bound protein on each strand at 61 kb from each CMV-GFP (green), G-CMV-GFP (blue) and random (grey box) insertion site. The distributions of the entire datasets are represented by box plots. Data were retrieved from ChIP-sequencing experiments performed in the genome of human CD34+/CD133+ HPCs [39]. Two-sample test statistics among CMV-GFP, G-CMV-GFP and random distributions for each modification indicate that compar- isons between CMV-GFP and G-CMV-GFP are not statistically significant (non parametric Mann–Whitney U test, not shown). All comparisons between LV and random distributions are statistically significant (*P,0.05; ****P,0.001; Random: n = 9974; CMV- GFP: n = 438; G-CMV-GFP: n = 305). (EPS) Figure S6 GATA1 and CBP occupancy at the endogenous LCR HS4. Chromatin from GLOBE- and G-GLOBE-trans- duced HEL was immunoprecipitated with antibodies against GATA1 and CBP and control rabbit IgG. The endogenous LCR HS4 served as a positive control for GATA1 and CBP binding in both GLOBE- and G-GLOBE-transduced bulk populations (n = 2). Error bars represent SEM. (EPS) Figure S7 Similar acetylation levels at the control AldolaseA gene. ChIP assay was performed as described in the legend to Figure 7. The levels of histone H3 and H4 acetylation are comparable at the positive control region from Aldolase A gene in both GLOBE- and G-GLOBE-transduced HEL cells (n = 4). The results are averages of 4 independent experiments. Error bars represent SEM. (EPS) Figure S8 Analysis of histone methylation patterns at the 59LTR and at the b-globin promoter-HS2 region of GLOBE and G-GLOBE vectors. ChIP assay was carried out in GLOBE- and G-GLOBE-transduced HEL cells using antibodies against mono- and trimethyl-H3K4 (H3K4me1 and H3K4me3) and against trimethyl-H3K9 and trimethyl-H3K27. qPCR was performed to amplify the 59LTR (A) and the b-globin promoter- HS2 region (B). Bars denote averages of 4 independent experiments. Error bars represent SEM. (EPS) References 1. Lucarelli G, Andreani M, Angelucci E (2002) The cure of thalassemia by bone marrow transplantation. Blood Rev 16: 81–85. 2. May C, Rivella S, Callegari J, Heller G, Gaensler KM, et al. (2000) Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 406: 82–86. 3. Persons DA, Hargrove PW, Allay ER, Hanawa H, Nienhuis AW (2003) The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number. Blood 101: 2175–2183. 4. Puthenveetil G, Scholes J, Carbonell D, Qureshi N, Xia P, et al. (2004) Successful correction of the human beta-thalassemia major phenotype using a lentiviral vector. Blood 104: 3445–3453. 5. Miccio A, Cesari R, Lotti F, Rossi C, Sanvito F, et al. 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Persistent Expression of a b-Globin Transgene Table S1 VCN and number of integrants in primary trans- planted mice used as donors for CFU-S assays. The table shows the number of integration sites retrieved from BM of primary transplanted mice, the average vector copy number per BM cell, and the number of CFU-S analyzed for each primary transplanted mouse. (DOC) Table S2 Human b-globin expression in mice transplanted with GLOBE- and G GLOBE-transduced BM. The table shows the percentage of human ß-globin positive RBC and the ratio between the areas of HPLC peaks corresponding to human ß and murine a globin chains in mice transplanted with GLOBE- and G GLOBE- transduced BM. (DOC) Table S3 List of GLOBE proviral integration sites in secondary CFU-S. The table shows b-globin expression, chromosomal location of integrated provirus, target gene symbol, and RefSeq identifier number in secondary CFU-S. 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