6370 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 5. PU.1 regulates expression of Elf-1. (A) PU.1 ChIP-chip assays. MVista representation of mouse/human sequence conservation is shown on the top together with all candidate regulatory elements. Annotations are the same as in Figure 1A. ChIP-chip for the myeloid progenitor cell line 416B across the 109 kb tiling path spanning the mouse Elf-1 locus. Enrichment values are expressed as fold above mean intensity across the locus. (B) Transactivation of candidate regulatory elements by PU.1. Transient co-transfections of 293T cells with luciferase reporter constructs containing Elf-1 candidate regulatory elements in combination with either pcDNA3mPU.1 (PU.1) or the empty vector pcDNA3 (control). Values are expressed relative to the control, pcDNA3. Mean and SEM for two independent transfections (each one performed in triplicate) are shown. were ﬂow sorted into four populations representing sequential phases of erythroid diﬀerentiation (31) and the levels of PU.1 and Elf-1 transcripts in each population determined. Expression levels of mPU.1 and mElf-1 were determined by real-time qPCR in CD71medTER119low, CD71highTER119med/low, CD71highTER119high and CD71med/low TER119high cells, corresponding to regions RI–RIV, respectively (Figure 6A and Supplementary Figure S11A). The CD71lowTER119high population are largely enucleated cells, and were therefore excluded from this analysis. Interestingly, a very similar pattern of downregulation was seen for PU.1 and Elf-1, which was consistent with our previous results identifying PU.1 as an upstream regulator of Elf-1 and also suggesting that downregulation of Elf-1 may be necessary for erythroid diﬀerentiation. To further explore the biological signiﬁcance of our results, we took advantage of an extensively validated in vitro culture system that supports terminal erythroblast proliferation and diﬀerentiation in a manner that closely mimics the in vivo terminal proliferation and maturation of erythroid cells (30). In this system, diﬀerentiation of CFU-E progenitors can be followed step by step and in a quantitative manner using ﬂow cytometry. Essentially, lineage negative E14.5 fetal liver cells are puriﬁed and cultured in vitro. Diﬀerentiation is then analysed at various time points by double labelling for TER119 and CD71, which makes it possible to distinguish at least ﬁve distinct populations of cells: CD71medTER119low that contains primitive progenitor cells and proerythroblasts; CD71highTER119low, proerythroblasts and early basophilic erythroblasts; CD71highTER119high, early and late basophilic erythroblasts; CD71medTER119high chromatophilic and orthocromatophilic erythroblasts; CD71lowTER119high late orthocromatophilic erythro- blasts and reticulocytes (Figure 6B and Supplementary Figure S11B, regions R1–R5, respectively). TER119high surface expressing cells constitute committed erythroid cells. During the course of erythroid diﬀerentiation, a decrease of cell size is also observed. We therefore overexpressed mElf-1 cDNA in puriﬁed TER119low cells with the use of a retroviral vector (MSCV-Elf-1-PGKprom-puro-IRES-GFP) that also encodes GFP. Thus, transduced cells were labelled with GFP and overexpressed mElf-1 (Supplementary Figure S11C); uninfected GFP cells served as internal controls. We then analysed erythroid-diﬀerentiation proﬁles in the in vitro culture system. Figure 6B–D show the results of a representative experiment. Overexpression of Elf-1 did not cause any signiﬁcant changes in FACS proﬁles after 24 h of diﬀerentiation. After 48 h of diﬀerentiation, however, overexpression of Elf-1 impaired terminal erythroid diﬀer- entiation, as evidenced by the statistically signiﬁcant (Student’s t-test, P 0.05) accumulation of more immature cells in R3 at the expense of more mature cells in R4 when compared to the empty vector control (Figure 6B and D). Moreover, a signiﬁcantly larger pro- portion of Elf-1 transduced cells displayed a larger cell size consistent with the presence of more immature cells (Figure 6C). These results indicate that downregulation of Elf-1 is important for terminal diﬀerentiation of erythrocytes.