and transactivation was performed on at least two diﬀer- ent days in triplicate. In vitro erythroid diﬀerentiation assay In vitro erythroid diﬀerentiation assays were performed as previously described (30,31). Brieﬂy, livers from E14.5 (C57Bl/6 CBA) F1 murine embryos were extracted by blunt dissection and homogenizsed. Lineage-negative haematopoietic progenitor cells were isolated using the MACS Murine Lineage Cell Depletion Kit (Miltenyi Biotec GmbH, Gladbach, Germany) according to the manufacturer’s instructions. 2 105 lineage-negative cells were plated onto ﬁbronectin coated 12-well plates (BD Discovery Labware, Bedford, MA, USA) and grown in Erythroid Diﬀerentiation Medium [IMDM sup- plemented with 15% fetal bovine serum, 1% detoxiﬁed bovine serum albumin (BSA) (both from Stem Cell Technologies, Vancouver, Canada), 600 mg/ml holo-transferrin (Sigma-Aldrich), 10 mg/ml recombinant human insulin (Sigma-Aldrich), 2 mM L-glutamine, 10 4 Mb-mercaptoethanol, and 2 U/ml human recombin- ant erythropoietin]. Retroviral transfection Plasmid containing murine whole-length Elf-1 cDNA (6) was digested with PstI, blunted with T4 polymerase, digested again with XhoI and subcloned into a retroviral overexpression vector containing MSCV pGKprom- Puro-IRES-EGFP (PIG). Retrovirus production was carried out using the pCL-Eco Retrovirus Packaging Vector (Imgenex, San Diego, CA, USA) and the 293T cell line. Murine fetal liver cells were infected with retro- virus by centrifugation at 2000 r.p.m. at 30 C for 1.5 h with 4 mg/ml polybrene (Sigma-Aldrich) after which the retroviral supernatant was replaced with Erythroid Diﬀerentiation Medium (30). Flow cytometry analysis and cell sorting Samples for FACS analysis were stained using antibodies against the surface markers Ter119 [APC conjugated, 1 in 100 dilution] (BD, Oxford, UK) and CD71 [PE conjugated, 1 in 100 dilution] (BD). Dead cells were excluded using the 7-aminoactinomycin (7AAD) stain. All analysis was performed using a FacsCalibur analyser (BD) and sorting was performed using a Dakocytomation MoFlo cell sorter. RESULTS Locus-wide comparative genomic analysis identiﬁes ﬁve non-coding conserved regions representing candidate Elf-1 regulatory elements Cross-species sequence conservation of non-coding regions has been successfully used in the past to identify regulatory elements (27,32). To locate candidate regula- tory elements within the mElf-1 gene locus, we compared the human, dog and opossum Elf-1 loci to 109 kb of the mouse locus containing the entire Elf-1 gene and extending into upstream and downstream Nucleic Acids Research, 2010, Vol. 38, No. 19 6365 ﬂanking genes. All coding regions were highly conserved between species analysed (Figure 1A). Non-coding sequence conservation was more limited but comparison with the opossum sequence revealed ﬁve highly conserved non-coding regions that represented candidate regulatory elements of mElf-1 gene. Four of those regions were located 55, 21, 14 and 8.5 kb upstream of the ATG start codon of the open reading frame and the ﬁfth region was located just upstream of the start codon which from here on will be referred to as the proximal region. Although Elf-1 is expressed in most haematopoietic cells (33), levels vary between cell types. In order to identify cellular systems for functional validation of can- didate regulatory elements, expression levels of Elf-1 were assessed in diﬀerent cell types (Figure 1B). We analysed two sources of primary cells (E13.5 fetal liver and whole adult thymus) and four diﬀerent cell lines: F4N (erythro- leukaemia), 416B (early myeloid progenitor), BW5147 (T-lymphoid) and MS1 (endothelial). Finally, we also included in the study the embryonic stem (ES) cell line HM-1 since ES cells express low levels of Elf-1 mRNA (Supplementary Figure S6). As shown in Figure 1B, all cell types expressed signiﬁcant levels of Elf-1 mRNA when compared to the ES cell line HM-1. Particularly, high expression was detected in the whole adult thymus and in the myeloid progenitor cell line 416B. Locus-wide ChIP-chip analysis identiﬁes ﬁve Elf-1 candidate regulatory elements We had previously shown that the combination of locus-wide comparative genomics and ChIP-chip analysis provides an eﬀective strategy for the identiﬁcation of bona ﬁde regulatory elements (16,27). We therefore performed ChIP-chip analysis using a 109 kb tiling array that spanned the Elf-1 locus and ﬂanking genes with chro- matin prepared from the four Elf-1 expressing cell lines (erythroleukaemia F4N, myeloid progenitor 416B, T-lymphoid BW5147 and endothelial MS1) as well as two sources of primary cells (E11.5 fetal liver and whole-adult thymus). Acetylation of histone H3 lysine 9 (H3AcK9) is a histone mark associated with an open chro- matin conformation and is generally observed at pro- moters and enhancers of active genes. The acetylation pattern across the Elf-1 locus in the highly expressing myeloid progenitor cell line 416B included an enriched region close to the ATG start codon of the gene. This region was also enriched in the endothelial and T-lymphoid cell lines but it was practically absent in the erythroid cell line. Myeloid and T-lymphoid cell lines pre- sented a broad enriched region that co-localized with the 21 and 14 regions identiﬁed by comparative genomic analysis, whereas the endothelial and erythroid cell lines only showed H3K9 acetylation around the 21 region. In the vicinity of the 50-ﬂanking gene Wbp4, around 55 kb upstream of the ATG start codon, another enriched region was detected in all cell types analysed. Therefore, with the exception of the 8.5 region all homology peaks identiﬁed by comparative genomics displayed histone H3K9 acetylation. Consistent with its predominant eryth- roid nature, the pattern of acetylation found in fetal liver