Nucleic Acids Research, 2010, Vol. 38, No. 19 6371 Figure 6. Overexpression of Elf-1 impairs terminal erythroid diﬀerentiation. (A) Expression of PU.1 and Elf-1 during in vivo fetal liver terminal erythroid diﬀerentiation. Real-time PCR analysis of mouse PU.1 and Elf-1 expression in three populations of murine E14.5 fetal liver cells: (RI) CD71medTER119low, (RII) CD71highTER119med/low and (RIII) CD71highTER119high; sorted by ﬂow cytometry and representing sequential stages of erythroid diﬀerentiation (Supplementary Figure S11A). m18s ribosomal RNA gene expression was used to normalize expression of each population. Values are expressed relative to the expression at RI stage. Error bars indicate SD. (B) Representative FACS plots for Ter119 and CD71 expression during in vitro diﬀerentiation in lineage negative E14.5 fetal liver cells overexpressing Elf-1 or a control (PIG) vector after 24 h (Day 1) or 48 h (Day 2). Five populations corresponding to stages of erythroid diﬀerentiation from progenitors to enucleated cells are gated: (R1) CD71medTER1119low, (R2) CD71highTER119low, (R3) CD71highTER1119high, (R4) CD71medTER1119high and (R5) CD71lowTER1119high (Supplementary Figure S11B). Percentage of GFP+ cells in each of these stages are indicated. (C) Representative FACS plots for cellular size in lineage negative E14.5 fetal liver cells overexpressing Elf-1 (black line) or a control (PIG) (grey shaded area) vector after 24 h (Day 1) or 48 h (Day 2) of in vitro culture. (D) Bar charts indicating the proportion of cells at each of the ﬁve stages of erythroid diﬀerentiation (R1–R5) after 24 h (Day 1) and 48 h (Day 2) of in vitro culture of lineage negative E14.5 fetal liver cells retrovirally transfected with an overexpressing Elf-1 or control (PIG) vector. Representative plot (mean and SD) for one of ﬁve independent experiments, each one performed in triplicate. P 0.05 (Student’s t-test) are indicated with an asterisk. DISCUSSION Reconstruction of regulatory networks is essential to understand the complex and dynamic interactions between the multiple regulators that control the develop- ment and subsequent diﬀerentiation of blood stem cells. Deﬁnition of transcriptional regulatory networks requires both knowledge of the transcription factors involved and also the DNA sequences to which they bind. Therefore, identiﬁcation and subsequent functional analysis of gene regulatory elements remains an essential area of research, in particular when it serves to clarify upstream hierarchies of key regulators thus providing ﬁrm anchor points for major regulatory network nodes. In this article, we have analysed the transcriptional regulation of Elf-1, which itself encodes a major transcriptional regulator of blood and endothelial cells. In this study, an integrated analysis of the Elf-1 locus using comparative genomic analysis, ChIP-chip and func- tional studies allowed us to deﬁne ﬁve distinct regulatory regions spread over 50 kb, including the identiﬁcation of a lineage-speciﬁc enhancer element. The comparative genomic analysis showed the presence of ﬁve regions conserved between mouse, human, dog and opossum. Of these conserved regions, four contained high levels of acetylation at H3K9, a chromatin mark associated with areas of open chromatin. One additional region, conserved between mouse, human and dog but not opossum, was also enriched for this chromatin mark. Further ChIP-chip assays were performed to analyse trimethylation of lysine 4 on the tail of histone H3, a modiﬁcation that is associated with recently transcribed regions. These experiments led to the discovery that four of the ﬁve regions identiﬁed by elevated histone acetyl- ation display promoter activity in functional assays. The ﬁfth region, which carried active chromatin marks but no transcription marks, was validated as an enhancer in func- tional analysis. We also demonstrate that PU.1 is a major upstream regulator of Elf-1 since PU.1 binding could be detected by ChIP-chip in one of the four promoter regions. We validated this result with functional analysis