The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation

Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5′ MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST–MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a K(D) of ∼3.3 × 10(–8) M was determined for the GST–MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT–VP16 transactivator construct.

原癌基因MLL（mixed lineage leukemia，混合谱系白血病）的变异在高比例急性白血病中具有特征性，尤其多见于婴幼儿患者。MLL的激活可通过两种途径实现：一是其5'端外显子发生内部串联重复，二是通过染色体易位形成嵌合蛋白，将MLL的N端与多种不同的伴侣蛋白融合。研究发现，MLL中一段与真核DNA甲基转移酶（eukaryotic DNA methyltransferases）具有显著同源性的结构域（MT结构域），是致癌性MLL变体发挥转化潜能的必需结构。本研究证实，该MT结构域可特异性识别带有未甲基化CpG序列的DNA。在凝胶迁移实验中，仅需CpG序列的存在即可使重组GST-MT融合蛋白与DNA发生结合。若在DNA单链或双链中引入5-甲基CpG，则会阻断二者的有效结合。通过表面等离子体共振（surface plasmon resonance, SPR）技术，我们测得GST-MT/DNA复合物形成的解离常数（K(D)）约为3.3×10^–8 M。位点筛选实验与DNase I足迹实验均证实，CpG序列为MT结构域的结合靶点。最后，我们构建了MT-VP16杂交反式激活因子构建体，利用其MT结构域的DNA结合特性开展体内报告基因实验，进一步验证了这一相互作用。