6368 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 3. Locus-wide ChIP-chip of trimethylation of histone H3 lysine 4 identiﬁes four potential promoter 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, whole E11.5 fetal murine liver and whole-adult murine thymus. Enrichment values are calculated as fold enrichment over the mean intensity across the whole locus and expressed as log base 2. possibility of transcriptional interference. However, no activity was detected in any of the four cell lines when stably transfected with this construct in an antisense orien- tation (data not shown). The Elf-1 14 region functions as a lineage-speciﬁc enhancer The 14 region is highly conserved between species and it is strongly acetylated in several cell types, which suggests an important function for this region in the regulation of the locus. As it only carried acetylation marks, we hypothesized it may function as an enhancer. Thus, we tested the enhancer activity of this element when cloned downstream of the luciferase gene under the control of the SV40 minimal promoter (pGL2 promoter). Stable trans- fection assays using 416B myeloid progenitor and BW5147 T-lymphoid cell lines demonstrated that the 14 element functioned as a very strong enhancer in the myeloid progenitor cell line 416B and showed moderate activity in the T-lymphoid cell line BW5147 (Figure 4B). In contrast, the 14 element showed no activity in the F4N erythroleukaemia and MS1 endothelial cell lines. These results were entirely consistent with the ChIP-chip analysis which had shown cell-type-speciﬁc acetylation and therefore demonstrate that the 14 region contains a lineage-speciﬁc transcriptional enhancer element. PU.1 binds to 21 and 14 elements in vivo and controls the activity of regulatory elements of Elf-1 We also explored which transcription factors could be re- sponsible for the activation of the newly identiﬁed regula- tory elements of Elf-1 and performed ChIP-chip using antibodies directed against known key haematopoietic transcription factors such as PU.1, SCL and GATA2. PU.1 is a transcription factor known to be important for haematopoiesis that has frequently been reported to co-operate with Elf-1 in gene regulation (6,35–37). In addition, interrogation of the Supplementary Data for a recent large-scale system biology analysis of myeloid diﬀerentiation (38) demonstrated that knock-down of PU.1 expression in the myeloid cell line THP-1 leads to a reduction of Elf-1 gene expression (Supplementary Figure S10A). In order to analyse whether PU.1 is directly involved in the transcriptional regulation of Elf-1, we performed ChIP-chip assays in the Elf-1 high-expressing cell line 416B using an antibody against PU.1 (Figure 5A). Binding of PU.1 can be detected at both the 21 and 14 elements. ChIP-chip analysis with antibodies against GATA-2 and SCL were also performed but no binding was detected across the Elf-1 locus even though clear binding peaks were observed in regions from the Scl locus included on the same array as positive controls (data not shown).