Table S1. Primers used in RT-PCR studies (all in 5 to 3 direction)

Transcription

1 Table S1. Primers used in RT-PCR studies (all in 5 to 3 direction) Epo Fw CTGTATCATGGACCACCTCGG Epo Rw TGAAGCACAGAAGCTCTTCGG Jak2 Fw ATCTGACCTTTCCATCTGGGG Jak2 Rw TGGTTGGGTGGATACCAGATC Stat5A Fw TTACTGAAGATCAAGCTGGGG Stat5A Rw TCATTGTACAGAATGTGCCGG Stat5B Fw CATTTTCCCATTGAGGTGCG Stat5B Rw GGGTGGCCTTAATGTTCTCC GAPDH Fw ACGGATTTGGTCGTATTGGG GAPDH Rw TGATTTTGGAGGGATCTCGC ALAS-E Fw CAACATCTCAGGCACCAGTA ALAS-E Rw CTCCACTGTTACGGATACCT Fech Fw ATCCAGCAGCTGGAGGGTCT Fech Rw TGAATCTTGGGGGTTCGGCG GATA1 Fw AGTTTGTGGATCCTGCTCTG GATA1 Rw GCAATGGGTACACCTGAAAG GATA2 Fw AGCGTCTCCAGCCTCATCTTCCGCG GATA2 Rw CGAGTCTTGCTGCGCCTGCTT EKLF Fw CCTGTTTGGTGGTCTCTTCACA EKLF Rw AGGGTCCATTCGTGGGAAA NF-E2p45 Fw AGTGTCAGCTCAGGCTCAGC NF-E2p45 Rw GCAGCTCGGTGATGGACATG p53 Fw CTGTCATCTTCTGTCCCTTC p53 Rw TGGAATCAACCCACAGCTGCA p21 Fw GTCACTGTCTTGTACCCTTGTG p21 Rw CGGCGTTTGGAGTGGTAGAAA p27 Rw AAGCGACCTGCAACCGACGATTCTT p27 Fw GCTCCACAGAACCGGCATTT alpha globin Fw GAGGCCCTGGAGAGGATGTTCC alpha globin Rw ACAGCGCGTTGGGCATGTCGTC beta globin Fw TACATTTGCTTCTGACACAAC beta globin Rw ACAGATCCCCAAAGGAC gamma globin Fw CTTCAAGCTCCTGGGAAATGT gamma globin Rw GCAGAATAAAGCCTACCTTGAAAG epsilon globin Fw GCCTGTGGAGCAAGATGAAT epsilon globin Rw GCGGGCTTGAGGTTGT SCL/Tal1 Fw GTTCTTTGGGGAGCCGGATG SCL/Tal1 Rw ACATTCTGCTGCCGCCATCG Beta-actin Fw CATCACCATTGGCAATGAGC Beta-actin Rw CATACTCCTGCTTGCTGATC GPIIB Fw CTCCACAACAATGGCCCTGG GPIIB Rw CTTGAGAGGGTTGACAGGAG Bcl-xL Fw GAATGACCACCTAGAGCCTTGG

2 Bcl-xL Rw Bcl-2 Fw Bcl-2 Rw IGF-1 Fw IGF-1 Rw Sox6 Fw Endogenous Sox6 Rw Transduced Sox6 Rw (designed on Flag epitope) SOCS3 Fw SOCS3 Rw TGTTCCCATAGAGTTCCACAAAAG ATGTGTGTGGAGAGCGTCAACC TGAGCAGAGTCTTCAGAGACAGCC ATGCTCTTCAGTTCGTGTGTG GCACTCCCTCTACTTGCGTTC GAGGCAGTTCTTTACTGTGG CCGCCATCTGTCTTCATAC CTTATCGTCGTCATCCTTGTA GGAGACTTCGATTCGGGACC GAAACTTGCTGTGGGTGACC

3 Table S2. Primer used to amplify of human genomic regions, and their cloning strategies SOCS3 promoter: Fw:5 ACGTGTCGACACGTGGTACCGCTCTCCCGAAGCGGCGCC 3 ; Rev:5 ACGTAAGCTTCAAGTCGGAGCCGCCGCGG 3, containing a SalI and HindIII restriction site (underlined), respectively, for further cloning into XhoI-HindIII of the pgl2 luciferase reporter vector (Promega, Fitchburg, WI). SOCS3 enhancer: The following oligonucleotides were annealed and then cloned upstream to SOCS3 promoter (SacI-NheI restriction sites, underlined). Mutations are shown in small letters. WT Fw 5 ACGTGAGCTCACTTGACTTGTGTCAGAGCATTGTAATTTACAAAGCACTTTCTCAT CCATGCTAGCACGT3 WT Rev: 5 ACGTGCTAGCATGGATGAGAAAGTGCTTTGTAAATTACAATGCTCTGACACAAGTC AAGTGAGCTCACGT 3 Mut Fw: 5 ACGTGAGCTCACTTGACTTGTGTCAGAGCAgTGTgATTTgCAggGCACTTTCTCATCC ATGCTAGCACGT 3 Mut Rev: 5 ACGTGCTAGCATGGATGAGAAAGTGCccTGcAAATcACAcTGCTCTGACACAAGTCA AGTGAGCTCACGT 3

4 Table S3. Primers used in chromatin immunoprecipitation Human ε-globin promoter Fw: 5 GTTGCAGATAGATGAGGAGCC 3 ; Rw 5 GTCAAGGCTGACCTGTGTCC 3 Human γ-globin promoter A Fw: 5 CCAAGGTCATGGATCGAGTT 3 ; Rw 5 ACACTGTGACAGCTGGGATG 3 Human γ-globin promoter B Fw 5 AAACGGTCCCTGGCTAAACT 3 ; Rw 5 GCTGAAGGGTGCTTCCTTTT 3 The two primer pairs covering 1kb region upstream to the γ transcriptional start site gave the same result. SOCS3 enhancer Fw 5 CTCTCGGCAGAGGTTTATGG 3 ; Rw 5 TCAAGGAATAGCCCTTGAGG 3 GAPDH locus Fw: 5 CGGAGTCAACGGATTTGGTCGTAT 3 ; Rw: 5 AGCCTTCTCCATGGTGGTGAAGAC 3 EMSA oligonucleotide probes: SOCS3 WT probe: Fw: 5 GTCAGAGCATTGTAATTTACAAAGCACTTTCTC3 Rw: 5 GAGAAAGTGCTTTGTAAATTACAATGCTCTGAC3 SOCS3 Mut probe: Fw: 5 GTCAGAGCAGTGTGATTTGCAGGGCACTTTCTC3 Rev: 5 GAGAAAGTGCCCTGCAAATCACACTGCTCTGAC3 Col2a1 probe Fw: 5 CTGTGAATCGGGCTCTGTATGCGCTTGAGAAAAGCCCCATTCATGAGA3 Rw: 5 TCTCATGAATGGGGCTTTTCTCAAGCGCATACAGAGCCCGATTCACAG3

5 Figure S1 (A) Real Time quantitation of Sox6 overexpression in K562cells, CD34 + -derived primary erythroblasts and HEL cells, relative to GAPDH expression (set equal to 100%). (B) Expression of endogenous Sox6 and of transduced Sox6 (Sox6FLAG) in primary erythroid cells. Histograms show the relative levels of expression (mean+ SEM of at least 3 independent experiments) compared to GAPDH, considered as 100%. Figure S2 (A) Real Time quantitation of erythroid genes expression in K562 cells overexpressing Sox6. The increased expression of erythroid genes globins, heme biosynthesis pathway is accompanied by a downregulation of the megacaryocitic gene GPIIB. Histograms show the fold increase in the expression levels induced by Sox6 (mean+ SEM of at least 3 independent experiments) using GAPDH as internal standard. (B) All globins chains normally expressed in K562 are upregulated in Sox6-K562 cells but the ratio of their expression level change significantly. Upper panel: fold increase of α/ζ (3.68) and γ/ε (2.19) ratios in Sox6-K562 versus EV-K562 (set equal to 1). Lower panel: fold decrease of ε/α and γ/α ratios in Sox6-K562 versus EV-K562 (set equal to 1). The increased γ/ε ratio indicates that ε-globin is less strongly induced than γ, in agreement with the known repressor role of Sox6 on the ε-globin gene. 13 However, ε/α and γ/α ratios are both decreased in Sox6-K562 vs EV-K562, suggesting a Sox6 repressive effect also on γ-globin, although less evident than that on ε (the ε/α ratio is almost 10 fold reduced, while the γ/α ratio is reduced by 5 fold. (C D) ChIP analysis confirms the ability of Sox6 to bind the human ε and γ-globin promoters. (C) Sequence comparison of the double Sox6 binding sites within mouse εy and human ε-globin promoters. Nucleotides positions are relative to the start site. (D) The anti-flag antibody or rabbit IgG were used to immunoprecipitate chromatin from EV-K562 or Sox6-K562 cells. Lanes 1 and 2: input chromatins. Lanes 3 and 4: normal rabbit IgG. Lane 5 and 6: anti-flag antibody (that recognizes the Sox6FLAG transduced protein) Lane 7: water. 40 : PCR cycles number. Figure S3 (A) Sox6 overexpression anticipates and induces the expression of the α, β, and γ but not of the ε globin genes in cord blood derived primary erythroid culture. Real time PCR quantification of globins mrna expression level on samples taken at d8, d10, d12, d14 of the culture. Histograms represent the mean expression level (relative to GAPDH); standard deviations refer to 3 independent experiments. Please note the different scale on the y axis: ε expression is marginal and does not increase upon Sox6 overexpression. (B) The ε/α and γ/α ratios (set equal to1 in untransduced cells) are decreased in Sox6-transduced cells. Figure S4. SOCS3 overexpression in K562 cells (A) a SOCS3-IRES-GFP plasmid was transfected in K562 cells and 48 hours after transfection GFP + cells were FACS sorted and seeded in parallel with GFP cells, and the increased expression of SOCS3 in GFP + cells was tested by Real Time PCR. (B) GFP + K562 (SOCS3- overexpressing) cells stop proliferating with kinetics similar to that observed upon Sox6 overexpression (Fig. 1F), y axis: number of cells, x axis: days after sorting. (C) The increased SOCS3 is not associated with K562 differentiation, as shown by Real-Time PCR on α, γ and ε- globin transcripts.

6 Figure S5. Stat5 phosphorylation does not change upon Sox6 overexpression (A) STAT5 phosphorylation was monitored by FACS analysis at 1h, 3h and 6h (and 72h, not shown) after Sox6 transduction in K562, in parallel with GFP expression. x axis: Mean Fluorescence Intensity, y axis: Phospho STAT5. Left panels: EV-K562; Right panels, Sox6- K562. Gray peaks: unstained cells. Black peaks: uninfected K562. Red peaks: GFP cells, not expressing the transduced vectors. Green peaks: GFP + cells, expressing the transduced vector (note that GFP positivity coincides with Sox6 expression thanks to the bicistronic Sox6-IRES- GFP cassette). (B) Semiquantitative PCR reveals non major changes in the level of EPOR, JAK2 and STAT5A/B transcripts in K562 cells overexpressing Sox6. GAPDH is used as internal standard. PCR cycles are indicated below the figure.

7 Supplementary Fig. 1 A Expression relative to GAPDH (%) Sox6FLAG expression Sox6-K562 Sox6-Erythroblasts Sox6-HEL B Expression relative to GAPDH (%) CB-derived Erythroblasts Endogenous 1 Sox6 Sox6FLAG 2

8

9

10

11