SUPPLEMENTAL LEGEND

Supplemental Figure 1.

Identification of the Flt3 transcriptional start site. (A) RNA samples from Pax5+/+ (Flt3-) and two Pax5-/- (Flt3+) pro-B cell lines and myeloid M1 cells were analyzed by RNase protection assay with a riboprobe spanning nt -431 to +107 of the Flt3 gene. The positions of 32P-labelled single-stranded DNA fragments of known size (in nt) are indicated (left). A riboprobe complementary to a transcript encoding the ribosomal S16 protein, expressed in all samples, was a loading control. (B) Alignment of mouse (Mus), human (Hum), rat, and dog genomic sequences adjacent to the Flt3 coding region (capitalized). Asterisks denote nucleotide identity. The positions of the Flt3 transcriptional start site determined by RNase protection or 5’-RACE are denoted by a square bracket or vertical arrow, respectively. Two putative Pax5 binding sites, Px1 and Px2 (in the reverse orientation), are boxed and the position of PCR primers used for the ChIP experiments is indicated (horizontal arrows). (C) The two Pax5 binding sites from the murine Flt3 promoter aligned to the Pax5 consensus sequence with matching nucleotides capitalized.

Supplemental Materials and methods.

Flt3 promoter analysis

RNase protection: A Flt3 genomic fragment (-431 to +107) was amplified by PCR and cloned into the plasmid vector pSP72 enabling the in vitro synthesis of a riboprobe complementary to the Flt3 mRNA and incorporating 32P-GTP using the SP6 riboprobe system (Promega). The RNase protection assay, using 10g total RNA from pro-B or M1 cell lines, was performed as previously described (Vitelli et al. 1988) except samples were hybridized at 60C.

5’-RACE: The 5’-end of the Flt3 transcript was determined by rapid amplification of cDNA ends, (RACE) using 1g poly (A)+ mRNA (extracted from total RNA using Oligotex, Qiagen) and two Flt3-specific primers (Supplementary table) corresponding to sequences near to the Flt3 translation start site, according to the manufacturer’s instructions (SMART RACE kit, BD Biosciences).

Oligonucleotide primers used in this study

Sequence / Reference
RT-PCR
CD19-F / GAGAGGCACGTGAAGGTCATTG
CD19-R / CATGGCTCTGAGCTCCAGTATC
Flt3-F / GTGACTGGCCCCCTGGATAACGAG
Flt3-R / TCCAAGGGCGGGTGTAACTGAACT
Hprt-F / GGGGGCTATAAGTTCTTTGC / (Hu et al. 1997)
Hprt-R / TCCAACACTTCGAGAGGTCC / (Hu et al. 1997)
5'-RACE
Flt-race1 / CTCTGTCACGTTCAAGATGGCCATG
Flt-race2 / AGCTGCACTTGCAGGGTGATGGAC
EMSA
Flt Px1 / CTGTGGTCAGTGACGCGCATCCTTCAGCGA
Flt Px2 / GCCGCTGGGACCGCATCACAGGCTGGGCCG
CD19 / GCAGACACCCATGGTTGAGTGCCCTCCAGG / (Kozmik et al. 1992)
CD19 mut / GCAGACACCCGTGATTAATTGCCCTCCAGG / (Kozmik et al. 1992)
ChIP
CD19-F / GGCCTAACCTAAGGTGTGACCAC
CD19-R / GTGGCTGCGCAGAGGATGCTG
Flt3-F / GGCCTCTGGAGAGAGGTTCCTC
Flt3-R / GCTGCGCCAACGCCCGCATG

Retroviral infection of bone marrow stem cells

The MigR1-Flt3 and parental MigR1 retroviral vectors were transfected into Phoenix packaging cells using the Fugene reagent (Roche Diagnostics). Supernatants were harvested after 40 hours. For stem cell cultures, C57BL/6-Ly5.2 mice were injected intraperitoneally with 5-Fluorouracil (150mg/kg, Sigma). After 4 days, the bone marrow was harvested, depleted for red blood cells and cultured for 3 days in HSC medium. Cells were transferred daily onto retronectin-coated (4g/cm2; Takara Biotechnology) tissue culture wells pre-loaded with viral supernatants (MigR1-Flt3 or MiR1) in the presence of 12.5g/ml polybrene. Cells were then either cultured further on OP9 stroma for 7 days or used for transplantation studies. Bone marrow chimeras were generated by intravenous transfer of 2-5x105 C57BL/6-Ly5.2 cultured stem cells into lethally irradiated C57BL/6-Ly5.1 recipients. Recipients were analyzed between 7-12 weeks post reconstitution.

SUPPLEMENTAL REFEREENCES

Hu, M., D. Krause, M. Greaves, S. Sharkis, M. Dexter, C. Heyworth, and T. Enver. 1997. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev11: 774-85.

Kozmik, Z., S. Wang, P. Dorfler, B. Adams, and M. Busslinger. 1992. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol Cell Biol12: 2662-72.

Vitelli, L., I. Kemler, B. Lauber, M.L. Birnstiel, and M. Busslinger. 1988. Developmental regulation of micro-injected histone genes in sea urchin embryos. Dev Biol127: 54-63.