Nucleic Acids Research, 2010, Vol. 38, No. 19 6367 Figure 2. Locus-wide ChIP-chip of acetylation of histone H3 lysine 9 identiﬁes ﬁve potential regulatory elements. MVista representation of mouse/ human sequence conservation is shown on the top together with all candidate regulatory elements. Annotations as in Figure 1A. ChIP-chip for the murine erythroleukaemia cell line F4N, the myeloid progenitor cell line 416B, the T-lymphoid cell line BW5147, the endothelial cell line MS1, E11.5 fetal murine liver and adult murine thymus. Enrichment values are calculated as fold enrichment over the mean intensity across the whole locus and expressed as log base 2. transcripts originating from this element have been reported so far. DNAse I hypersensitivity analysis by ChIP-chip performed in the primary tissues fetal liver and thymus showed that this region was highly hypersen- sitive (Supplementary Figure S8). Taken together, these results are consistent with the hypothesis that this region could represent an as yet unidentiﬁed promoter involved in Elf-1 regulation. Finally, one region ( 14) only carried acetylation marks, suggesting that it may function as an enhancer. Four of these regions overlapped with regions identiﬁed in the comparative genomic analysis, further validating these results. The Elf-1 locus contains four distinct regions with promoter activity To validate the results obtained by integration of the com- parative genomic sequence analysis and ChIP-chip approaches, the four candidate promoter regions were cloned into the pGL2 basic luciferase vector and stably transfected into the erythroleukaemia cell line F4N, myeloid progenitor cell line 416B, the T-lymphoid cell line BW5147 and endothelial cell line MS1. In three of the four cell lines, the proximal element showed the highest promoter activity. However, diﬀerent proﬁles were observed according to the proportional activity of the diﬀerent regions (Figure 4A). In the high-expressing myeloid cell line 416B and in T-cell line BW5147, proximal and 21 elements presented higher activity than the other two elements. Proximal and 55 elements showed strong activity in the F4N cell line deﬁning a diﬀerent proﬁle. Finally, in the endothelial cell line MS1, proximal, 21 and 55 regions had similar activity. The activity of the 49 element was low in all four cell lines analysed, although this region was acetylated and methylated in our ChIP-chip assays. Genome-wide ChIP-Seq binding proﬁles for GATA-1 have been reported in erythroid cells (34). We extracted the data cor- responding to the Elf-1 locus that demonstrated speciﬁc binding of GATA-1 to the 49 region (Supplementary Figure S9). We therefore tested whether this element might in fact be an enhancer and generated reporter con- structs with the 49 element cloned up- and downstream of the luciferase gene under the control of a minimal region of the SV40 promoter (pGL2 promoter). No sig- niﬁcant enhancer activity could be detected in any of the cell lines when this element was present (data not shown). However, a reproducible decrease in the activity can be detected in F4N when this element was located down- stream of the reporter gene. Then, antisense promoter activity of this element was also tested to explore the