E-­‐03 up 2.39 340.91 142.59 serine  (or  cysteine)  peptidase  inhibitor,  clade  E,  member  217321263 Adcy6 7.080E-­‐05 up 2.39 91.48 38.27 adenylate  cyclase  617404585 Zmat3 9.470E-­‐04 up 2.39 234.78 98.26 zinc  finger  matrin  type  317356288 Ctsf 2.044E-­‐03 up 2.39 88.24 36.95 cathepsin  F17430373 Zbtb8a 6.070E-­‐04 up 2.37 173.74 73.28 zinc  finger  and  BTB  domain  containing  8a17497980 Muc6 4.111E-­‐03 up 2.37 111.75 47.23 mucin  6,  gastric17249898 Galnt10 1.340E-­‐05 up 2.35 256.43 108.92 UDP-­‐N-­‐acetyl-­‐alpha-­‐D-­‐galactosamine:polypeptide  N-­‐acetylgalactosaminyltransferase  1017221197 Tcf24 9.709E-­‐03 up 2.34 94.37 40.24 transcription  factor  2417468828 Plxna1 2.830E-­‐04 up 2.34 172.45 73.77 plexin  A117267471 Ppm1e 2.188E-­‐03 up 2.33 94.68 40.59 protein  phosphatase  1E  (PP2C  domain  containing)17503254 Klf1 1.033E-­‐03 up 2.33 113.54 48.72 Kruppel-­‐like  factor  1  (erythroid)17247852 Bcl11a 6.772E-­‐03 up 2.32 372.53 160.75 B  cell  CLL/lymphoma  11A  (zinc  finger  protein)17367163 Cacnb2 6.070E-­‐04 up 2.30 163.70 71.08 calcium  channel,  voltage-­‐dependent,  beta  2  subunit17508691 Rbpms 1.770E-­‐04 up 2.30 112.82 49.03 RNA  binding  protein  gene  with  multiple  splicing17538628 Fgd1 1.314E-­‐03 up 2.24 91.35 40.71 FYVE,  RhoGEF  and  PH  domain  containing  117411961 Tmem64 1.710E-­‐04 up 2.23 300.33 134.93 transmembrane  protein  6417345728 Mdfi 7.480E-­‐04 up 2.22 200.78 90.35 MyoD  family  inhibitor17325892 Cd200r3 4.561E-­‐03 up 2.22 128.60 58.00 CD200  receptor  317248288 Sh3pxd2b 9.540E-­‐04 up 2.21 111.51 50.36 SH3  and  PX  domains  2B17313705 Parvb 3.683E-­‐03 up 2.19 162.38 74.20 parvin,  beta17548468 Rbpms2|Gm34708.890E-­‐04 up 2.18 1032.58 472.62 RNA  binding  protein  with  multiple  splicing  2  |  predicted  gene  347017310912 Laptm4b 1.380E-­‐04 up 2.18 788.10 361.37 lysosomal-­‐associated  protein  transmembrane  4B17223985 Ikzf2 1.395E-­‐03 up 2.18 856.15 393.03 IKAROS  family  zinc  finger  217377144 Slc24a3 2.810E-­‐04 up 2.18 186.35 85.58 solute  carrier  family  24  (sodium/potassium/calcium  exchanger),  member  317348138 9430020K01Rik1.373E-­‐03 up 2.15 271.12 125.89 RIKEN  cDNA  9430020K01  gene17301365 Fbxo16 4.740E-­‐04 up 2.15 217.18 100.88 F-­‐box  protein  1617502789 Gypa 2.965E-­‐03 up 2.15 49.83 23.17 glycophorin  A17355189 Spire1 3.514E-­‐03 up 2.14 179.69 83.80 spire  homolog  1  (Drosophila)17429191 Mpl 7.370E-­‐05 up 2.13 106.78 50.02 myeloproliferative  leukemia  virus  oncogene

17248263 Hba-­‐a2|Hba-­‐a11.569E-­‐03 up 2.13 69.93 32.81 hemoglobin  alpha,  adult  chain  2  |  hemoglobin  alpha,  adult  chain  117376728 Plcb4 6.370E-­‐04 up 2.12 237.61 112.21 phospholipase  C,  beta  417249542 8-­‐Sep 2.566E-­‐03 up 2.12 245.79 116.14 septin  817522958 Ctdspl 1.530E-­‐04 up 2.12 638.37 301.66 CTD  (carboxy-­‐terminal  domain,  RNA  polymerase  II,  polypeptide  A)  small  phosphatase-­‐like17334638 Prss34 3.186E-­‐03 up 2.11 90.28 42.71 protease,  serine,  3417388999 Prrg4 5.770E-­‐04 up 2.11 234.55 111.06 proline  rich  Gla  (G-­‐carboxyglutamic  acid)  4  (transmembrane)17227348 Nav1 3.330E-­‐04 up 2.11 351.89 166.88 neuron  navigator  117464455 Far2 3.109E-­‐03 up 2.10 1221.21 582.56 fatty  acyl  CoA  reductase  217212211 Il1rl1 6.809E-­‐03 up 2.09 55.47 26.53 interleukin  1  receptor-­‐like  117462373 Cecr2 2.869E-­‐03 up 2.09 175.55 84.06 cat  eye  syndrome  chromosome  region,  candidate  217369368 Ncs1 8.230E-­‐04 up 2.09 373.90 179.07 neuronal  calcium  sensor  117228751 Rabgap1l 1.380E-­‐03 up 2.08 123.59 59.47 RAB  GTPase  activating  protein  1-­‐like17468298 Exoc6b 1.360E-­‐05 up 2.07 330.15 159.35 exocyst  complex  component  6B17384021 Stxbp1 1.320E-­‐04 up 2.05 342.15 166.63 syntaxin  binding  protein  117414212 Fsd1l 6.460E-­‐04 up 2.05 83.84 40.92 fibronectin  type  III  and  SPRY  domain  containing  1-­‐like17329636 Gp5 1.320E-­‐04 up 2.05 62.00 30.32 glycoprotein  5  (platelet)17429206 Tie1 1.466E-­‐03 up 2.04 97.92 47.94 tyrosine  kinase  with  immunoglobulin-­‐like  and  EGF-­‐like  domains  117271168 Prkca 1.757E-­‐03 up 2.04 257.32 126.28 protein  kinase  C,  alpha17518616 Rbpms2|Gm34704.070E-­‐04 up 2.03 393.24 193.57 RNA  binding  protein  with  multiple  splicing  2  |  predicted  gene  347017416616 Echdc2 1.060E-­‐04 up 2.01 204.96 101.75 enoyl  Coenzyme  A  hydratase  domain  containing  217367852 Nrarp 3.320E-­‐04 up 2.01 207.06 102.93 Notch-­‐regulated  ankyrin  repeat  protein17512221 Cmtm4 2.950E-­‐04 up 2.01 220.88 110.03 CKLF-­‐like  MARVEL  transmembrane  domain  containing  417305221 5730469M10Rik8.250E-­‐03 up 2.00 196.53 98.13 RIKEN  cDNA  5730469M10  gene17543870 Zdhhc15 1.302E-­‐03 up 2.00 57.45 28.75 zinc  finger,  DHHC  domain  containing  1517338136 Cul7 3.614E-­‐03 up 1.99 183.93 92.30 cullin  717271281 Fam20a 1.986E-­‐03 up 1.99 150.77 75.68 family  with  sequence  similarity  20,  member  A17467171 Vopp1 2.186E-­‐03 up 1.99 219.02 109.99 vesicular,  overexpressed  in  cancer,  prosurvival  protein  117399266 Pklr 3.302E-­‐03 up 1.98 123.23 62.19 pyruvate  kinase  liver  and  red  blood  cell17396162 Car2 1.020E-­‐04 up 1.97 297.08 150.44 carbonic  anhydrase  217411103 Lphn2 2.869E-­‐03 up 1.97 65.25 33.11 latrophilin  217276020 Frmd6 2.293E-­‐03 up 1.97 139.61 70.91 FERM  domain  containing  617310872 Sdc2 6.050E-­‐04 up 1.97 85.67 43.55 syndecan  217217666 Tmem9 3.600E-­‐04 up 1.96 373.35 190.49 transmembrane  protein  9

17303722 Kcnk5 1.314E-­‐03 up 1.95 62.80 32.15 potassium  channel,  subfamily  K,  member  517396492 Eif5a2 3.784E-­‐03 up 1.95 244.37 125.14 eukaryotic  translation  initiation  factor  5A217254948 Cuedc1 8.275E-­‐03 up 1.94 658.66 339.81 CUE  domain  containing  117487759 Cd177 1.395E-­‐03 up 1.93 180.19 93.24 CD177  antigen17302092 Tsc22d1|Gm195974.070E-­‐04 up 1.93 82.48 42.71 TSC22  domain  family,  member  1  |  predicted  gene,  1959717349926 Pcdhb17 1.302E-­‐03 up 1.93 63.17 32.74 protocadherin  beta  1717236623 Usp44 4.560E-­‐05 up 1.93 139.69 72.49 ubiquitin  specific  peptidase  4417366953 Prkcq 1.569E-­‐03 up 1.93 60.45 31.38 protein  kinase  C,  theta17543691 Phka1 1.986E-­‐03 up 1.92 63.57 33.04 phosphorylase  kinase  alpha  117443539 Ephb4 4.900E-­‐04 up 1.92 101.61 52.94 Eph  receptor  B417492196 Slco3a1 6.630E-­‐04 up 1.90 195.92 103.29 solute  carrier  organic  anion  transporter  family,  member  3a117526776 Ncam1 7.236E-­‐03 up 1.90 965.58 509.14 neural  cell  adhesion  molecule  117290865 Epdr1 8.438E-­‐03 up 1.89 67.56 35.71 ependymin  related  protein  1  (zebrafish)17351027 Pdgfrb 2.597E-­‐03 up 1.88 146.88 77.99 platelet  derived  growth  factor  receptor,  beta  polypeptide17408414 Fam46c 4.084E-­‐03 up 1.88 64.22 34.23 family  with  sequence  similarity  46,  member  C17450618 Tgfbr3 2.740E-­‐05 up 1.88 74.50 39.73 transforming  growth  factor,  beta  receptor  III17425248 Tmem246 9.316E-­‐03 up 1.87 81.71 43.74 transmembrane  protein  24617427033 Mllt3 5.000E-­‐05 up 1.87 208.53 111.66 myeloid/lymphoid  or  mixed-­‐lineage  leukemia  (trithorax  homolog,  Drosophila);  translocated  to,  3

17417146 Pdzk1ip1 8.300E-­‐04 up 1.86 107.64 57.74 PDZK1  interacting  protein  117374098 Lgr4 3.320E-­‐04 up 1.86 71.85 38.67 leucine-­‐rich  repeat-­‐containing  G  protein-­‐coupled  receptor  417339973 Pkdcc 7.310E-­‐04 up 1.86 81.02 43.67 protein  kinase  domain  containing,  cytoplasmic17363025 Stx3 1.022E-­‐03 up 1.85 105.70 57.08 syntaxin  317241512 Tet1 5.584E-­‐03 up 1.85 162.88 88.03 tet  methylcytosine  dioxygenase  117427147 Cdkn2a 2.487E-­‐03 up 1.84 161.93 87.89 cyclin-­‐dependent  kinase  inhibitor  2A17235243 Efna2 6.101E-­‐03 up 1.84 122.30 66.41 ephrin  A217456710 Tspan33 5.550E-­‐04 up 1.83 79.31 43.24 tetraspanin  3317548458 1.270E-­‐04 up 1.83 57.37 31.4117255335 Samd14 5.550E-­‐04 up 1.82 72.20 39.58 sterile  alpha  motif  domain  containing  1417318312 Nrbp2 4.876E-­‐03 up 1.82 62.76 34.42 nuclear  receptor  binding  protein  217457694 Trbv13-­‐2 4.449E-­‐03 up 1.82 52.05 28.55 T  cell  receptor  beta,  variable  13-­‐217509758 Csgalnact1 2.346E-­‐03 up 1.82 78.15 42.97 chondroitin  sulfate  N-­‐acetylgalactosaminyltransferase  117379606 Mmp9 5.048E-­‐03 up 1.82 71.47 39.33 matrix  metallopeptidase  917311179 Fzd6 5.000E-­‐05 up 1.81 54.00 29.81 frizzled  homolog  6  (Drosophila)

17214053 Xrcc5 1.580E-­‐04 up 1.81 143.01 78.96 X-­‐ray  repair  complementing  defective  repair  in  Chinese  hamster  cells  517470930 Leprel2 3.130E-­‐04 up 1.80 157.73 87.41 leprecan-­‐like  217338617 Emr4 1.405E-­‐03 up 1.80 78.46 43.59 EGF-­‐like  module  containing,  mucin-­‐like,  hormone  receptor-­‐like  sequence  417358375 Smarca2 9.530E-­‐05 up 1.80 493.24 274.02 SWI/SNF  related,  matrix  associated,  actin  dependent  regulator  of  chromatin,  subfamily  a,  member  2

17512732 Nqo1 1.326E-­‐03 up 1.80 82.99 46.20 NAD(P)H  dehydrogenase,  quinone  117430894 Smpdl3b 8.890E-­‐04 up 1.80 140.99 78.50 sphingomyelin  phosphodiesterase,  acid-­‐like  3B17397740 Mab21l1 2.232E-­‐03 up 1.79 75.04 41.86 mab-­‐21-­‐like  1  (C.  elegans)17466921 Jazf1 1.460E-­‐03 up 1.79 73.37 41.01 JAZF  zinc  finger  117498897 Tnfsf13b 3.320E-­‐04 up 1.79 107.06 59.86 tumor  necrosis  factor  (ligand)  superfamily,  member  13b17522971 Vill 1.302E-­‐03 up 1.79 58.11 32.53 villin-­‐like17391094 Hdc 9.991E-­‐03 up 1.78 1113.18 624.21 histidine  decarboxylase17284065 Cdc42bpb 2.330E-­‐04 up 1.78 261.50 146.68 CDC42  binding  protein  kinase  beta17325159 Mylk 8.550E-­‐04 up 1.78 117.34 65.83 myosin,  light  polypeptide  kinase17349922 Pcdhb16 5.048E-­‐03 up 1.78 57.64 32.37 protocadherin  beta  1617302523 Slain1 3.486E-­‐03 up 1.77 345.77 194.86 SLAIN  motif  family,  member  117322525 Glis2 1.410E-­‐04 up 1.77 486.45 275.05 GLIS  family  zinc  finger  217299954 2.526E-­‐03 up 1.77 203.97 115.3717299877 2.572E-­‐03 up 1.77 203.85 115.4217425466 Ctnnal1 3.317E-­‐03 up 1.76 103.61 58.82 catenin  (cadherin  associated  protein),  alpha-­‐like  117255663 Copz2 3.717E-­‐03 up 1.76 128.44 73.12 coatomer  protein  complex,  subunit  zeta  217234339 Adora2a 1.118E-­‐03 up 1.76 184.93 105.30 adenosine  A2a  receptor17382203 Hnmt 2.107E-­‐03 up 1.75 73.47 41.89 histamine  N-­‐methyltransferase17378359 Acss2 3.476E-­‐03 up 1.75 571.24 326.50 acyl-­‐CoA  synthetase  short-­‐chain  family  member  217214476 Des 3.614E-­‐03 up 1.75 108.36 61.96 desmin17264792 Atp1b2 1.314E-­‐03 up 1.74 86.61 49.78 ATPase,  Na+/K+  transporting,  beta  2  polypeptide17368171 Bmyc 4.590E-­‐04 up 1.74 2594.22 1490.96 brain  expressed  myelocytomatosis  oncogene17396439 Tnik 2.693E-­‐03 up 1.74 473.00 271.95 TRAF2  and  NCK  interacting  kinase17402193 Arhgap29 1.920E-­‐04 up 1.73 49.80 28.75 Rho  GTPase  activating  protein  2917480030 Rab38 6.710E-­‐04 up 1.73 745.17 430.34 RAB38,  member  of  RAS  oncogene  family17293362 Ctla2a 3.830E-­‐04 up 1.73 199.50 115.27 cytotoxic  T  lymphocyte-­‐associated  protein  2  alpha17342829 Tead3 6.460E-­‐04 up 1.73 107.58 62.36 TEA  domain  family  member  317464803 Ica1 5.190E-­‐04 up 1.72 143.79 83.49 islet  cell  autoantigen  117228234 Npl 3.804E-­‐03 up 1.72 852.65 495.80 N-­‐acetylneuraminate  pyruvate  lyase

17475801 Eid2 7.380E-­‐03 up 1.72 76.72 44.64 EP300  interacting  inhibitor  of  differentiation  217506356 Zfpm1 4.913E-­‐03 up 1.72 993.16 578.26 zinc  finger  protein,  multitype  117429057 Ptprf 8.890E-­‐04 up 1.71 80.00 46.71 protein  tyrosine  phosphatase,  receptor  type,  F17403514 Ssx2ip 2.615E-­‐03 up 1.71 239.75 140.27 synovial  sarcoma,  X  breakpoint  2  interacting  protein17257361 Mrc2 3.943E-­‐03 up 1.70 59.47 34.92 mannose  receptor,  C  type  217516462 Thy1 6.421E-­‐03 up 1.70 68.89 40.52 thymus  cell  antigen  1,  theta17239546 D10Bwg1379e2.274E-­‐03 up 1.70 143.90 84.74 DNA  segment,  Chr  10,  Brigham  &  Women's  Genetics  1379  expressed17371873 Rapgef4 3.292E-­‐03 up 1.69 70.64 41.75 Rap  guanine  nucleotide  exchange  factor  (GEF)  417239113 Sash1 6.070E-­‐04 up 1.69 82.47 48.78 SAM  and  SH3  domain  containing  117527520 Scamp5 4.990E-­‐03 up 1.69 225.45 133.36 secretory  carrier  membrane  protein  517253478 Rab34 1.520E-­‐03 up 1.69 153.27 90.67 RAB34,  member  of  RAS  oncogene  family17417138 Tal1 1.033E-­‐03 up 1.69 186.92 110.65 T  cell  acute  lymphocytic  leukemia  117443613 Tfr2 6.570E-­‐04 up 1.69 511.32 302.94 transferrin  receptor  217281971 Sgpp1 1.320E-­‐04 up 1.69 1012.56 599.94 sphingosine-­‐1-­‐phosphate  phosphatase  117228588 Ralgps2 1.130E-­‐04 up 1.69 68.11 40.36 Ral  GEF  with  PH  domain  and  SH3  binding  motif  217235990 1500009L16Rik1.341E-­‐03 up 1.69 156.07 92.54 RIKEN  cDNA  1500009L16  gene17499117 Atp11a 1.090E-­‐03 up 1.68 479.15 284.74 ATPase,  class  VI,  type  11A17306477 Slc7a8 1.825E-­‐03 up 1.68 3876.29 2311.11 solute  carrier  family  7  (cationic  amino  acid  transporter,  y+  system),  member  817215576 Agap1 2.010E-­‐04 up 1.67 539.81 322.31 ArfGAP  with  GTPase  domain,  ankyrin  repeat  and  PH  domain  117460634 Gata2 6.421E-­‐03 up 1.67 2724.11 1627.01 GATA  binding  protein  217300064 2.237E-­‐03 up 1.67 205.23 122.6817325329 Gm5483 4.579E-­‐03 up 1.67 36.74 21.97 predicted  gene  548317285097 Dip2c 7.810E-­‐04 up 1.67 57.61 34.47 DIP2  disco-­‐interacting  protein  2  homolog  C  (Drosophila)17398274 Ppm1l 1.380E-­‐04 up 1.67 115.31 69.06 protein  phosphatase  1  (formerly  2C)-­‐like17308361 Bmp1 7.550E-­‐05 up 1.67 79.61 47.79 bone  morphogenetic  protein  117353032 Fam59a 3.130E-­‐04 up 1.66 86.61 52.08 family  with  sequence  similarity  59,  member  A17450338 Klhl8 1.986E-­‐03 up 1.66 59.01 35.61 kelch-­‐like  8  (Drosophila)17268120 Pdk2 4.860E-­‐04 up 1.65 173.40 104.79 pyruvate  dehydrogenase  kinase,  isoenzyme  217364474 Pdlim1 8.150E-­‐04 up 1.65 1758.84 1063.09 PDZ  and  LIM  domain  1  (elfin)17441396 Vsig10 3.490E-­‐04 up 1.65 95.75 57.88 V-­‐set  and  immunoglobulin  domain  containing  1017339801 Crim1 4.449E-­‐03 up 1.65 155.52 94.20 cysteine  rich  transmembrane  BMP  regulator  1  (chordin  like)17526102 Oaf 7.580E-­‐04 up 1.65 77.38 46.91 OAF  homolog  (Drosophila)17456001 Glcci1 4.560E-­‐05 up 1.65 407.28 247.04 glucocorticoid  induced  transcript  1

17246284 Suox 1.656E-­‐03 up 1.65 300.04 182.03 sulfite  oxidase17495373 Plekha7 1.302E-­‐03 up 1.65 86.91 52.78 pleckstrin  homology  domain  containing,  family  A  member  717323675 Arvcf 9.920E-­‐04 up 1.65 122.33 74.31 armadillo  repeat  gene  deleted  in  velo-­‐cardio-­‐facial  syndrome17329236 Tmem41a 2.237E-­‐03 up 1.64 1242.74 758.88 transmembrane  protein  41a17306968 Mcpt8 3.044E-­‐03 up 1.64 1372.90 838.56 mast  cell  protease  817279562 Vipr2 3.850E-­‐04 up 1.63 56.78 34.73 vasoactive  intestinal  peptide  receptor  217479183 Fam174b 1.144E-­‐03 up 1.63 310.73 190.11 family  with  sequence  similarity  174,  member  B17319823 1700001L05Rik1.278E-­‐03 up 1.63 109.53 67.09 RIKEN  cDNA  1700001L05  gene17307354 Atp8a2 1.302E-­‐03 up 1.63 65.58 40.31 ATPase,  aminophospholipid  transporter-­‐like,  class  I,  type  8A,  member  217371019 Tanc1 8.550E-­‐04 up 1.63 97.40 59.88 tetratricopeptide  repeat,  ankyrin  repeat  and  coiled-­‐coil  containing  117414009 5730528L13Rik3.320E-­‐04 up 1.62 65.18 40.29 RIKEN  cDNA  5730528L13  gene17373930 0610012H03Rik5.151E-­‐03 up 1.61 134.12 83.13 RIKEN  cDNA  0610012H03  gene17544078 Itm2a 6.574E-­‐03 up 1.61 111.92 69.41 integral  membrane  protein  2A17272770 Timp2 2.869E-­‐03 up 1.61 457.64 283.93 tissue  inhibitor  of  metalloproteinase  217278188 Otub2 8.890E-­‐04 up 1.61 113.55 70.52 OTU  domain,  ubiquitin  aldehyde  binding  217535607 Slc6a8 1.432E-­‐03 up 1.61 113.29 70.50 solute  carrier  family  6  (neurotransmitter  transporter,  creatine),  member  817385574 Ly75 2.214E-­‐03 up 1.61 114.44 71.23 lymphocyte  antigen  7517363572 Tjp2 3.302E-­‐03 up 1.60 237.89 148.31 tight  junction  protein  217442834 Gpr133 6.591E-­‐03 up 1.60 89.17 55.64 G  protein-­‐coupled  receptor  13317433055 Kif1b 1.190E-­‐04 up 1.60 242.41 151.27 kinesin  family  member  1B17467742 Vamp5 1.390E-­‐04 up 1.60 1354.63 845.76 vesicle-­‐associated  membrane  protein  517366670 Itih5 2.717E-­‐03 up 1.60 632.89 395.89 inter-­‐alpha  (globulin)  inhibitor  H517381330 Optn 6.410E-­‐03 up 1.60 199.17 124.60 optineurin17539004 Klf8 2.725E-­‐03 up 1.60 154.96 96.95 Kruppel-­‐like  factor  817218349 Glul 1.860E-­‐03 up 1.60 485.16 303.57 glutamate-­‐ammonia  ligase  (glutamine  synthetase)17470540 Phc1 3.364E-­‐03 up 1.60 322.86 202.17 polyhomeotic-­‐like  1  (Drosophila)17501633 Lpl 6.497E-­‐03 up 1.60 53.26 33.37 lipoprotein  lipase17217500 Kdm5b 5.343E-­‐03 up 1.59 509.72 319.76 lysine  (K)-­‐specific  demethylase  5B17457876 Gstk1 6.460E-­‐04 up 1.59 1630.14 1025.91 glutathione  S-­‐transferase  kappa  117430680 Ptpru 2.021E-­‐03 up 1.59 122.30 77.12 protein  tyrosine  phosphatase,  receptor  type,  U17355463 Ctif|Gm105323.136E-­‐03 up 1.59 177.06 111.68 CBP80/20-­‐dependent  translation  initiation  factor  |  predicted  gene  1053217445525 Rundc3b 2.395E-­‐03 up 1.58 95.29 60.17 RUN  domain  containing  3B17390823 Slc30a4 6.542E-­‐03 up 1.58 49.74 31.44 solute  carrier  family  30  (zinc  transporter),  member  4

17403463 Syde2 1.130E-­‐04 up 1.58 68.45 43.27 synapse  defective  1,  Rho  GTPase,  homolog  2  (C.  elegans)17438969 Pf4 3.420E-­‐03 up 1.58 103.40 65.37 platelet  factor  417323649 Rtn4r 4.327E-­‐03 up 1.58 386.26 244.29 reticulon  4  receptor17506991 Nrp1 5.048E-­‐03 up 1.58 133.32 84.42 neuropilin  117500535 Dusp4 6.118E-­‐03 up 1.57 565.41 359.20 dual  specificity  phosphatase  417404601 Gnb4 4.979E-­‐03 up 1.57 78.63 50.04 guanine  nucleotide  binding  protein  (G  protein),  beta  417404835 Bbs7 6.070E-­‐04 up 1.57 63.21 40.26 Bardet-­‐Biedl  syndrome  7  (human)17327720 Adcy9 3.660E-­‐04 up 1.57 224.55 143.08 adenylate  cyclase  917356216 Pcx 6.460E-­‐04 up 1.57 252.48 160.94 pyruvate  carboxylase17392937 Sox12 8.713E-­‐03 up 1.57 63.77 40.69 SRY-­‐box  containing  gene  1217537936 Tceal1 2.738E-­‐03 up 1.56 51.12 32.67 transcription  elongation  factor  A  (SII)-­‐like  117451297 Adrbk2 3.053E-­‐03 up 1.56 268.63 171.79 adrenergic  receptor  kinase,  beta  217537895 Ngfrap1 6.460E-­‐04 up 1.56 1861.55 1193.08 nerve  growth  factor  receptor  (TNFRSF16)  associated  protein  117260618 2.738E-­‐03 up 1.56 41.82 26.8217480880 Pde2a 8.410E-­‐04 up 1.56 376.29 241.36 phosphodiesterase  2A,  cGMP-­‐stimulated17311533 Deptor 3.136E-­‐03 up 1.56 215.27 138.15 DEP  domain  containing  MTOR-­‐interacting  protein17274415 Asap2 3.850E-­‐04 up 1.56 95.96 61.70 ArfGAP  with  SH3  domain,  ankyrin  repeat  and  PH  domain  217438963 Ppbp 6.530E-­‐04 up 1.55 80.49 51.92 pro-­‐platelet  basic  protein17292871 Dbn1 2.777E-­‐03 up 1.55 352.14 227.37 drebrin  117377464 Cst7 1.072E-­‐03 up 1.55 1948.45 1260.79 cystatin  F  (leukocystatin)17500716 Slc7a2 7.693E-­‐03 up 1.54 46.66 30.23 solute  carrier  family  7  (cationic  amino  acid  transporter,  y+  system),  member  217296084 Zswim6 2.293E-­‐03 up 1.54 574.96 372.79 zinc  finger,  SWIM  domain  containing  617467806 Tcf7l1 1.460E-­‐03 up 1.54 74.57 48.39 transcription  factor  7  like  1  (T  cell  specific,  HMG  box)17463973 Plekha5 4.111E-­‐03 up 1.54 225.37 146.40 pleckstrin  homology  domain  containing,  family  A  member  517228349 Ier5 3.850E-­‐04 up 1.54 136.15 88.46 immediate  early  response  517519481 Gnb5 2.359E-­‐03 up 1.54 143.30 93.29 guanine  nucleotide  binding  protein  (G  protein),  beta  517369613 Aif1l 8.756E-­‐03 up 1.53 83.70 54.54 allograft  inflammatory  factor  1-­‐like17215097 B3gnt7 4.617E-­‐03 up 1.53 177.10 115.56 UDP-­‐GlcNAc:betaGal  beta-­‐1,3-­‐N-­‐acetylglucosaminyltransferase  717374880 Tyro3 1.238E-­‐03 up 1.53 75.29 49.14 TYRO3  protein  tyrosine  kinase  317287536 Tspan17 8.030E-­‐04 up 1.53 381.96 249.27 tetraspanin  1717510994 Il27ra 2.235E-­‐03 up 1.53 121.14 79.06 interleukin  27  receptor,  alpha17547877 Deptor 3.660E-­‐04 up 1.53 140.94 92.14 DEP  domain  containing  MTOR-­‐interacting  protein17272877 Tbc1d16 7.613E-­‐03 up 1.53 187.88 122.97 TBC1  domain  family,  member  16

17273984 Wdr35 2.274E-­‐03 up 1.53 116.35 76.23 WD  repeat  domain  3517543264 Maged1 1.569E-­‐03 up 1.53 46.09 30.20 melanoma  antigen,  family  D,  117360942 Gal 3.859E-­‐03 up 1.52 50.62 33.22 galanin17447979 Gpr125 7.236E-­‐03 up 1.52 644.86 423.29 G  protein-­‐coupled  receptor  12517458403 3.364E-­‐03 up 1.52 157.64 103.6617345475 Ptk7 8.890E-­‐04 up 1.52 76.55 50.36 PTK7  protein  tyrosine  kinase  717341589 Paqr4 1.118E-­‐03 up 1.52 466.74 307.33 progestin  and  adipoQ  receptor  family  member  IV17213446 Abi2 1.610E-­‐03 up 1.52 528.72 348.76 abl-­‐interactor  217422659 B930041F14Rik3.610E-­‐04 up 1.51 351.63 232.51 RIKEN  cDNA  B930041F14  gene17248304 Ubtd2 2.021E-­‐03 up 1.51 895.12 592.50 ubiquitin  domain  containing  217354810 Afap1l1 9.540E-­‐04 up 1.51 479.49 317.40 actin  filament  associated  protein  1-­‐like  117300686 Nynrin 8.520E-­‐04 up 1.50 356.45 236.86 NYN  domain  and  retroviral  integrase  containing17313000 Kdelr3 4.913E-­‐03 up 1.50 77.57 51.55 KDEL  (Lys-­‐Asp-­‐Glu-­‐Leu)  endoplasmic  reticulum  protein  retention  receptor  317264984 Nlgn2 9.479E-­‐03 up 1.50 445.41 296.15 neuroligin  217349304 Tslp 2.253E-­‐03 up 1.50 50.45 33.57 thymic  stromal  lymphopoietin17357072 Pla2g16 7.700E-­‐03 up 1.50 65.31 43.51 phospholipase  A2,  group  XVI

Transcripts  Cluster  Id

Gene  Symbol

p  value  (Corrected)  

Regulation

Fold  change  (FC)

Average  Expression  -­‐DMSO  

Average  Expression  -­‐UNC1999  

Average  Expression  -­‐UNC2400 Gene  description

17233273 Dcbld1 5.550E-­‐04 up 2.731 45.93 125.46 46.22 discoidin,  CUB  and  LCCL  domain  containing  117293103 Gkap1 5.550E-­‐04 up 3.048 131.72 401.45 117.88 G  kinase  anchoring  protein  117427147 Cdkn2a 5.550E-­‐04 up 3.088 67.46 208.34 70.00 cyclin-­‐dependent  kinase  inhibitor  2A17276674 Gphn 5.990E-­‐04 up 1.579 717.49 1132.89 712.85 gephyrin17356288 Ctsf 5.990E-­‐04 up 2.578 44.70 115.25 46.18 cathepsin  F17415606 Nfia 5.990E-­‐04 up 1.830 76.96 140.80 79.47 nuclear  factor  I/A17215576 Agap1 8.040E-­‐04 up 2.193 224.26 491.87 194.58 ArfGAP  with  GTPase  domain,  ankyrin  repeat  and  PH  domain  117223283 Satb2 9.420E-­‐04 up 2.059 176.97 364.46 185.34 special  AT-­‐rich  sequence  binding  protein  217310912 Laptm4b 1.381E-­‐03 up 2.737 266.56 729.69 272.39 lysosomal-­‐associated  protein  transmembrane  4B17290420 Zmynd11 2.368E-­‐03 up 1.521 515.00 783.08 535.94 zinc  finger,  MYND  domain  containing  1117240226 Marcks 2.416E-­‐03 up 2.478 48.54 120.30 55.72 myristoylated  alanine  rich  protein  kinase  C  substrate17218349 Glul 2.733E-­‐03 up 1.716 350.97 602.40 396.75 glutamate-­‐ammonia  ligase  (glutamine  synthetase)17277170 Acot6 2.733E-­‐03 up 1.714 46.74 80.13 44.77 acyl-­‐CoA  thioesterase  617422659 B930041F14Rik2.733E-­‐03 up 2.286 164.15 375.29 178.04 RIKEN  cDNA  B930041F14  gene17220059 Cdc42bpa 3.076E-­‐03 up 3.038 30.14 91.58 30.03 CDC42  binding  protein  kinase  alpha17236288 Igf1 3.874E-­‐03 up 3.994 44.67 178.40 48.27 insulin-­‐like  growth  factor  117520886 Ryk 3.874E-­‐03 up 3.527 79.64 280.92 62.06 receptor-­‐like  tyrosine  kinase17257405 Tanc2 3.910E-­‐03 up 2.394 36.60 87.60 37.69 tetratricopeptide  repeat,  ankyrin  repeat  and  coiled-­‐coil  containing  217249542 septin  8 4.579E-­‐03 up 1.645 104.84 172.45 110.20 septin  817397497 Foxo1 4.710E-­‐03 up 1.546 108.14 167.17 112.88 forkhead  box  O117228751 Rabgap1l 4.976E-­‐03 up 1.773 114.45 202.98 109.62 RAB  GTPase  activating  protein  1-­‐like17363025 Stx3 4.976E-­‐03 up 2.755 92.91 255.97 102.57 syntaxin  317404585 Zmat3 5.126E-­‐03 up 2.858 119.13 340.46 134.30 zinc  finger  matrin  type  317433977 Agrn 5.235E-­‐03 up 1.748 97.53 170.43 97.25 agrin17235243 Efna2 5.463E-­‐03 up 1.532 56.08 85.91 58.06 ephrin  A217318950 Csf2rb2 5.463E-­‐03 up 1.925 1029.97 1982.32 1131.06 colony  stimulating  factor  2  receptor,  beta  2,  low-­‐affinity  (granulocyte-­‐macrophage)

Supplemental  Table  S4.  List  of  the  transcripts  found  commonly  up  regulated  after  combining  the  studies  done  in  Supplemental  Table  S2  with  new  treatment  studies  (an  additional  MLL-­‐ENL  line  treated  with  3uM  of  compounds  for  4-­‐5  days  and  the  MLL-­‐AF9-­‐GFP  line  treated  with  2uM  of  

compounds  for  3  days)  in  the  microarray  analysis,  with  a  cut-­‐off  of  >1.5-­‐fold  and  p  <0.01.  

17535607 Slc6a8 5.463E-­‐03 up 2.520 133.44 336.31 156.05 solute  carrier  family  6  (neurotransmitter  transporter,  creatine),  member  817545785 Cdkl5 7.182E-­‐03 up 1.584 31.07 49.23 30.86 cyclin-­‐dependent  kinase-­‐like  517371019 Tanc1 7.196E-­‐03 up 1.834 68.18 125.04 55.10 tetratricopeptide  repeat,  ankyrin  repeat  and  coiled-­‐coil  containing  117376541 Prnp|Prnd 7.196E-­‐03 up 1.756 58.65 103.02 70.63 prion  protein  |  prion  protein  dublet17417551 Zswim5 7.196E-­‐03 up 2.913 53.39 155.52 58.47 zinc  finger,  SWIM  domain  containing  517529398 Me1 7.791E-­‐03 up 1.870 36.58 68.40 40.91 malic  enzyme  1,  NADP(+)-­‐dependent,  cytosolic17294458 Rhobtb3 8.629E-­‐03 up 3.662 66.08 242.03 67.51 Rho-­‐related  BTB  domain  containing  317402193 Arhgap29 8.629E-­‐03 up 1.895 34.06 64.54 33.59 Rho  GTPase  activating  protein  2917313000 Kdelr3 8.720E-­‐03 up 2.170 87.84 190.61 92.00 KDEL  (Lys-­‐Asp-­‐Glu-­‐Leu)  endoplasmic  reticulum  protein  retention  receptor  317224771 Serpine2 9.319E-­‐03 up 3.880 64.49 250.24 60.37 serine  (or  cysteine)  peptidase  inhibitor,  clade  E,  member  217308361 Bmp1 9.319E-­‐03 up 1.609 38.59 62.12 42.13 bone  morphogenetic  protein  117355722 Sall3 9.319E-­‐03 up 1.583 38.07 60.29 40.22 sal-­‐like  3  (Drosophila)17414277 Zfp462 9.319E-­‐03 up 1.775 60.33 107.11 55.38 zinc  finger  protein  46217540059 Porcn 9.408E-­‐03 up 1.656 75.98 125.80 81.77 porcupine  homolog  (Drosophila)17548987 2810025M15Rik9.841E-­‐03 up 2.561 105.75 270.79 90.46 RIKEN  cDNA  2810025M15  gene17326510 Pros1 9.880E-­‐03 up 2.011 44.74 89.97 54.09 protein  S  (alpha)17339013 Emr1 9.880E-­‐03 up 1.943 256.73 498.79 337.20 EGF-­‐like  module  containing,  mucin-­‐like,  hormone  receptor-­‐like  sequence  117370807 Fmnl2 9.880E-­‐03 up 2.616 34.32 89.79 32.88 formin-­‐like  217405746 Rarres1 9.880E-­‐03 up 1.869 49.89 93.23 48.42 retinoic  acid  receptor  responder  (tazarotene  induced)  1

Supplementary Table S5. Summary of ChIP-Seq reads that are uniquely aligned.

Cell treatmet Epitope Total Raw Reads

Uniquely aligned reads (by BWA)

Input 25,565,885 13,855,275Suz12 21,763,346 18,353,657

H3K27me3 35,999,158 21,671,310Input 45,104,771 34,519,509

UNC1999 H3K27me3 16,596,001 8,945,791Suz12 8,016,615 7,112,901H3K27ac 35,875,595 30,451,729Input 46,841,361 33,996,489

UNC2400 H3K27me3 27,155,935 21,597,799Suz12 13,710,323 12,033,327H3K27ac 35,987,395 29,501,658

Mock

Supplemental  Table  S6.  Information  of  primers  used  in  this  study

Gene  ID Forward  primer Reverse  primer Note/ReferenceRT-­‐qPCR  of  murine  genesp16Ink4a   GTGTGCATGACGTGCGGG GCAGTTCGAATCTGCACCGTAG Agger  K,  et  al  Genes  &Devp19Arf   GCTCTGGCTTTCGTGAACATG TCGAATCTGCACCGTAGTTGAG Agger  K,  et  al  Genes  &DevEgr1 CCTATGAGCACCTGACCACA TCGTTTGGCTGGGATAACTC Tanaka  et  al.  Blood  Ezh2 AGACGTCCAGCTCCTCTGAA ATCCTCAGTGGGAACAGGTG Hunkapiller,  J.  et  al.  PLoS  genetics  8,  e1002576  (2012).Suz12 GTGCACTCTGAACTGCCGTA CCGGTCCATTTCGACTAAAA Hunkapiller,  J.  et  al.  PLoS  genetics  8,  e1002576  (2012).HoxA9 ACAATGCCGAGAATGAGAGC CAGCGTCTGGTGTTTTGTGTKdm5b CATGATCGAGGACGAGAAAG ATACACTGCCGCTCATCATCBcl11a AACCCCAGCACTTAAGCAAA ACAGGTGAGAAGGTCGTGGTTet1 CCTCACAGGCACAGGTTACA ATTTGGGGCCATTTACTGGTGata1 CAGGTTCAACCCCAGTGTTCC CCCTCCATACTGTTGAGCAGTGGNFI-­‐A TGGCATACTTTGTACATGCAGC ACCTGATGTGACAAAGCTGTCCβ-­‐actin ACCAACTGGGACGACATGGA GGTCTCAAACATGATCTGGGTCATGapdh TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG

ChIP-­‐qPCR  of  murine  genesChr8  intragenic   AAGGGGCCTCTGCTTAAAAA AGAGCTCCATGGCAGGTAGA H3K27me3+;  Wang  et  al  2009Actb  tss TTGATAGTTCGCCATGGATGACGA ATCGATCCCCAAGAAAACCCCA H3K27me3-­‐;  Wang  et  al  2009Cdkn2a  -­‐  a AAAACCCTCTCTTGGAGTGGG   GCAGGTTCTTGGTCACTGTGAG p19ARF  TSS Tanaka  et  al.  Blood  Cdkn2a  -­‐  b CTTAGAGTTACAGAAAGGGCTGGA   GAATTTCAAGGAAGTGCTACCCTA p19ARF_TSS_+2Kb Tanaka  et  al.  Blood  Cdkn2a  -­‐  d AACAGGGGAACGGAGAGTTT TCCAGACACACAAATGCACA p16Ink4a_TSS_-­‐2KbCdkn2a  -­‐  e GATGGAGCCCGGACTACAGAAG   CTGTTTCAACGCCCAGCTCTC   p16Ink4a_TSS Tanaka  et  al.  Blood  Cdkn2a  -­‐  f AGGGAATACACTGTAAGCCTGTGT   TTAACTACTCGGATCAGACATCCA   near  p16Ink4a_exon2 Tanaka  et  al.  Blood  Cdkn2a  -­‐  g GCCACATGCTAGACACGCTA   AGAGCAGAGCTAAATCCGGC   p16Ink4a_exon3Cdkn2a  -­‐  h AAGACCATATACCGGCTGGA CCTCTCCCATTGAGACCAGA p16Ink4a_TES_+2KbHoxA1_tss gactccctttggtcccagtg tgatggatcaccgtttcagtgHoxA7_tss AACCCTTCCCCTAAACGCCTC AAAAGGTCGCCAGTCTTCCAGHoxA9_tss ATCTGTATGCCTAGTCCCGCTCC TTGATGTTGACTGGCGATTTTCHoxA13_tss CTATGACAGCCTCCGTGCTC CCCCTTCCATGTTCTTGTTG