Transcriptional Regulation of the LMO2 T-Cell Leukaemia Oncogene.

Transcriptional control has long been identified as a key mechanism regulating the formation and subsequent behaviour of haematopoietic stem cells. We have used a comparative genomics approach to identify transcriptional regulatory elements of the LMO2 gene, a transcriptional cofactor originally identified through its involvement in T-cell leukaemia and subsequently shown to be critical for the formation of haematopoietic stem cells and endothelial development. An initial stringent search for homology between evolutionary distant species demonstrated that, apart from the coding exons, high level of identity between mammalian, amphibian and fish sequences was restricted to the proximal promoter region of LMO2. Real-time RT-PCR expression analysis identified this promoter as the predominant source of transcription in haematopoietic tissue. Transient and stable transfections indicated that the proximal promoter was active in haematopoietic progenitor and endothelial cell lines and this activity was shown to depend on three conserved Ets sites which were bound in vivo by Elf1, Fli1 and Ets1. Transgenic analysis demonstrated that the LMO2 proximal promoter was sufficient for expression in endothelial cells in vivo. However, no haematopoietic expression was observed indicating that additional enhancers are required to mediate transcription from the proximal promoter in haematopoietic cells.

To identify additional elements involved in haematopoietic expression of LMO2, we have performed a less restrictive search for conserved sequences by comparing the human, dog, rat and mouse LMO2 loci to the marsupial opossum LMO2 locus. The addition of the opossum locus, and removal of the more distant fish and amphibian sequences from the alignment, resulted in the discovery of eleven conserved regions. These sequences represent candidate haematopoietic regulatory regions as they contain conserved transcription factor binding sites (E boxes, Ets and Gata sites) previously shown to regulate several other haematopoietic genes. We will present results from a systematic analysis of these regions for enhancer activity in both haematopoietic cell lines and transgenic mice, which suggest that several of these elements indeed act as enhancers. Taken together, our experiments will provide a framework for the transcriptional hierarchies within which LMO2 exerts its function in normal haematopoietic cells. Moreover, the current studies will serve as a platform to examine potential molecular mechanisms that can cause ectopic expression of LMO2 in T-cell progenitors with the ultimate consequence of developing T-ALL.