The 3’ myeloid enhancer activity of element -25 is dependent on GATA binding.

<p>A) DNaseI-Seq profile of <i>Lmo2</i> locus region in mouse myeloid progenitors 416B cells. Elements previously described to be involved in the regulation of <i>Lmo2</i> expression are indicated. The strongest DHS in the <i>Lmo2</i> locus co-localises with element -25. A magnification of the region corresponding to element -25 is shown. Diagram of element -25 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131577#pone.0131577.g002" target="_blank">Fig 2A</a>. DNaseI profile shows a strong protection in the region corresponding to conserved GATA motif (location shown in grey). B, C) ChIP-Seq profile of RUNX1 and GATA2 in <i>LMO2</i> locus region in murine haematopoietic progenitors HPC7 (B) and human CD34+ (C) cells. Elements previously described to be involved in the regulation of <i>LMO2</i> expression are indicated. Strong binding for GATA2 and RUNX1 can be detected at element -25. A magnification of the region corresponding to element -25 is shown. Diagram of element -25 as previously. Binding of RUNX1 and GATA2 specifically takes place at the 3‘ myeloid enhancer module of element -25. D) Myeloid enhancer activity depends on a conserved GATA motif. Conserved RUNT and GATA motifs present in the myeloid enhancer module of element -25 were mutated (3’mRUNT -25 el. and mGATA -25 el., respectively). Mutated constructs were stably transfected together with <i>LMO2</i> proximal promoter element (pPex) in myeloid progenitor 416B cells and luciferase activity was measured. Mean and standard error of the means (SEM) for at least two independent stable transfections (each one performed at least in triplicate) are shown. Values are expressed relative to empty vector, pGL2 basic. **P < 0.01; ***P < 0.001.