Huang, et al. Supplementary Materials

Supplemental Materials and Methods

Shp2 shRNA knockdown

Shp2 shRNAs constructs were designed to target murine Shp2 using RNAi Codex (http://

http://cancan.cshl.edu/cgi-bin/Codex/Codex.cgi).

The shp2 shRNA 1 (Exon3) sequence is 5'-

TGCTGTTGACAGTGAGCGCGGGACTACTATGACCTCTATTTAGTGAAGCCAC

AGATGTAAATAGAGGTCATAGTAGTCCCTTGCCTACTGCCTCGGA-3'.

The Shp2 shRNA 2 (Exon13) sequence is 5'-

TGCTGTTGACAGTGAGCGCGGCACAGTACCGGTTTATCTTTAGTGAAGCCAC

AGATGTAAAGATAAACCGGTACTGTGCCTTGCCTACTGCCTCGGA-3'.

These shRNAs were cloned into the LMPIG vector.

RUNX1 multiprotein complex purification and proteomic analysis

L8057 clones stably expressing FLAG-bioRUNX1 were induced with TPA (50 nM) for 3

days. Crude nuclear extracts were harvested and then dialyzed, centrifuged and pre-

cleared using agarose beads. The FLAG-BioRUNX1 complexes were immunoprecipitated

using anti-FLAG M2-agarose beads (Sigma). The beads and bound material were washed

with BC139K buffer and eluted with excess FLAG peptide (Sigma). The eluted material

was incubated with streptavidin-agarose beads, washed and centrifuged. The pellet was

then boiled with SDS sample buffer and loading onto SDS-PAGE gel. After colloidal

Coomassie blue staining (Invitrogen), the lanes were divided into three sections and each

section was cut into ~ 1 mm3 pieces. In situ trypsinization was performed and the

Huang, et al. Supplementary Materials

peptides were eluted and separated using a nano-scale reverse-phase HPLC capillary

column. As each peptide was eluted they were subjected to electrospray ionization and

then they entered into an LTQ linear ion-trap mass spectrometer (Thermo Scientific, San

Jose, CA). Eluting peptides were detected, isolated, and fragmented to produce a tandem

mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence

protein identity) were determined by matching protein or translated nucleotide databases

with the acquired fragmentation pattern by the software program, Sequest (Thermo

Scientific, San Jose, CA).

Retroviral infection of primary Mks

E13.5 fetal livers from C57BL/6 or RUNX1fl/fl, Vav-Cre mice were harvested in 2%

FCS/PBS on ice, and passed serially through a 1 ml pipette tip, 18-, 20-, and 23-gauge

needles, and a 40 μm cell strainer to generate single cell suspensions. These were then

cultured in DMEM with 10% FCS and1% TPO and 1% SCF conditional medium for 24

hrs or 4 days as indicated in the figure legends. Primary fetal liver cells were

resuspended with medium contained 1% TPO conditioned medium, 5 μg/ml polybrene

(Sigma) and fresh retroviral particles, which were generated by transfection of PLAT-E

cells with MIG, MIG-RUNX1, MIG-RUNX15F, or MIG-RUNX15D DNA constructs

using FuGene 6 and incubated for 72 hrs. The cells were centrifuged at 600 x g for 90

min at room temperature and then incubated in 5% CO2 at 37°C for 90 min before

replacing viral supernatant medium with normal culture medium (DMEM with 10% FCS

and 1% TPO conditioned medium) for additional days as indicated in the figure legends.

Cells were then stained with CD41, CD42b, or c-mpl antibodies, and analyzed using a

Huang, et al. Supplementary Materials

FACSCalibur flow cytometer (BD Biosciences). CD41+GFP+ cells were sorted using a

FACSAria flow cytometer (BD Biosciences) for gene expression analysis.

Analysis of RUNX1 tyrosine phosphorylation by mass spectrometry

The lyophilized peptides were resuspended in 5 % acetonitrile and 5 % formic acid

before it was directly injected into the LC/MS system comprising a micro-autosampler,

Suvery HPLC pump and a linear ion trap (LTQ) mass spectrometer (all: Thermo

Scientific, San Jose, CA). The LC-system featured an in-house packed reversed phase

column using C18 Magic (3 mm, 200 Å; Michrom Bioresource) packing material and

PicoTip Emitters (New Objective, Woburn, MA). The peptides were eluted with a 30 min

linear gradient and data acquired in a data dependent fashion, i.e. the 6 most abundant

species for selected for automated fragmentation. If the fragmentation of an MS

spectrum resulted in an immediate neutral loss (NL) mimicking the mass of a M/Z value

of a phosphate group in the MS2, then an MS3 fragmentation was performed on the NL

MS2 peak. Mass spectrometric data was searched against the mouse international protein

index (IPI mouse 3.39) database using the protein identification software Mascot (v2.2.04,

Matrix Sciences, London, UK). Search criteria included tryptic peptide specificity with

one missed cleavage, Carbamidomethyl (C) as a fixed modification, Deamidated

(NQ),Gln->pyro-Glu (N-term Q), Phospho (STY) and Oxidation (M) as variable

modifications for MS2 spectra. MS3 spectra (triggered by the NL in the MS2) were

searched with the following criteria; tryptic peptide specificity with one missed cleavage,

Carbamidomethyl (C) as a fixed modification, Deamidated (NQ), Gln->pyro-Glu (N-term

Q), Dehydrated (ST), Phospho (Y) and Dethiomethyl (M) as variable modifications. A

1.5 Da peptide tolerance for the MS and a 0.8 Da peptide tolerance for MS/MS spectra

Huang, et al. Supplementary Materials

were implemented along with a Mascot ion cut off score of 30. Spectra sequences

identified by Mascot to have a phosphorylation were validated manually to ensure proper

identification of the amino acid location of the modification site and access overall

quality of the data.

Flow cytometry

For cell surface marker analysis, murine cells were treated with Fc-blocker for 10 min on

ice. CD42b, CD41, CD8a, CD4, CD3e, or CD45.1 antibodies (see Supplemental Table

S2) were incubated with the cells for 30 min on ice. The cells were washed with 2% FCS

in PBS and centrifuged. The cell pellet was resuspended and stained with 7AAD or DAPI

to exclude dead cells, and analyzed using FACSCalibur or LSR II instruments (BD

Biosciences), or sorted using a FACSAria instrument (BD Biosciences). For DNA ploidy

determination, the cells were resuspended in 200 μl of staining solution (50 μg/ml

propidium iodide, 4 mM sodium citrate, 0.2 mg/ml Rnase A, 0.1% Triton X-100),

incubated for 30 min and analyzed on a FACSCalibur flow cytometer.

qRT-PCR

The sequences of the primers used for qRT-PCR analysis are as follows. RUNX1: 5’-

acctaccatagagccatcaaaatc-3’ (forward) and 5’-ttggtctgatcatctagtttctgc-3’ (reverse); c-mpl:

5’-acaagtcaccagccaagatgt-3’ (forward) and 5’-gtcctcaaatgtttgggagaag-3’ (reverse); GPIbα:

5’-cttagaatccaccaccacaataatc-3’ (forward) and 5’-aaggactgctttcagaagtatcaga-3’ (reverse);

β-actin: 5’-caccacaccttctacaatgagc-3’ (forward) and 5’-aacatgatctgggtcatcttttc-3’ (reverse).

Huang, et al. Supplementary Materials

AChE staining

0.12 million cells were resuspended in 120 ul PBS and cytospun at 400 rpm for 4 min.

The slides were air-dried. Staining solution was prepared by dissolving 10 mg of

acetylthiocholine iodide (Sigma) in 15 ml 0.1M sodium phosphate buffer (pH 6.0). One

milliliter of 0.1M sodium citrate (pH 6.0), 2 ml 30 mM copper sulfate and 2 ml of 5 mM

potassium ferricyanide. The slides were staining at room temperature for 3-6 hrs. The

reaction was stopped by fixing the cells in 95% ethanol for 5 min. The cells were

counterstained with Harris’ haematoxylin for 30 seconds and then washed with tap water

for several times. Saturated lithium carbonate solution was added to produce a blue color

to the counterstain. The slides were rinsed in tap water and air-dried.

Huang, et al. Supplementary Materials

1

Supplemental References Bakshi R, Hassan MQ, Pratap J, Lian JB, Montecino MA, van Wijnen AJ, Stein JL, Imbalzano

AN, Stein GS. 2010. The human SWI/SNF complex associates with RUNX1 to control transcription of hematopoietic target genes. J Cell Physiol.

Elagib KE, Racke FK, Mogass M, Khetawat R, Delehanty LL, Goldfarb AN. 2003. RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation. Blood 101: 4333-4341.

Fujimoto T, Anderson K, Jacobsen SE, Nishikawa SI, Nerlov C. 2007. Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. The EMBO journal 26: 2361-2370.

Huang H, Yu M, Akie TE, Moran TB, Woo AJ, Tu N, Waldon Z, Lin YY, Steen H, Cantor AB. 2009. Differentiation-dependent interactions between RUNX-1 and FLI-1 during megakaryocyte development. Mol Cell Biol 29: 4103-4115.

Lutterbach B, Westendorf JJ, Linggi B, Isaac S, Seto E, Hiebert SW. 2000. A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia. J Biol Chem 275: 651-656.

Nguyen LA, Pandolfi PP, Aikawa Y, Tagata Y, Ohki M, Kitabayashi I. 2005. Physical and functional link of the leukemia-associated factors AML1 and PML. Blood 105: 292-300.

Palii CG, Perez-Iratxeta C, Yao Z, Cao Y, Dai F, Davison J, Atkins H, Allan D, Dilworth FJ, Gentleman R et al. Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineages. 2011. Embo J 30: 494-509.

Pardali E, Xie XQ, Tsapogas P, Itoh S, Arvanitidis K, Heldin CH, ten Dijke P, Grundstrom T, Sideras P. 2000. Smad and AML proteins synergistically confer transforming growth factor beta1 responsiveness to human germ-line IgA genes. J Biol Chem 275: 3552-3560.

Wang S, Wang Q, Crute BE, Melnikova IN, Keller SR, Speck NA. 1993. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol Cell Biol 13: 3324-3339.

Wilson NK, Foster SD, Wang X, Knezevic K, Schutte J, Kaimakis P, Chilarska PM, Kinston S, Ouwehand WH, Dzierzak E et al. Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. 2010. Cell Stem Cell 7: 532-544.

Zhao X, Jankovic V, Gural A, Huang G, Pardanani A, Menendez S, Zhang J, Dunne R, Xiao A, Erdjument-Bromage H et al. 2008. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev 22: 640-653.

Huang, et al. Supplementary Materials

1

Supplementary Tables

Table S1. Identification of RUNX1 associated proteins in TPA-induced L8057 cells by mass spectrometry. FLAG-BioRUNX1 containing multiprotein complexes were purified by tandem anti-FLAG, streptavidin, or single streptavidin affinity chromatography from crude nuclear extracts of L8057 cells induced with TPA for 72 hrs. Co-purified proteins were separated by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and the entire lane was subjected to in situ trypsinization. Trypsinized peptides were separated by microcapillary liquid chromatography and identified by tandem mass spectrometry. See (Huang et al. 2009) for additional details. The number of recovered peptides corresponding to each protein is given from five independent experiments. Table adapted from (Huang et al. 2009).

Protein Identity

Exp. 1a

Exp. 2a

Exp. 3b

Exp. 4b

Exp. 5b

Total Peptidesc

Total Peptides

in controld

RUNX1

Known proteinse

CBFβ GATA1 GATA2

SCL/TAL1 PML

PRMT1 Sin 3a Smad2 CDK6 FLI1

Swi/Snf Complex

BAF60 BAF57

BAF155 BAF170

Brg1 SNF2 SNF5

Novel Proteins

Shp2 c-src kinase

67

32 1 5 5

20 0 0 0 1

22

0 0 0 0 0 0 0

0 0

25

2 0 0 0 6 0 0 0 0 0

0 0 0 0 0 0 0

0 0

9

2 2 0 1 0 4 3 0

11 5

3 3 6 2 1 1 0

2 2

3

3 0 0 0 0 3 0 1 9 3

2 3 7 2 3 0 3

5 3

19

3 0 0 2 0 0 0 0 2 1

1 0 0 0 0 0 0

0 0

123

42 3 5 8

26 7 3 1

23 31

6 6

13 4 4 1 3

7 5

0

1 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0

Total Proteins

Total Specific Proteinsf 183 152

46 14

358 219

243 143

73 22

a Tandem anti-FLAG, streptavidin affinity purification. b Single streptavidin affinity purification. c Experiment-to-experiment variation in the number of peptides obtained for each of the proteins likely reflects differences in the amount of input material used, purification strategy, and/or sampling and detection issues related to the LC/MS/MS analysis. d From cells expressing birA alone.

Huang, et al. Supplementary Materials

2

e CBFβ (Wang et al. 1993), GATA-1 (Elagib et al. 2003), GATA-2 (Wilson et al. 2010), TAL1/SCL (Palii et al. 2011), Sin3A (Lutterbach et al. 2000), PRMT1 (Zhao et al. 2008), PML (Nguyen et al. 2005), Smad2 (Pardali et al. 2000), CDK6 (Fujimoto et al. 2007), SWI/SNF complex (Bakshi et al. 2010), FLI1 (Huang et al. 2009). f Proteins not identified in cells expressing birA alone.

Huang, et al. Supplementary Materials

3

Table S2. Antibody source information

Name Company Clone # Catalogue # Application

RUNX1 Santa Cruz H-65 sc-28679 WB

RUNX1 Santa Cruz N-20 sc-8563 WB

RUNX1 Abcam N/A Ab23980 IF

Brg1 Santa Cruz H-88 sc-10768 WB

pY Millipore 4G10 05-1050 WB

β-actin Sigma AC-74 A2228 WB

CBF-β Santa Cruz FL182X sc-20693x WB

FLAG-HRP Sigma M2 A8592 WB

SA-HRP GE Healthcare N/A RPN1231V WB

Shp1 Santa Cruz C-19 sc-287 WB

Shp2 Santa Cruz C-18 sc-280 WB, IF

YY1 Santa Cruz H-10 sc-7341 WB

Csk Santa Cruz C-20 sc-286 WB

Src Santa Cruz 17AT28 sc-130124 IF

FLI1 Santa Cruz C-19 sc-356 WB

GATA1 Santa Cruz N-6 sc-265 WB

vWF DAKO N/A A0082 IHC

SNF5 Bethyl N/A A301-087A WB

CD45.1 BD A20 560580 FACS (Percp Cy5.5)

CD4 eBioscience GK1.5 17-0041-81 FACS (APC)

CD8a BD 53-6.7 557654 FACS (APC-Cy7)

CD3e BD 145-2C11 553063 FACS (PE)

CD41

CD41

GPIb

eBioscience

BD

Emfret

eBioMWReg30

MWReg30

N/A

17-0411-82

558040

R300

FACS (APC)

FACS (PE)

Platelet depletion

CD42b Emfret Xia.G5 M040-2 FACS (PE)

α−rabbit IgG Jackson IR 191/1.0 111-116-144 FACS (PE)

c-mpl1 FACS 1 Gift of Wei Tong, Children’s Hospital of Philadelphia.

WB, western blot; IF, immunofluorescence; IHC, immunohistochemistry; FACS, flow cytometry; APC, allophycocyanin; PE, phycoerythrin; N/A, not available;

Huang, et al. Supplementary Materials

1

Supplementary Figure Legends

Figure S1. Peptides recovered for PTPN11 (Shp2) (top) and c-Src kinase (bottom) from combined mass spectrometry experiments. The recovered peptides are underlined and in bold red type. The percentage of total protein coverage is shown below each protein sequence. Figure S2. Interaction between RUNX1 and Csk (c-Src tyrosine kinase). Streptavidin precipitation (SA-IP) of FLAG-BioRUNX1 from nuclear extracts of uninduced L8057 cells and western blot of eluted material with α-Csk antibody. 1% of input is shown. bir A, biotin ligase. Figure S3. C-Src and RUNX1 subcellular localization analysis by confocal indirect immunofluorescence microscopy in L8057 cells with and without TPA, PP2 or Na3VO4 treatment. DAPI stains DNA (see Figures 2 and 7 of main paper). Figure S4. Amino acid sequence alignment of RUNX1, -2, and -3 from multiple species showing regions encompassing Group 2 and Group 5 tyrosine residues (see Figure 3 of main paper). Functional domains are indicated: NRBD, negative regulatory DNA binding domain; AD, activation domain; ID, autoinhibitory domain. Red arrows indicate tyrosine residues that are the most highly conserved among species and in all RUNX family members. Blue arrows indicate tyrosine residues only present in some species, and/or a subset of RUNX family members. Figure S5. Starting material and recovered peptides from mass spectrometry analysis of RUNX1 tyrosine phosphorylation (see Figure 3 of main paper). (A) Western blot analysis of SA-purified FLAG-BioRUNX1 from uninduced L8057 cells treated with or without 1.25 mM of sodium orthovanadate. The sodium orthovanadate treated sample was used for the analysis. Bands corresponding to this region of the SDS-PAGE gel were excised, digested with trypsin in situ, and analyzed by tandem mass spectrometry. (B) Peptide recovery. The peptides recovered are shown in red. Tyrosine 260, isoform 3 numbering (tyrosine 274, isoform 1 numbering), which was found to be tyrosine phosphorylated is indicated by the boxed residue. The underlined sequence indicates a peptide that was also phosphorylated, but only 4 peptides were recovered. This low recovery did not allow assignment of specific residues within this region. Green “S” and “T”’s represent serine and threonine residues found to be phosphorylated by mass spectrometry. Figure S6. Enhanced megakaryopoiesis by overexpression of RUNX1Y375F, Y378F, Y379F, Y386F

(“RUNX14F”), and RUNX1Y379F, Y386F (“RUNX12F”) mutants (see Figure 4 of main paper). (A) Schematic diagram showing experimental procedure. Embryonic day 13.5 (e13.5) fetal liver cells from wild type mice were cultured in the presence of TPO and SCF, retrovirally transduced with MIG retroviral vectors expressing wild type RUNX1, RUNX14F, or RUNX12F. After 5 additional days in culture with TPO alone, GFP+ cells were analyzed for CD42b expression by flow cytometry (B) or gene expression analysis of CD41+ GFP+ cells by qRT-PCR (C). For qRT-PCR, levels of the tested mRNAs were normalized to β-actin mRNA levels. The fold-change relative to MIG-RUNX1 (GPIbα, c-mpl, and PF4), or endogenous RUNX1 is indicated above each column.

Huang, et al. Supplementary Materials

2

Figure S7. Partial nuclear localization of Shp2 in TPA-induced L8057 megakaryoblastic cells and primary murine fetal liver cells (see Figure 6 of main paper). Indirect immunofluoresence microscopy images are shown for Shp-2 in: (a-c) primary megakaryocytes cultured from murine e.13.5 fetal liver and enriched over an albumin step-gradient; (d-f) uninduced L8057 cells; and (g-i) L8057 cells induced with 50 nM TPA for 5 days. DAPI, nuclear stain (blue). Shp2, red. Original magnification 200x.

Huang_Fig S1

α-Csk50 kDa-

bir AFLAG-BioRUNX1

+ + + +

+ +

Input SA-IP

Huang_FigS2

Na3VO4

PP2

TPA

DMSO

Untreated

DAPI Src RUNX1 Src + RUNX1 DAPI + Src + RUNX1

Huang_Fig S3

Huang_FigS4

NRBD AD ID

Less conserved Highly conserved

Y254

Y260 Y258

Y375

Y379

Y386

Y378

α- pY

α-RUNX1

Na3VO4

- +

SA-IP (3%SDS)

50-

50-

kDa

A

B

Huang_Fig S5

IAFKVVALGD VPDGTLVTVM AGNDENYSAE LRNATAAMKN QVARFNDLRF

RUNX1 (isoform 1)

MASDSIFESF PSYPQCFMRD ASTSRRFTPP STALSPGKMS EALPLGAPDG

GAALASKLRS GDRSMVEVLA DHPGELVRTD SPNFLCSVLP THWRCNKTLP

VGRSGRGKSF TLTITVFTNP PQVATYHRAI KITVDGPREP RRHRQKLDDQ

TKPGSLSFSE RLSELEQLRR TAMRVSPHHP APTPNPRASL NHSTAFNPQP

QSQMQDARQI QPSPPWSYDQ SYQ Y LGSITS SSVHPATPIS PGRASGMTSL

SAELSSRLST APDLTAFGDP RQFPTLPSIS DPRMHYPGAF TYSPPVTSGI GIGMSAMSSA SRYHTYLPPP YPGSSQAQAG PFQTGSPSYH LYYGASAGSY

QFSMVGGERS PPRILPPCTN ASTGAALLNP SLPSQSDVVE TEGSHSNSPT

1

51

101151

201

251

301351

401

E13.5Fetal liver cell Liquid

Culture with TPO and SCF

Retrovirus infectionMIG, MIG-RUNX1, MIG-RUNX1 mut

Culture 5 days with TPO

4 days

A B

C0

2

4

6

8

10

12

14

16

% C

D42

b+ c

ells

in G

FP+

gate

s

MIGMIG-Runx1MIG-Runx1 4FMIG-Runx1 2F

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

00.0020.0040.0060.008

0.010.0120.0140.016

00.10.20.30.40.50.60.70.80.9

MIG-RUNX1

MIG-RUNX1 4F

MIG-RUNX1 2F

2.31 2.26

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

C57BL/6

MIGMIG-RUNX1

MIG-RUNX1 4F

MIG-RUNX1 2F

61

45

77

1.9

11

5.2

12

7.5

GPIbα c-mpl PF4

RUNX1

4F

2F

4F

2F

1.6

4.6 4.4

Rel

ativ

e m

RN

A le

vel

Rel

ativ

e m

RN

A le

vel

Huang_Fig S6

MIG

MIG-RUNX1

MIG-RUNX14F

MIG-RUNX12F

ba c

d e f

g h i

DAPI α-Shp2 Merged

Primary fetal liver Mks

UninducedL8057 cells

InducedL8057 cells

Huang_Fig S7