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