Mueller Dorothee, Bach Christian, Zeisig Deniz, Garcia-Cuellar Maria-Paz, Monroe Sara, Sreekumar Arun, Zhou Rong, Nesvizhskii Alexey, Chinnaiyan Arul, Hess Jay L, Slany Robert K
Department of Genetics, University Erlangen, Erlangen, Germany.
Blood. 2007 Dec 15;110(13):4445-54. doi: 10.1182/blood-2007-05-090514. Epub 2007 Sep 12.
Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.
将组蛋白甲基转移酶MLL与各种融合伴侣连接的嵌合蛋白会引发独特的淋巴样和髓样白血病。在此，我们免疫纯化了与ENL相关的蛋白，ENL是一种常与MLL融合的蛋白。通过质谱鉴定这些ENL相关蛋白（EAP），发现了在转录延伸中起已知作用的酶（RNA聚合酶II C端结构域激酶[RNAPolII CTD]正向转录延伸因子b [pTEFb]）、在染色质修饰中起作用的酶（组蛋白H3甲基转移酶DOT1L）以及其他常见的MLL伴侣（AF4、AF5q31和LAF4）和多梳蛋白家族成员（RING1、CBX8和BCoR）。通过共免疫沉淀、双杂交分析、下拉和共定位实验进一步验证了EAP的组成。纯化的EAP显示出组蛋白H3赖氨酸79特异性甲基化酶活性，表现出强大的RNAPolII CTD激酶功能，并抵消了pTEFb抑制剂5,6-二氯苯并咪唑核糖苷的作用。在体内，敲低ENL会减少全基因组以及基因特异性的H3K79二甲基化，降低整体连续转录延伸，并抑制瞬时转录报告活性。根据结构功能数据，DOT1L的募集对于MLL-ENL融合衍生物的转化很重要。这些结果表明ENL在组蛋白修饰和转录延伸中具有功能。
