Persistent Expression of a b-Globin Transgene Figure 7. Increased CBP-mediated histone acetylation levels at GATA1-HS2-containing LV. ChIP assay was carried out in GLOBE- and G-GLOBE-transduced HEL cells using antibodies against pan-acetylated histone H3, acetylated histone H3K18, acetylated histone H4K5, and acetylated histone H4K8. (A) Map of GLOBE and G-GLOBE proviruses Arrows indicate the primer pairs used to amplify the 59LTR and the b- globin promoter-HS2 region. The acetylation levels are significantly higher at the 59 LTRs (B) and at the b-globin promoter region (C) of GATA1-HS2-containing LV compared to the GLOBE vector. (n = 4). *P,0.05; **P,0.01 by Student t test. Error bars represent SEM. doi:10.1371/journal.pone.0027955.g007 Figure 6. GATA1 and CBP bind to GATA1-HS2-containing 59 LTRs. HEL cells were transduced with GLOBE and G-GLOBE and ChIP assay was performed using antibodies against GATA1 and CBP. (A) Schematic map of GLOBE and G-GLOBE vectors. Arrows indicate the primer pairs used to amplify by PCR the viral 59 LTR. (B) Chromatin was immunoprecipitated with antibodies against GATA1 and CBP and isotype-matched control antibodies (Rabbit IgG). GATA1 and CBP binding to the 59LTR of GLOBE and G-GLOBE vectors was measured by real-time PCR. GATA1 and CBP are detected only at the GATA1-HS2-containing 59LTRs (n = 2). *P,0.05 by Student t test. Error bars represent SEM. doi:10.1371/journal.pone.0027955.g006 induces open chromatin conformations by acetylating histones at specific residues. The GATA1-HS2 element contains a high-affinity, palindromic GATA1-binding element, and binds the transcription factors CP2 [31] and BCL11A [32]. We previously showed that GATA1-HS2 acts as an efficient, erythroid-specific enhancer in the context of both retroviral and lentiviral vectors [33,34]. In this study, we probed the activity of GATA1-HS2 in reducing silencing and position-dependent variability of transgene expression in the context of self-inactivating LV in which GATA1-HS2 was inserted in both LTRs. The modification had no influence on the target site selection of the vector, which maintained the typical lentiviral preferences for active genes, enriched in H3K36me3, H4K20me1, H3K9me1, and H3K27me1 histone modifications. Nevertheless, GATA1-HS2-flanked transgenes showed more persistent and position-independent expression characteristics, indicating an active role of the element in maintaining a permissive chromatin structure in the target cells. Differentiation of HSCs into erythroblasts is marked by a gradual increase in chromatin condensation, which is associated with gene silencing at most loci. We therefore tested whether GATA1-HS2 could prevent inactivation during erythroid differentiation of PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e27955