The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression
The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression
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DOI：
10.1073/pnas.0506464102
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发表时间：
2005-09-27
影响因子：
11.1
通讯作者：
Zeleznik-Le, NJ
中科院分区：
文献类型：
--
作者：
Xia, ZB;Popovic, R;Zeleznik-Le, NJ
MLL涉及与急性髓性和淋巴性白血病相关的许多染色体易位，具有> 50个已知的伴侣基因，其能够与之形成框内融合。对MLL及其融合蛋白下游重要靶基因的研究可能为MLL相关白血病的治疗提供合理的治疗策略。我们探索了最普遍的MLL融合蛋白MLL-AF 4的下游靶基因。为此，我们开发了不同背景的诱导型MLL-AF 4融合细胞系。MLL-AF 4的过表达不会导致任一细胞系中增殖的增加，相反，与诱导表达截短的MLL的类似细胞系相比，细胞生长减慢。我们发现，在MLL-AF 4诱导的细胞系中，细胞周期蛋白依赖性激酶抑制剂基因CDKN 1B的表达在RNA和蛋白质（p27（kip 1））水平上都发生了显着变化。相反，CDKN 1A（p21）和CDKN 2A（p16）的表达水平没有变化。为了探索CDKN 1B是否可能是MLL和MLL-AF 4的直接靶点，我们使用染色质免疫沉淀（ChIP）测定和荧光素酶报告基因测定。MLL-AF 4在体内结合CDKN 1B启动子并调节CDKN 1B启动子活性。此外，我们在MLL-AF 4以及MLL-AF 9白血病细胞系中证实了ChIP与CDKN 1B启动子的结合。我们的研究结果表明，CDKN 1B是MLL和MLL-AF 4的下游靶点，并且根据背景细胞类型，MLL-AF 4抑制或激活CDKN 1B表达。这一发现可能对MLL-AF 4白血病中白血病干细胞对化疗的耐药性产生影响。
MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has > 50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27(kip1)) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.