A Study Of Physiologic And Pathologic Function And Regulation Targets Of Proto-Oncogene Lmo2

The human proto-oncogene lmo2, which locates in 11p13 in the genome, was first cloned from an acute T lymphocyte leukemia patient sample with t(11;14)(p13;q11) translocation. The lmo2 gene is a critical gene in hematopoiesis. lmo2 null mutation in mice leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5, and the mosaic mouse model shows that lmo2 is essential for the development of all hematopoietic lineages. However, the expression of LMO2 in hematopoietic cells is tightly regulated, it is specifically unexpressed in committed T cells, whereas its ectopic expression immediately causes acute T lymphocyte leukemia (T-ALL). Moreover, over-expression of LMO2 exists in more the 50% T-ALL samples.LMO2, which mainly affects the transcription of its target genes, belongs to the Zinc-finger transcriptional regulators, but interestingly, it has no direct DNA binding ability. It always functions as bridge molecules to assemble their partners, including LDB1, GATA1, TAL1 and E47, together to form a complex that recognizes and binds to specific DNA sequences of the target genes, which consist of a GATA site and an E-box, with 9~12 bp in between or two E-boxes as the pathogenic targets.In this study, the main work was focused on revealing the function pattern of LMO2 and identifying its targets in both physiological and pathological conditions.Firstly, the lmo2 gene has three transcripts Lmo2-a/b/c, in which Lmo2-a/b encode a same protein product termed LMO2-L and Lmo2-c encodes a shorter protein isoform, termed LMO2-S, which has 14 amino acids different in the N-terminal region compared to LMO2-L. In this study, the combination of MBP pulldown assay and mammalian two hybrid assay were used to confirm the homo-binding character of LMO2-L/-S isoforms. Further, a precisely different regulatory pattern of LMO2-L/-S isoforms on their targets was revealed. The results showed that LMO2-L could either positively or negatively regulate the transcription of its different target genes, depending on the arrangement and strand location of the two elements GATA site and E-box, LMO2-S, however, performed constitutively transcriptional inhibiting function on all target genes. These results suggest that LMO2 isoforms have independent functions while there is no interaction between each other and they could play synergetic or antagonistic roles precisely in regulating their different genes involved in normal and aberrant hematopoiesis.Secondly, miR-142 and miR-223 have been identified as hematopoietic specific microRNAs and have crucial functions in hematopoiesis. In this study, we found that both miR-142 and miR-223 attenuated the proliferation of hematopoietic cells, and that miR-223 up-regulated miR-142 expression through the LMO2-L/-S isoforms and CEBP-β. miR-223 negatively regulated both LMO2-L/-S isoforms and CEBP-βpost-transcriptionally, while CEBP-βpositively regulated the LMO2-L/-S isoforms and both of the LMO2-L/-S isoforms negatively regulated miR-142. These results reveal a miR-223—CEBP-β—LMO2—miR-142 regulatory pathway in hematopoiesis. Our data also indicated that ectopic expression of both LMO2 isoforms accelerated cell proliferation in T cells, whereas over-expression of miR-142 eliminated LMO2 function and further arrested proliferation, implying miR-142 an executor of LMO2 physiologic and pathologic function by affecting cell cycle and identifying miR-142 as a key pathologic target that negatively regulated by LMO2 and plays pivotal role for the onset of T lymphocyte leukemia in the aspect of changing the feature of cell proliferation.Thirdly, deltaEF1, which belongs to the Zinc finger-homeodomain family transcription factor, exhibits crucial function in promoting T cell differentiation. In this study, we found that deltaEF1 was positively regulated by T lineage specific transcriptional regulator GATA3, while ectopically expressed LMO2 targeted to deltaEF1 promoter by interaction with GATA3 and inhibited deltaEF1 expression in transcriptional level. Moreover, LMO2 interacted with the N-terminal Zinc finger (NZF) domain of DeltaEF1 protein and inhibited its positive transcriptional regulatory function by this interaction. These findings revealed that ectopically expressed LMO2 impaired the function of deltaEF1 in both transcriptional and protein levels and identified deltaEF1 as a novel pathogenic target of LMO2 in T cell leukemia.Taken together, our research concluded that the two isoforms of lmo2 gene product regulate their both physiologic and pathologic target gene expression by a precise mechanism with synergetic or antagonistic patterns, through which down-regulate miR-142 and accelerate cell proliferation while inhibit deltaEF1 function and block T cell development. These results give a draft of LMO2 pathologic function involved in T cell leukemia.