Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Exp Hematol. 2011 Jan;39(1):77-86.e1-5. doi: 10.1016/j.exphem.2010.09.003. Epub 2010 Sep 18.
The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia.
The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation.
Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented.
The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci.
本研究旨在更好地了解混合谱系白血病（MLL）融合蛋白如何调节白血病关键基因的表达。
在髓样白血病 M1 细胞中表达后，免疫纯化最常见的 MLL 融合伙伴之一 AF9 的转化结构域，并通过质谱分析鉴定相关蛋白。采用染色质免疫沉淀结合定量聚合酶链反应的方法，确定在具有和不具有 MLL 融合蛋白的细胞系中，与 Hoxa9 和 Meis1 相比，结合蛋白在何处结合，以及在基因下调和分化过程中结合如何改变。
与之前从 293 细胞中纯化 ENL 和 AF4 的结果一致，AF9 的 90 个氨基酸 C 端结构域与许多其他 MLL 易位伙伴（包括 Enl、Af4、Laf4、Af5q31、Ell 和 Af10）结合。该复合物被称为延伸辅助蛋白（EAPs），还包含 RNA 聚合酶 II C 端结构域激酶 Cdk9/Cyclin T1/T2（pTEFb）和组蛋白 H3 赖氨酸 79 甲基转移酶 Dot1L。由 MLL 融合转化的髓样细胞在白血病关键基因中显示出更高水平和更广泛分布的 EAP 成分。抑制 EAP 成分 pTEFb 和 Dot1l 表明它们都显著促进了 Hoxa9 和 Meis1 表达的激活。EAP 在造血细胞中与 Hoxa9 和 Meis1 基因座动态结合，并在诱导分化过程中迅速解离。在存在 MLL 融合蛋白的情况下，其解离被阻止。
这些发现表明，MLL 融合蛋白通过过度募集和 EAP 从靶基因座的异常解离来调节白血病关键基因的表达。
