PAF1复合物介导白血病原癌蛋白——MLL融合蛋白定位的分子基础研究

MLL (mixed lineage leukemia) is a histone methyltransferase which methylates histone H3 lysine 4 in MLL complex. Rearrangements of MLL gene are one of the most common genetic alterations in human leukemia. The most common MLL rearrangements result in oncogenic fusion protein of N terminal 1400 residues of MLL in frame with a variety of different translocation partners. MLL fusion proteins are among the most potent oncoproteins driving leukemia development. Recent studies reported that MLL and its fusion proteins could be recruited to chromatin through a direct interaction between an N-terminal fragment of MLL and the PAF1 complex (RNA Polymerase II Associated Factor 1 complex, PAF1c), and disruption of this interaction eliminated MLL-AF9 (an MLL fusion protein) mediated immortalization and leukemogenesis. Therefore, this MLL-PAF1c interaction provides an attractive venue to screen therapeutic compounds for treating MLL associated leukemia. However, the molecular mechanism of this interaction is still elusive. In this study, we will pursue the structural and functional study of this interaction. We will conduct the protein-protein interaction mapping between MLL and components of PAF1c by Pull-down and ITC (Isothermal titration calorimetry). In fact, we have finished the interaction mapping between MLL and PAF1, one component of PAF1c, and found that the fragment of PAF1 covering residues 164-380 interacts with MLL (residues 1115-1201). To elucidate the molecular basis of MLL-PAF1c interaction, we would like to solve the crystal structure of binary complexes between MLL and associated components of PAF1c. Based on the solved crystal structures, we will carry out site-directed mutagenesis study to identify critical residues affecting the interaction of the MLL N-terminal fragment and the PAF1c components. Once we have identified functionally important residues, we will validate the biological relevance of these critical residues in MLL complex recruitment by cell based experiments. Overall, our study will shed light on the molecular mechanisms of MLL-PAF1c interaction and greatly aid in drug discovery for leukemia.

MLL是一种组蛋白甲基转移酶，参与催化组蛋白H3K4甲基化，从而激活下游基因的表达。该基因常与多种易位基因形成重组基因，表达的MLL融合蛋白是驱动白血病发展的重要原癌蛋白。研究表明，MLL融合蛋白可通过与PAF1复合物（PAF1c）相互作用而被定位于调控基因，然而该相互作用的分子基础尚不清楚。本项目在前期工作的基础上，对PAF1c如何介导MLL融合蛋白定位的分子基础展开研究：利用Pull-down和ITC等方法定位MLL与PAF1c组分的结合位点；通过解析MLL与PAF1c组分复合物晶体结构，揭示MLL与PAF1c相互作用的结构特点；依据结构设计突变体，通过体内外实验验证突变体对MLL融合蛋白的生物学功能的影响。本项目研究结果将展示MLL融合蛋白-PAF1c互作的分子机制，为阻断MLL-PAF1互作的小分子药物的开发提供理论基础，进而为治疗由MLL融合蛋白所致的白血病奠定基础。

MLL是一种组蛋白甲基转移酶，参与催化组蛋白H3K4甲基化，从而激活下游基因的表达。该基因常与多种易位基因形成重组基因，表达的MLL融合蛋白是驱动白血病发展的重要原癌蛋白。研究表明，MLL融合蛋白可通过其N端的RD1-RD2结构域与PAF1复合物（PAF1c）的主要组分PAF1、CTR9和CDC73相互作用而被定位于调控基因，然而该相互作用的分子基础尚不清楚。因此，本项目在前期已经确认MLL与PAF1相互作用的基础上，对MLL和PAF1c主要组分间的相互作用展开了较为全面的研究。我们克隆并表达纯化了MLL、CTR9、CDC73不同区段的蛋白，并通过ITC检测MLL与CTR9、CDC73不同区段的蛋白的相互作用，检测结果提示MLL的RD1-RD2结构域与CTR9、CDC73之间不直接相互作用，我们推测该相互作用可能通过PAF1间接介导；我们获得了MLL-PAF1复合物晶体，尽管晶体为针状，且分辨率较低，尚未能解析结构，但通过后期的优化有望解析复合物的结构，从而揭示MLL-PAF1间相互作用的分子机制。此外，根据研究期间工作开展的实际情况，我们增加了MLL同源蛋白MLL3/MLL4被募集至作用靶点的分子机制研究，以及其他一些蛋白的结构和功能研究，并取得了一些研究进展。我们的研究发现MLL的同源蛋白MLL3/MLL4可以通过其ePHD6结构域与组蛋白H4H18区段特异性相互作用而被募集到作用靶点而发挥功能。为了解释了该相互作用的分子机制，我们解析了MLL3 ePHD6结构域与组蛋白H4包含H4H18区段小肽的复合物晶体结构，我们的研究结果表明MLL3/MLL4蛋白通过其ePHD6结构域以一种全新的模式识别并结合组蛋白H4，从而反式调节组蛋白H3K4甲基化修饰。又如我们系统地研究了含有CW结构域的蛋白家族与组蛋白相互作用的分子机理，为进一步研究CW结构域蛋白的功能奠定了分子基础。