6366 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 1. Identiﬁcation of ﬁve non-coding conserved regions by comparative analysis. (A) Sequence conservation of the Elf-1 locus. MVista rep- resentation of sequence conservation across the mouse Elf-1 locus showing mouse/human, mouse/dog and mouse/opossum alignments. The conser- vation plots show regions with at least 50% of conservation (y-axis) across the 109 kb tiling path spanning the mouse Elf-1 locus. Peaks of sequence conservation in coding regions of the genome are shown in dark grey, those in transcribed but not translated regions (30UTR and 50UTR) are shown in pale grey. Neighbouring genes are also depicted. Arrows indicate the direction of transcription. Positions of the conserved regions are named relative to the ATG of Elf-1. (B) Elf-1 expression in haematopoietic cell lines. Real-time PCR analysis of mouse Elf-1 expression in the murine erythroleukaemia cell line F4N, the myeloid progenitor cell line 416B, the T-lymphoid cell line BW5147, the endothelial cell line MS1, the ES cell line HM-1 as well as in two sources of primary cells (fetal liver and whole-adult thymus). Values are expressed relative to m18s expression. Error bars indicate SD. was most similar to erythroid F4N cells and, similarly, the pattern for the thymus resembled that of the T-lymphoid BW5147 cells. Interestingly, a region 49 kb upstream of the Elf-1 ATG ( 49 region) showed substantial levels of acetylation in low-expressing Elf-1 cells, while it was largely absent in highly expressing cells. This region is conserved in human and dog but it is not conserved in opossum. Taken together therefore, comparative genomics combined with histone acetylation ChIP-chip analysis identiﬁed ﬁve candidate regulatory regions, namely the 55, 49, 21 14 and proximal regions (Figure 2). Four of the ﬁve Elf-1 histone acetylation peaks represent candidate alternative promoters Interrogation of EST databases as well as the database of transcriptional start sites (DBTSS) suggested that the acetylation peaks identiﬁed above might represent al- ternative promoters of the Elf-1 gene (Supplementary Figure S7). We therefore also assessed trimethylation of histone H3 lysine 4 (H3Me3K4) by ChIP-chip analysis (Figure 3). This chromatin modiﬁcation is speciﬁcally associated with active promoter regions and the results correlated very closely to those previously obtained using antibodies against acetylation. When the high Elf-1 expressing myeloid cell line 416B was analysed, a high enrichment could be detected for the regions corres- ponding to the 55, 21 and proximal elements but not for the 14 element, suggesting that this element could act as an enhancer within this locus. As in the acetylation analysis, the trimethylation patterns of fetal liver and erythroid F4N cells were similar, showing robust enrich- ments at the 55 and 49 regions. T-lymphoid cells and adult thymus showed similar patterns, with peaks at the 55 and 21 regions and the endothelial MS1 cell line only showed major enrichment at the 55 region. In summary, the ChIP-chip survey allowed us to identify ﬁve candidate regulatory elements: 55, 49, 21, 14 and proximal regions. Four of these regions ( 55, 49, 21 and proximal) displayed both acetylation and trimethylation marks suggesting that they represent alternative Elf-1 promoters. Transcripts that could be promoted by three of these regions ( 55, 21 and proximal) have been previously reported in mouse or rat (Supplementary Figure S7). Although the region 49 also displayed both acetylation and trimethylation marks, no