Persistent Expression of a b-Globin Transgene Figure 3. GATA1-HS2 is able to sustain transgene expression in the erythroid compartment differentiating from human HPCs. (A–B) BFU-E and CFU-GM derived from CMV-GFP- and G-CMV-GFP-transduced CD34+ cells were scored by PCR for vector integration and for GFP expression under fluorescence microscopy. The percentages of GFP+ (dark grey) and GFP2 (light grey) colonies among the vector-containing BFU-E are represented (for each sample set, n is indicated above the corresponding bar). The proportion of vector-containing BFU-E that do not express the transgene is significantly higher for CMV-GFP compared to the percentage observed in G-CMV-GFP-transduced BFU-E (A; P,0.005 Fisher exact test). (B) No significant difference in the proportions of GFP+ CFU-GM was observed between the two groups. (C–D) NOD SCID mice were transplanted with CMV-GFP- and G-CMV-GFP-transduced CB-derived CD34+ cells. 8–11 weeks after transplantation mice were sacrificed and BM cells were ex vivo cultured in a medium allowing the growth of human erythroid and myeloid colonies. BFU-E and CFU-GM derived from CMV-GFP- and G-CMV-GFP- transduced SRCs (SCID repopulating cells) were scored by PCR for vector integration. The proportion of vector-positive BFU-E that do not express the transgene is higher for CMV-GFP vector compared to the percentage observed in G-CMV-GFP-transduced BFU-E (P,0.05; Fisher exact test). The proportions of GFP+ CFU-GM were similar for both vectors (P.0.1). doi:10.1371/journal.pone.0027955.g003 GATA1-HS2 reduces proviral silencing: exploring the mechanism GATA1 regulates all known erythroid-specific genes, often through the association with the CBP histone acetyltransferase [40]. To investigate whether GATA1 binds to the HS2-containing LTRs and recruits CBP, ChIP experiments were carried out in GATA1-expressing HEL cells transduced with either GLOBE or G-GLOBE LV, using antibodies against GATA1 and CBP. PCR primer pairs were used to amplify sequences corresponding to the 59 LTR by qPCR analysis (Figures 6A). As expected, GATA1 was highly enriched at GATA1-HS2-containing LTRs (Figures 6B). Accordingly, CBP showed occupancy above PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e27955