Nucleic Acids Research, 2010, Vol. 38, No. 19 6369 Figure 4. Functional validation of candidate regulatory elements. Promoter (A) and enhancer (B) activity of candidate regulatory regions was assayed. Stable transfection of the murine erythroleukaemia cell line F4N, the myeloid progenitor cell line 416B, the T-lymphoid cell line BW5147, the endothelial cell line MS1 with luciferase reporter constructs containing Elf-1 candidate regulatory elements. Mean and SEM for two (A) or three (B) independent transfections (each one performed in triplicate) are shown. (A) 55, 49, 21 and proximal elements of Elf-1 were inserted upstream of a luciferase reporter gene. Values are expressed relative to empty vector, pGL2 basic. (B) A 550-bp length of DNA corresponding to the Elf-1 14 element was inserted downstream of a luciferase reporter gene under the transcriptional control of the minimal SV promoter. Values are expressed relative to empty vector, pGL2 promoter. To validate whether binding of PU.1 signiﬁed direct activation of Elf-1 by PU.1, transactivation assays were performed in human embryonic kidney 293T cells where a PU.1 expressing plasmid was co-transfected with plasmids carrying the four promoter elements and the enhancer region. No transactivation could be detected on the proximal, 49 or 55 elements but a signiﬁcant increase in luciferase activity was detected when the 21 element and PU.1 were co-transfected (Figure 5B). We failed to detect transactivation on the 14 element (data not shown), likely because additional factors and/or integra- tion into chromatin may be required. The transactivation assays were therefore not only consistent with the ChIP-chip data by showing speciﬁc activation by PU.1 at the only promoter identiﬁed by ChIP-chip to be bound by PU.1, but also importantly validated PU.1 as an upstream activator of Elf-1 expression. As an addition- al validation of the regulation of Elf-1 by PU.1, we performed transient co-transfections of the 21 element with a PU.1 knock-down or control construct in the myeloid progenitor cell line 416B. These experiments revealed a speciﬁc reduction of the activity of the Elf-1 21 promoter when PU.1 levels are reduced thus corroborating further the role of PU.1 as an upstream regulator of Elf-1 expression (Supplementary Figure S10B). Elf-1 downregulation is important for erythroid diﬀerentiation The enrichment levels obtained when using H3Me3K4 antibodies for the erythroleukaemia cell line F4N and fetal liver were modest, especially when compared to myeloid progenitor cells. Since downregulation of other Ets factors is important for erythroid development (39,40), we therefore investigated whether this may also be true for Elf-1. Erythroid diﬀerentiation can be moni- tored by labelling fetal liver cells for erythroid-speciﬁc TER119 and non-erythroid-speciﬁc transferrin receptor (CD71; 30). To investigate Elf-1 expression during murine fetal liver erythropoiesis, E14.5 fetal liver cells