Supplementary information

GATA-1 and GATA-2 binding to 3’ enhancer of WT1 gene is essential for its transcription in acute leukemia and solid tumor cell lines by Ayako Furuhata et al.

Rapid amplification of 5’ cDNA ends (5’ RACE) The transcription initiation point of

WT1 was determined with the RNA ligase-mediated rapid amplification method of

5’cDNA ends (5’RACE) using a Gene Racer kit (Invitrogen). The reverse WT1 gene- specific primer was 5’-GACCCGTAAGCCGAAGCGCCC-3’. The reverse WT1 gene- specific nested primer was 5’-TCCCTGCTGCTCTGGCTGCTGTA-3’.

Cloning of WT1 promoter and enhancer region

The upper primers for truncated luciferase vectors of -907, -301, -224, -203, and -92 bp 5’ promoter/luc were 5’-CAAACAGAGACTAGACGAAGG-3’, 5’-

TCATGGCCACTCCCCTAC-3’, 5’-CCTGAAGTTCCCGCCCTC-3’, 5’-

GAGCCTACCTGCCCCTCCCTCCA-3’, and 5’-TGAGACGAGGCTCCCACA-3’, respectively. The lower primer was pGL primer 2 of the pGL3 basic vector. The fragment was ligated into the SmaI and NcoI sites of pGL3 basic vector. The introduction of mutated Sp1 site into -224/luc was performed by PCR using the following forward primer, 5’-

CCTGAAGTTCCCTAAATCTTGGAGCCTACCTGCCCCT-3’ (underline: mutated sequence). The lower primer was pGL primer 2. The fragment was also ligated to the

SmaI and NcoI sites of pGL3 basic vector.

The intron 3- and 3’enhancers of the WT1 were also obtained by PCR using the primer sets described below. Intron 3 enhancer primers were forward, 5’-

GGAGATCACAAGGCCAGCTTCATTCC-3’; reverse, 5’-

GGGAATTCCCTTTCCAGGGCAACTGA-3’. 3’ enhancer primers were forward (A),

5’-GGGAATTCCCTTTCCAGGGCAACTGA-3’; reverse (B), 5’-

GGGTCGACCCTGGCTCTTTCCGACTC-3’. A single, double or dotted underline denotes an added BglII, EcoRI, or the SalI enzyme site, respectively. After obtaining intron 3 enhancer and 3’ enhancers, each fragment was subcloned into the pBluescriptII

KS(+) vector, and these constructs were digested by BamHI/EcoRI [in cases of intron 3 enhancer] or EcoRI/ SalI [in cases of 3’ enhancer]. Both fragments were ligated to the

BamHI and SalI sites of pGL3 basic vector (pro-enh/Luc). Preparing truncated and mutated enhancer

Truncation of 3’ enhancer region was performed using promoter-enhancer/Luc as the starting material. The primer sets for producing truncated luciferase vectors of e1, e2, e3, e4, e5, e6 and e7 (illustrated in Fig 4b) were described below. e1 forward, 5’-GGGAATTCTTTGGTGCAGCCCCTCTC-3’ named (C); reverse, (B) described above. e2 forward, 5’-GGGAATTCTAAAAGTCAGGTCCAGAGGC-3’ named (D); reverse,

(B). e3 forward, (A); reverse, 5’-GGGTCGACTGTAAAACAGTCTCCATAGCT-3’ named

(E). e4 forward, 5’-GGGAATTCTTTGGTGCAGCCCCTCTC-3’ named (F); reverse, 5’-

GGGTCGACTGTAAAACAGTCTCCATAGCT-3’ named (G). e5 forward, (A); reverse, 5’-GGGTCGACTGAGAGGGGCTGCACCAA-3’ named

(H).

A single or double underline denotes the added SalI enzyme site or EcoRI enzyme site, respectively. These fragments were inserted into SalI and BamHI sites of pro- enh/Luc, and used for enhancer analysis. To introduce mutated GATA binding sites (Figure 4c), the following primer sets were prepared, and PCR was performed using the e1 as a template.

Primer (I) (for proximal GATA binding site), 5’-

GGGAATTCTAAAAGTCAGGTCCAGAGGCCCCTCTTCCTTTAACACTGGCTCT

TGCATC-3’.

(J) and (K) (for middle GATA binding site), 5’-

TGACTCATTTCCAAAAGCCGTTTTTATCTTTTCCTGC-3’ and 5’-

GCAGGAAAAGATAAAAACGGCTTTTGGAAATGAGTCA-3’.

(L) and (M) (for distal GATA binding site), 5’-TGACTCATTTATATCAGCCGT-

TCCAAACTTTTCCTGCC-3’ and 5’-

GGCAGGAAAAGTTTGGAACGGCTGATATAA-3’. A double or dotted underline denote the added EcoRI enzyme site or mutated GATA site, respectively. To introduce the mutated middle and distal GATA sites, a two-step PCR was performed. Briefly, to obtain a middle GATA site mutation, PCR amplification was performed using the two primer sets, (D and K, and B and J, respectively) with e1 as the template. Each PCR product was purified, and the mixture of these two PCR products was used for the second PCR template with the primer set of B and D. Similarly, distal GATA mutation was obtained using the same method. For the first PCR, two primer sets, D and M, and

B and L, were used. Then the second primer set, B and D, was used with the first PCR products as the template.

Collection of stem cell and common progenitor cell fraction by flowcytometry

Separation of hematopoietic stem cell (HSC) and progenitor cell fraction from normal bone marrow mononuclear cells was started by staining with lineage-associated PE-

Cy5.5-conjugated antibodies including CD2, CD3, CD4, CD8, CD14, CD19, CD20 and

CD56 from Caltag (South San Francisco, CA). The cells with the lineage cocktail antibodies were further incubated with HSC-associated antibodies consisting of APC- conjugated anti-CD34 (HPCA-2; BD Pharmingen, San Diego, CA) and Cy7PE- conjugated anti-CD38 (BD Pharmingen). Unstained samples and isotype controls were included to assess the background fluorescence. After staining, HSC identified as

CD34+CD38–Lin– or progenitor cells identified as CD34+CD38+ Lin– were analyzed and sorted by using FACSAria (BD Immunocytometry Systems, San Jose, CA). Using each fraction, RNA was purified and cDNA was prepared as described in the Materials and methods.