Additional Information for Michaud et al. 2008

I. Additional Material and Methods

II. Additional Table 1 legend: Summary of the gene expression profiling results, oPOSSUM and corresponding mouse Affymetrix data for each clone Accession number present on the human cDNA array.

III. Additional Table 2 legend: Gene expression profiling data for E8.5 and E12 Runx1 knockout embryos.

IV. Additional Table 3: Genes differentially expressed in the FPD-AML samples and also differentially expressed in t(8;21) and Inv(16) samples

V. Additional Table 4: Set of genes used for the Gene Set Enrichment analysis

VI. Additional Table 5: List of genes differentially expressed in FPD and CBF, which are associated to cellular proliferation

VII. Additional Table 6: List of genes differentially expressed following overexpression of the CBF complex and associated to the cytoskeleton

VIII. Additional Figure 1: Level of RUNX1 expression in FPD-AML cells

IX. Additional Figure 2: protein overexpression of RUNX1 and CBFb

X. Additional Figure 3: Correspondence analysis between gene expression profiles.

XI. Additional Figure 4: Supporting graphs for the Gene Set Enrichment analysis

XII. Additional Figure 5: Expression pattern of RUNX1 and a subset of differentially expressed genes

XIII. References for Additional Information

I. Additional Material and Methods

Adenovirus production

Recombinant adenoviruses were generated as described[1], except that VmRL-CMV1 and pSCOT were used as the adenovirus backbone and transfer vector respectively. VmRL-CMV1 is identical to VmAdcDNA3 except that it contains a beta-globin polyadenylation signal. The pSCOT vector contains two Tet operators flanking the TATA box allowing suppression of expression in the presence of Tet Repressors. The RUNX1 p49 isoform[2] and CBFb coding sequences were amplified from fetal lung cDNA and cloned into the pSCOT vector using EcoRV. The EGFP coding sequence was subcloned into pSCOT using NotI and SalI. Recombinant adenovirus was transfected into E1-complementing packaging cells HER911[3] and high titer of infectious particles were produced as described.[1] The titer of each adenovirus was measured by optical densitometry.

Identification of the genes and combination of human and mouse platforms

The genes corresponding to each cDNA clone were determined using Unigene (Build 181, 9/3/05). The EntrezGene IDs were obtained for each Unigene and Homologene (Build 39.2) was used to identify the mouse orthologous genes. Similarly Unigene cluster ID was assigned to each mouse Affymetrix probe (Affymetrix annotations 14 March 2005). Refseq IDs (UCSC May 2004) corresponding to each Unigene cluster were identified. More than one Unigene or Refseq number can correspond to the same cDNA clone.

Glycophorin A assay

Spheroplasts were prepared and frozen until required. Three to five technical replicates were generated for each sample. After thawing, spheroplasts were labeled with anti-GPA-M and anti-GPA-N antibodies and subjected to flow cytometry analysis [4], recording 2.5x10⁶ gated events for each sample. Spheroplasts with phenotypes N0 and NN were gated to have intensity £ 10% of the geometric mean M-labelled intensity for the wild-type MN population. N0 cells were gated to be centred on the MN geometric mean N intensity with gate width encompassing 95% of all cells.  NN cells were centred around twice the N0 mean with gates at the equivalent geometric fluorescence intensity width.

oPOSSUM analysis (www.cisreg.ca/cgi-bin/oPOSSUM/opossum)

The oPOSSUM analysis was performed using 1) the top 30% of conserved regions with a minimum of 60% of conservation between human and mouse sequences, 2) a matrix match score of 80% and 3) non-coding sequences 5000bp upstream or downstream of the transcription start.

cDNA panel production

The human cDNA panel was generated as described.[5] Briefly cDNA was synthesized from 1mg of polyA RNA and normalized according to GAPDH level. A normalized dilution of 1:500 of the initial cDNA sample was used in PCR amplification. The human hematopoietic cell line cDNA panel was generated following a similar protocol except that 5mg of total RNA was reverse transcribed and the relative amount of each cDNA was normalized according to GAPDH and HPRT housekeeping gene levels determined using the QuantiTect SYBR Green PCR kit and the Lightcycler PCR machine. A normalized dilution of 1:250 of the initial cDNA sample was used in PCR amplification.

II.

Additional Table 1: Human datasets. A summary of all the human data obtained in this study and the corresponding mouse Affymetrix probes are indicated in this table. The Unigene cluster ID, gene symbol and gene name are indicated for each clone accession number present on the array according to Unigene Build 181. Each human dataset (FPD and CBF) is represented by three columns: the first column is a classification column representing whether the gene is up- (1), down- (-1) or not differentially expressed in the mutant following statistical analysis as described above. The second and third columns represent the M-value (log₂ of the fold change) and the moderated t-statistics, respectively. The following column contains the corresponding Refseq numbers. The presence or absence of a RUNX1 binding site in the regulatory region of the gene is then indicated by “1” or “0” respectively (oPOSSUM). ND means that the regulatory region of the gene could not be identified in the oPOSSUM database. The next column gives the corresponding Human Gene IDs (March 2005). The corresponding mouse Affymetrix probes were identified using Gene IDs and Homologene (Build 39.2). The Unigene cluster IDs corresponding to each Affymetrix probe were obtained from the latest Affymetrix annotation file (March 2005). M and t are also showed for the mouse probesets.

III.

Additional Table 2: Mouse datasets. The M (log₂ of the fold change) and moderated t-statistics for each Affymetrix probe is indicated as well as the identity of the probes according to the latest Affymetrix annotation file (March 2005).

IV.

Additional Table 3: Top 14 differentially expressed genes in the FPD-AML cells that are also differentially expressed in t(8;21) and Inv(16) samples.

FPD_t(8;21)
Gene Symbol	Gene Title	FPD M	Probe Set ID	rank in t(8;21) samples
TAF9	TAF9 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 32kDa	0.22	203893_at	84
SILV	silver homolog (mouse)	-0.27	209848_s_at	133
SHOX2	short stature homeobox 2	0.69	210134_x_at	140
CD83	CD83 molecule	0.39	204440_at	166
PPIB	peptidylprolyl isomerase B (cyclophilin B)	-0.29	200967_at	245
ARPC5	actin related protein 2/3 complex, subunit 5, 16kDa	0.24	211963_s_at	276
HLA-DQB1	major histocompatibility complex, class II, DQ beta 1	0.35	211654_x_at	299
MT1G	metallothionein 1G	-0.73	210472_at	397
LIPG	lipase, endothelial	-0.26	219181_at	574
TCF12	transcription factor 12 (HTF4, helix-loop-helix transcription factors 4)	0.17	215611_at	630
CALR	calreticulin	-0.38	214315_x_at	792
PHYH	phytanoyl-CoA 2-hydroxylase	-0.24	203335_at	797
IMPA2	inositol(myo)-1(or 4)-monophosphatase 2	-0.47	203126_at	829
KDELR2	KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 2	-0.23	200698_at	903
FPD_Inv(16)
Gene Symbol	Gene Title	FPD M	Probe Set ID	rank in Inv(16) samples
NFKBIA	nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha	0.24	201502_s_at	536
CD2BP2	CD2 (cytoplasmic tail) binding protein 2	-0.26	202257_s_at	919
SNAP25	synaptosomal-associated protein, 25kDa	-0.37	202508_s_at	965
RGS1	regulator of G-protein signalling 1	0.2	202988_s_at	1110
GOLPH4	golgi phosphoprotein 4	0.35	204322_at	911
ACAT1	acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme A thiolase)	0.3	205412_at	389
CD38	CD38 molecule	-0.67	205692_s_at	801
UBD	ubiquitin D	0.67	205890_s_at	944
CLTB	clathrin, light polypeptide (Lcb)	0.23	206284_x_at	469
MT1X	metallothionein 1X	-0.69	208581_x_at	447
IGF1	insulin-like growth factor 1 (somatomedin C)	-0.41	209540_at	746
ELF1	E74-like factor 1 (ets domain transcription factor)	-0.38	212418_at	1060
PRKD3	protein kinase D3	-0.4	218236_s_at	207
UNC13B	Unc-13 homolog B (C. elegans)	-0.36	221130_s_at	148

V.

Additional Table 4. Set of genes used for the Gene Set Enrichment analysis. Reference and description of each study, the format of the available data, the platform used in the study and the number of corresponding Unigene cluster ID (Build 181) are indicated. DEG: differentially expressed genes. M: Log₂ of the fold change. A: Log₂ of the overall intensity of the probe.

Gene set name	Reference	Study	Data format	Arrays used in the study	Number of Unigene clusters
Megakaryopoiesis
Mekagaryocyte differentiation	[6]	Identification of genes involved in the differentiation of megakaryocytes. DEGs between stem cells and differentiated megakaryocytes	List of DEGs	U95Av2	103
Platelets	[7]	Transcription profiling of human blood platelet	Top 50 DEGs	U95-Av2	36
Normal megakaryocytes	[8]	Genes highly expressed in megakaryocytes	Top 200 genes with high expression	U133	113
ET megakaryocytes	[8]	Genes highly expressed in essential thrombocytopenia megakaryocytes	Top 200 genes with high expression	U133	117
ET vs normal	[8] (GDS552)	DEGs between normal megakaryocytes and megakaryocytes derived from patients with essential thrombocytopenia (ET)	Genes with a |M|>2 and A>4	U133	128
Cell cycle
Cytokinesis proteome	[9]	Identification of proteins present in the midbody during cytokinesis	List of proteins	Mass spectrometry	155
Spindle checkpoint	[10]	Review			12
Genomic instability
DNA repair	[11]	Review			127
Lymphoblast irradiation; high dose	[12] (GDS479)	Effect of ionising radiation on lymphoblasts	List of DEGs	U95A	625
Lymphoblast irradiation; low dose	[12] (GDS479)	Effect of ionising radiation on lymphoblasts	List of DEGs	U95A	243
Cancer associated genes
Genes DE in cancer	[13]	Meta-analysis of cancer microarray data to identify genes consistently DE in tumours	List of genes	Many	67

VI.

Additional Table 5: List of genes differentially expressed in FPD and CBF, which are associated to cellular proliferation using Gene Ontology annotation GO:0008283. The M-values are shown for both FPD and CBF datasets. Genes in bold are those identified as differentially expressed in both datasets.

Accession	Symbol	RefSeq	FPD M	CBF M
W69954	AIF1	NM_001623	0.68	0.11
AA857163	AREG	NM_001657	-0.36	-0.01
T69273	ATPIF1	NM_178191	0.25	0.09
AI341427	BCAT1	NM_005504	0.37	-0.14
AA132086	C6ORF108	NM_199184	0.31	-0.01
AI375736	CD28	NM_006139	0.86	0.03
AA973397	CD86	NM_006889	-0.67	-0.02
N62245	CDC7	NM_003503	0.27	-0.07
AA873604	CRIP1	NM_001311	-0.3	0.08
AA878880	CXCL10	NM_001565	1.34	-0.03
AA450009	EDNRA	NM_001957	0.5	0.11
R54846	FGFR1	NM_015850	1.17	0.12
AA448277	FOXO1A	NM_002015	-0.43	0.07
N67876	IGF1	NM_000618	-0.41	0.01
AA977194	IL12RB2	NM_001559	0.3	-0.07
AA479795	ISG20	NM_002201	-0.54	-0.01
AA045731	KLF10	NM_005655	-0.35	0.18
AA826328	MALT1	NM_006785	-1.38	0.08
AA705886	MXI1	NM_130439	-0.33	-0.01
W56300	NFKBIA	NM_020529	0.24	-0.01
AA935262	NODAL	NM_018055	-0.82	-0.01
AA701502	PDGFA	NM_002607	0.36	-0.06
AA863383	PIM2	NM_006875	-0.62	0.01
N89985	PURB	NM_033224	-0.3	-0.02
AA458996	SLAMF1	NM_003037	-0.03	0.4
W72201	SMAD3	NM_005902	-0.31	-0.07
AA481026	SMARCA2	NM_003070	0.57	0.01
AA007444	TLX1	NM_005521	-0.7	0.07
AA166695	TNFSF13B	NM_006573	0.27	0
AA486088	TOB2	NM_016272	-0.26	0.06
AI950056	TPX2	NM_012112	0.23	0.01
H63077	ANXA1	NM_000700	0.93	-0.4
AA670438	UCHL1	NM_004181	-1.12	-0.25
AA490477	MYH10	NM_005964	0.45	0.12
AA858175	RUNX2	NM_004348	-0.46	0.53
AA968896	MDK	NM_002391	-0.1	0.3
W15277	TBC1D8	NM_007063	0.1	0.18
AA287300	CDC2L1	NM_033489	-0.08	0.29
N45138	TGFB2	NM_003238	-0.05	0.37
AA865712	FGFR1OP	NM_194429	-0.1	0.26
AA167269	NAP1L1	NM_139207	0.24	0.13
H07899	VEGFC	NM_005429	0.03	0.12
T59334	CSRP2	NM_001321	-0.06	0.46
AI828088	CDKN1C	NM_000076	-0.04	0.08
AA447661	SESN1	NM_014454	-0.27	0.26
AA102526	IL8	NM_000584	0.24	0.17
AA996024	HDAC4	NM_006037	-0.14	0.1
N72115	CDKN2C	NM_001262	0.25	0.2
N20203	BMPR2	NM_001204	-0.24	0.38
AA130714	PGF	NM_002632	0.2	0.27
N20338	HGS	NM_004712	-0.09	0.14
R43576	BLZF1	NM_003666	-0.14	0.33
AA040427	CLK1	NM_004071	-0.19	0.16

VII.

Additional Table 6: Cytoskeleton–related differentially expressed genes in CBF dataset. 57 differentially expressed genes following overexpression of CBF complex are associated to the cytoskeleton using Gene Ontology annotation GO:0005875 (cellular component). The corresponding gene symbols and gene names are indicated (Unigene Build 181). The direction of the difference in expression observed following overexpression of CBF is also indicated.

Accession	Gene symbol	Gene name	Difference in expression in CBF
AA001749	MAPRE1	Microtubule-associated protein, RP/EB family, member 1	Down
AA151125	TMOD3	Tropomodulin 3 (ubiquitous)	Down
AA180742	TUBA1	Tubulin, alpha 1 (testis specific)	Down
AA406601	ABLIM1	Actin binding LIM protein 1	Down
AA424824	DSTN	Destrin (actin depolymerizing factor)	Down
AA426374	TUBA2	Tubulin, alpha 2	Down
AA430574	PXN	Paxillin	Down
AA431967	LATS2	LATS, large tumor suppressor, homolog 2 (Drosophila)	Down
AA432066	SGCE	Sarcoglycan, epsilon	Down
AA436460	KIFC3	Kinesin family member C3	Down
AA446462	BUB1	BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast)	Down
AA449336	PRC1	Protein regulator of cytokinesis 1	Down
AA459400	ARHGDIA	Rho GDP dissociation inhibitor (GDI) alpha	Down
AA460685	BIRC5	Baculoviral IAP repeat-containing 5 (survivin)	Down
AA485959	KRT7	Keratin 7	Down
AA488676	BASP1	Brain abundant, membrane attached signal protein 1	Down
AA496691	DAG1	Dystroglycan 1 (dystrophin-associated glycoprotein 1)	Down
AA496785	ABL1	V-abl Abelson murine leukemia viral oncogene homolog 1	Down
AA504128	RAE1	RAE1 RNA export 1 homolog (S. pombe)	Down
AA599145	ZW10	ZW10 homolog, centromere/kinetochore protein (Drosophila)	Down
AA621315	CTNNAL1	Catenin (cadherin-associated protein), alpha-like 1	Down
AA630298	PTK2	PTK2 protein tyrosine kinase 2	Down
AA634006	ACTA2	Actin, alpha 2, smooth muscle, aorta	Down
AA634289	ACTL7B	Actin-like 7B	Down
AA676955	RHOA	Ras homolog gene family, member A	Down
AA865469	TUBA3	Tubulin, alpha 3	Down
AA873060	STMN1	Stathmin 1/oncoprotein 18	Down
AA888148	TUBB2	Tubulin, beta, 2	Down
H63077	ANXA1	Annexin A1	Down
H98666	PCOLN3	Procollagen (type III) N-endopeptidase	Down
N74524	TUBB4	Tubulin, beta 4	Down
R16712	ANLN	Anillin, actin binding protein (scraps homolog, Drosophila)	Down
R76544	HAX1	HS1 binding protein	Down
R77252	MAP7	Microtubule-associated protein 7	Down
T60048	ACTG2	Actin, gamma 2, smooth muscle, enteric	Down
T61428	NEDD9	Neural precursor cell expressed, developmentally down-regulated 9	Down
T77733	TUBG1	Tubulin, gamma 1	Down
W72207	CSTA	Cystatin A (stefin A)	Down
W95389	SHD1	Sac3 homology domain 1 (S. cerevisiae)	Down
AA001897	SPTA1	Spectrin, alpha, erythrocytic 1 (elliptocytosis 2)	Up
AA025276	CTNND1	Catenin (cadherin-associated protein), delta 1	Up
AA057796	MYO1B	Myosin IB	Up
AA284634	JAK1	Janus kinase 1 (a protein tyrosine kinase)	Up
AA461325	ADD3	Adducin 3 (gamma)	Up
AA482231	MARCKS	Myristoylated alanine-rich protein kinase C substrate	Up
AA490477	MYH10	Myosin, heavy polypeptide 10, non-muscle	Up
AA495981	ARHGAP6	Rho GTPase activating protein 6	Up
AA679180	PTPN13	Protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase)	Up
AA774983	TPM4	Tropomyosin 4	Up
AI261600	WASL	Wiskott-Aldrich syndrome-like	Up
AW005820	PTPN4	Protein tyrosine phosphatase, non-receptor type 4 (megakaryocyte)	Up
AW075457	CCT3	Chaperonin containing TCP1, subunit 3 (gamma)	Up
R56096	WASF1	WAS protein family, member 1	Up
T55835	HDAC6	Histone deacetylase 6	Up
T57805	ROCK1	Rho-associated, coiled-coil containing protein kinase 1	Up
W42849	APP	Amyloid beta (A4) precursor protein (protease nexin-II, Alzheimer disease)	Up
W86182	PNN	Pinin, desmosome associated protein	Up

VIII.

Additional Figure 1: The level of the RUNX1 transcript was measured by quantitative RT-PCR using the lightcycler PCR machine (Roche). The concentration of the RUNX1 transcript is relative to the level of the housekeeping gene PSMB2. The two affected individuals (3, 4) show a decrease of expression compared to the unaffected individuals (1, 2). 1: IV:1, 2: V:3, 3: V:1, 4: V:2, as described in [14].

IX.

Additional Figure 2: protein overexpression of RUNX1 and CBFb.

Levels of exogenous proteins were determined by western blot in Hela cells following infection with a range of multiplicity of infection (MOI) of adenovirus. The antibodies used in this assay do not recognize the endogenous proteins.

X.

Additional Figure 3: Correspondence between datasets of differentially expressed genes (DEGs). A mean-rank gene set enrichment test was performed to determine the rank of the DEGs in the ranked data (absolute t-statistics) of the other approaches. Significant p-values mean that a high correlation was observed between these approaches. The p-values are corrected for multiple testing. FPD: FPD-AML dataset, CBF: overexpression dataset, E8.5 and E12: embryonic stages for the mouse datasets, DEG: differentially expressed genes.

XI.

Additional Figure 4. Gene Set Enrichment analysis. Histograms of normalized rank for each gene set. Sets with adjusted p-value lower than 0.05 are plotted in red. Normalized ranks were calculated by dividing the rank of each gene by the total number of genes. The value plotted on the y-axis is the percentage of genes, which lies in each bin, and is calculated by dividing the counts in the bin by the total number of genes in the gene set.

XII.

Additional Figure 5. Expression patterns of RUNX1 and a subset of differentially expressed genes. The left panel represents a cDNA panel containing 20 human tissues and the right panel contains hematopoietic cell lines. RUNX1 expression is shown at the top. A number of RUNX1 isoforms were identified. The genes present in the first half of the figure are strongly expressed in the hematopoietic cells whereas the second half shows a low expression in these cells. Actin expression is shown at the bottom to illustrate the presence of an equal amount of template in each well.

XIII. References for Additional Information

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2. Levanon D, Glusman G, Bangsow T, Ben-Asher E, Male DA, Avidan N, Bangsow C, Hattori M, Taylor TD, Taudien S et al: Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1. Gene 2001, 262(1-2):23-33.

3. Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, Van Ormondt H, Hoeben RC, Van Der Eb AJ: Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 1996, 7(2):215-222.

4. Jensen RH, Bigbee WL: Direct immunofluorescence labeling provides an improved method for the glycophorin A somatic cell mutation assay. Cytometry 1996, 23(4):337-343.

5. Michaud J, Kudoh J, Berry A, Bonne-Tamir B, Lalioti MD, Rossier C, Shibuya K, Kawasaki K, Asakawa S, Minoshima S et al: Isolation and characterization of a human chromosome 21q22.3 gene (WDR4) and its mouse homologue that code for a WD-repeat protein. Genomics 2000, 68(1):71-79.

6. Shim MH, Hoover A, Blake N, Drachman JG, Reems JA: Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage. Exp Hematol 2004, 32(7):638-648.

7. Gnatenko DV, Dunn JJ, McCorkle SR, Weissmann D, Perrotta PL, Bahou WF: Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 2003, 101(6):2285-2293.

8. Tenedini E, Fagioli ME, Vianelli N, Tazzari PL, Ricci F, Tagliafico E, Ricci P, Gugliotta L, Martinelli G, Tura S et al: Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells. Blood 2004, 104(10):3126-3135.

9. Skop AR, Liu H, Yates J, 3rd, Meyer BJ, Heald R: Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science 2004, 305(5680):61-66.

10. Lew DJ, Burke DJ: The spindle assembly and spindle position checkpoints. Annu Rev Genet 2003, 37:251-282.

11. Wood RD, Mitchell M, Sgouros J, Lindahl T: Human DNA repair genes. Science 2001, 291(5507):1284-1289.

12. Jen KY, Cheung VG: Transcriptional response of lymphoblastoid cells to ionizing radiation. Genome Res 2003, 13(9):2092-2100.

13. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, Chinnaiyan AM: Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression. Proc Natl Acad Sci U S A 2004, 101(25):9309-9314.

14. Michaud J, Wu F, Osato M, Cottles GM, Yanagida M, Asou N, Shigesada K, Ito Y, Benson KF, Raskind WH et al: In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis. Blood 2002, 99(4):1364-1372.