analysis of leukemogenesis by MELS in MEL-EVI1 gene family

MDS1/ evi1样基因1 （MEL1）被鉴定为转录调控因子，是t (1; 3) （p36; q21）阳性急性髓系白血病发病的致病因子。然而，其转录功能的分子基础仍然是难以捉摸的。最近，我们发现生态亲和性病毒整合位点-1 （EVI1）与位于GATA-2 IS外显子上游约6-7 kb区域的DNA一致序列结合，并在小鼠多能祖细胞系EML-C1中上调GATA-2基因的表达（Yuasa等，2005）。相反，在il -3依赖性小鼠髓系L-G3中，PR结构域缺陷短形式MEL1S的过表达可阻断g - csf诱导的髓系分化，并下调GATA-2基因的表达。为了确定MEL1S在GATA-2基因调控中是否需要与CtBP结合，我们创建了MEL1S的CtBP结合缺陷突变体（PFAST/PLASS）。报告基因位于GATA-2 IS外显子上游0 ~ 7 kb区域，该野生型和突变型MELlS分别在上述L-G3细胞和293T、HeLa、COS7、CHO细胞以及真实成纤维细胞系中异位表达。当野生型MEL1S和突变型MEL1S在L-G3细胞中短暂表达时，野生型MEL1S显著降低了70%的gta2 IS启动子活性，但在表达突变型MEL1S的细胞中，抑制作用大大降低（降低40%）。另一方面，在测试的成纤维细胞中，当野生型MEL1S略微降低30%时，GATA-2 IS启动子的活性未被不结合CtBP的突变体MEL1S抑制。这些发现突出了MEL1S调控的GATA-2基因表达与MEL1S与HDAC相互作用的关系，以及这些因素如何影响MEL1S在白血病细胞中的基因表达和白血病发生。

The MDS1/EVI1-like gene 1 (MEL1) was identified as transcriptional regulator that was the causative factor in pathogenesis of the t (1 ; 3) (p36 ; q21)-positive acute myeloid leukemia. However, the molecular basis of their transcriptional functions has remained elusive. Recently, we have shown that the ecotropic virus integration site-1 (EVI1) binds to its DNA consensus sequence located around 6-7 kb upstream region of the GATA-2 IS exon and up-regulates the expression of GATA-2 gene in EML-C1, a murine multipotent progenitor cell line (Yuasa, et. al., 2005). In contrast, Overexpression of MEL1S, PR domain-defective short form in L-G3, a IL-3-dependent murine myeloid cell line, blocks G-CSF-induced myeloid differentiation and down-regulate the expression of GATA-2 gene. To determine whether the binding of MEL1S to CtBP is required for action of MEL1S in GATA-2 gene regulation, We created CtBP-binding-deficient mutant of MEL1S (PFAST/PLASS).With the reporter gene containing 0〜7 kb upstream region of the GATA-2 IS exon, This wild-type and the mutant of MELlS were ectopically expressed in L-G3 cells as above and 293T, HeLa, COS7, CHO cells, authentic fibroblast cell lines, respectively. When wild-type and the mutant of MEL1S was transiently expressed in L-G3 cells, wild-type MEL1S significantly reduced the activity of GATA-2 IS promoter by 70%, but the repression was greatly reduced in cells expressing mutant MELlS (40% reduction). By the other hand, the activity of GATA-2 IS promoter was not suppressed by the mutant MEL1S that does not bind CtBP in fibroblast cells tested, when wild-type MEL1S slightly reduced by 30%. These findings highlight the relationship between the expression of GATA-2 gene regulated by MEL1S and the interaction of MEL1S and HDAC and how these factors affect the gene expression and leukemogenesis of MEL1S in leukemia cells.