6364 Nucleic Acids Research, 2010, Vol. 38, No. 19 expression. Consistent with this notion, Elf-1 has been shown to be a direct upstream regulator of genes import- ant for haematopoiesis such as Scl, Fli-1, Lyl-1, Runx1 and Lmo2 (6–11). Like other members of the Ets family such as Ets-1 and Ets-2, no major haematopoietic pheno- types have been observed in Elf-1 / mice (12). However, given that Elf-1, like Ets-1, is a member of a small sub- family, a likely explanation for the lack of a strong pheno- type in loss of function assays is compensation by other Ets proteins which are also known to be expressed in the haematopoietic system. Elf-1 has also been shown to be important for blood vessel development, a process that is closely linked to early haematopoiesis during embryonic development. Elf-1 has been reported to take part in the transcriptional control of major regulators of blood vessel development such as Tie1, Tie2, angiopoietin-2, the vascular endothelial growth factor receptor 1 (VEGFR1), the endothelial nitric-oxide synthase (eNOS) and endoglin (3,13–16). Moreover, a recent landmark study demonstrated that Elf-1 constitutes a potentially very attractive target for the development of new cancer therapies as systemic ap- plication of an ELF-1 blocking peptide was able to inhibit tumour angiogenesis in a mouse model of melanoma (17). Functional activity of Ets proteins is modulated at multiple levels. For example, it is known that ELF-1 appears in the cytoplasm as a 80 KDa protein that is O-glycosylated and phosphorylated in order to be translocated into the nucleus where it can be detected as a 98 KDa protein. After dephosphorylation, the protein is degraded through the proteasome pathway (18–20). In resting T cells, ELF-1 binds to the underphosphorylated form of the retinoblastoma protein pRb and after T-cell activation, the phosphorylation of pRb results in the release of ELF-1 (21). Despite its function as an important transcriptional regulator in both blood and endothelium, nothing is known about the transcriptional regulation of the Elf-1 gene itself. Consequently, the position of Elf-1 within the wider regulatory networks controlling blood cells had remained obscure. In this study, we have identiﬁed and characterized ﬁve regulatory elements within the Elf-1 locus. Four regulatory regions functioned as pro- moters and one region as a lineage-speciﬁc enhancer. Furthermore, we showed that the transcription factor PU.1 plays a major role in the regulation of Elf-1 and that downregulation of Elf-1 is important for terminal erythroid diﬀerentiation. MATERIALS AND METHODS Sequence analysis and custom arrays Genomic Elf-1 sequences were downloaded from Ensembl, aligned using multi-Lagan (22) and displayed using mVista (23). Primers to generate the Elf-1 oligo tiling array were designed using Primer3 (24) on repeat masked sequence spanning Elf-1 and ﬂanking genes (chr14:74 160 804–74 269 970, coordinates in build mm7). Oligos were spotted in triplicate using a BioRobotics MicroGrid II Total Array System. Array design ﬁles have been submitted to ArrayExpress. ChIP-chip assays ChIP assays were performed as previously described (25). Brieﬂy, cells were treated with formaldehyde and cross-linked chromatin was sonicated to 300 bp averaged size. Immunoprecipitations were performed using anti-acetyl histone H3 antibody (Millipore, 06-599), anti-H3 trimethyl lysine 4 (Millipore, 04-745 and gift from Dr L. O’Neill, Birmingham, UK) and anti-PU.1 (Santa Cruz, sc-352X). ChIP material was labelled with Cy3 and Cy5 ﬂuorochromes and hybridized as described (26). Microarrays were scanned using an Agilent scanner and median spot intensities quantiﬁed using GenePix Pro 6.0 with background subtraction. A Perl script was de- veloped to normalize the resulting data and calculate mean ratios of normalized ChIP signals over input (27) using the triplicate values on the array. Resulting data were plotted using the Variable WidthBar Graph Drawer (http://hscl.cimr.cam.ac.uk/genomic_tools.html). All experiments have been deposited in ArrayExpress with accession numbers E-MEXP-2664, E-MEXP-2665 and E-MEXP-2666. Gene expression analysis Total RNA was isolated using Tri-Reagent (Sigma-Aldrich, Dorset, UK) according to the manufac- turer’s instructions. Contaminating genomic DNA was removed by DNAseI treatment with Turbo DNA free (Applied Biosystems/Ambion, Warrington, UK). Total RNA (125 ng) was used for cDNA synthesis, using oligo(dT) 17-mer primer and MMLV reverse transcriptase (Sigma-Aldrich). Mouse Elf-1, mouse PU.1 and mouse 18s mRNA levels were measured by real-time PCR using the primer sets listed in Supplementary Table S1. Relative ex- pression was calculated as a ratio to the 18 s signal. Cell culture, reporter constructs and transfection assays The myeloid progenitor cell line 416B, murine erythroleu- kaemia cell line F4N, endothelial cell line MS1 and the T-cell line BW5147 were maintained as previously described (28,29). Fetal liver and adult thymus cell suspen- sions were obtained by direct pipetting of freshly dissected tissues from mice. Elf-1 luciferase reporter constructs were ampliﬁed from mouse genomic DNA using primers listed in Supplementary Table S1, conﬁrmed by sequencing and cloned into pGL2basic (Promega, Southampton, UK). These regions were selected based on the combined results of comparative genomics and ChIP-chip experi- ments (Supplementary Figures S1–S5). Cell lines were stably transfected by electroporation as described (29). G418 or puromycin was added 24 h post-transfection and cells were assayed 7–27 days later, depending on the cell line. For transactivation assays, 293T cells were trans- fected with 1 mg ‘luciferase’ construct in combination with 3mg pcDNA3mPU.1 (generous gift from Dr J. Frampton, Birmingham, UK) or the empty vector pcDNA3 as control using ProFection Mammalian Transfection System-Calcium Phosphate (Promega). Each transfection