Critical functions specified by the MLL CXXC domain determine leukemogenic capaci

描述（由申请人提供）：混合谱系白血病（MLL）基因参与染色体易位，产生融合蛋白，导致预后不良的侵袭性急性白血病。所得到的嵌合蛋白保留了MLL的氨基末端部分，包括CXXC dna结合域，而癌蛋白的羧基末端部分由融合伙伴组成。CXXC结构域位于一个较大的抑制结构域内，与非甲基化的CpG DNA结合。这个CXXC dna结合域包含一个特定的8个半胱氨酸簇，它们协调两个锌离子，对表达MLL融合蛋白的细胞的转化能力至关重要。与MLL最接近的同源物MLL2（也称为MLL4）含有类似的CXXC结构域，但结合的dna结合亲和力较低。有趣的是，人工MLL2-ENL在体外不能转化造血祖细胞。MLL和MLL2的CXXC结构域或更大的抑制结构域之间的氨基酸差异必然导致了这些功能差异。沿着DNA接触界面的特定氨基酸可能在差异DNA结合亲和力中起作用。此外，我们之前已经证明MLL抑制结构域（包含CXXC结构域）与协同抑制蛋白结合。这些抑制蛋白与MLL和MLL2的差异结合可能会影响整体功能，但也可能有助于CXXC dna结合亲和力。半胱氨酸1188是CXXC结构域中唯一的非锌配位半胱氨酸残基，它位于dna结合表面，与一个参与锌配位的保守半胱氨酸相邻。当引入MLL-AF9时，Cys1188的天冬氨酸突变完全取消了CXXC结构域结合DNA的能力，并阻止了小鼠白血病的体外永生化和发展。相比之下，Cys1188向丙氨酸的突变保留了MLL结合CpG DNA的能力，以及在MLL- af9融合的情况下在体外和体内的永生化能力。由于CXXC结构域位于DNA结合表面的关键位置，并且巯基易被修饰，我们假设Cys1188的侧链可能被生理改变以调节DNA结合亲和力，从而使该残基作为调节MLL与其非甲基化CpG DNA靶标结合的分子开关。本应用程序提出了两个具体目标，即探讨MLL和MLL2 CXXC结构域的特定氨基酸残基如何改变DNA结合、共抑制因子结合以及对翻译后修饰的易感性。我们假设这些是MLL在急性白血病中作用的关键决定特征。

DESCRIPTION (provided by applicant): The Mixed Lineage Leukemia (MLL) gene is involved in chromosomal translocations which generate a fusion protein causing aggressive, acute leukemia with poor prognosis. The resulting chimeric protein retains the amino-terminal portion of MLL including the CXXC DNA-binding domain while the carboxy-terminal portion of the oncoprotein is comprised of the fusion partner. The CXXC domain is found within a larger repression domain and binds to non- methylated CpG DNA. This CXXC DNA-binding domain contains a specific clustering of eight cysteines which coordinate two zinc ions and is essential for the transformation capacity of cells expressing an MLL fusion protein. The closest homolog of MLL, MLL2 (alternatively named MLL4), contains a similar CXXC domain but binds with lower DNA-binding affinity. Interestingly, artificial MLL2-ENL does not transform hematopoietic progenitor cells in vitro. Amino acid differences between the CXXC domain or the larger repression domain of MLL and MLL2 must contribute to these functional differences. Specific amino acids along the DNA contact interface may play a role in differential DNA binding affinity. In addition, we have previously shown that the MLL repression domain (containing the CXXC domain) binds to co-repressor proteins. Differential binding of these repressor proteins to MLL and MLL2 may affect overall function, but might also contribute to CXXC DNA-binding affinity. Cysteine 1188, the only non-zinc-coordinating cysteine residue within the CXXC domain, is critically positioned on the DNA-binding surface and adjacent to one of the conserved cysteines involved in zinc coordination. Mutation of Cys1188 to aspartate completely abrogated the ability of the CXXC domain to bind DNA and prevented both in vitro immortalization and development of leukemia in mice when introduced in the context of MLL-AF9. In contrast, mutation of Cys1188 to alanine retained MLL's ability to bind CpG DNA, as well as to immortalize in vitro and in vivo in the context of the MLL-AF9 fusion. With a critical location on the DNA-binding surface of the CXXC domain and a thiol group susceptible to modification, we hypothesize that the side chain of Cys1188 may be physiologically altered to regulate DNA-binding affinity, allowing this residue to function as a molecular switch that regulates the binding of MLL to its non-methylated CpG DNA targets. Two specific aims are proposed in this application which will explore how specific amino acid residues of the MLL and MLL2 CXXC domains function to alter DNA binding, co-repressor binding, and susceptibility to post-translational modification. We hypothesize that these are critical determination features for MLL's role in acute leukemia. Keywords: MLL, MLL2, leukemia, hematopoiesis