Etiology of translocations in hematopoietic cells

描述（由申请人提供）：本研究的长期目标是了解造血特异性发育程序对一种DNA损伤修复的影响-双链断裂(DSB)-以及导致易位的初始分子事件，这是白血病，淋巴瘤和软组织肉瘤的标志。染色体dsb是在发育中的淋巴样细胞的正常代谢过程中形成的，包括DNA复制和抗原受体重排，以及暴露于DNA损伤剂后。某些化疗药物与继发性白血病之间存在关联，例如拓扑异构酶II抑制剂治疗和涉及1lq23的重排，断点的序列分析表明DNA损伤参与了易位过程。然而，在重排形成的初始阶段，特定易位在发育中的造血亚群中发生的机制尚不清楚。这些问题可以通过在小鼠胚胎干细胞中开发的基于稀有切割酵母内切酶I- sce I的遗传系统来解决，该系统可以在定义的基因组位点上引入DSBs，并在分子水平上分析重组修复事件。该系统还对可能导致的易位、重复和删除进行评分。该建议将采用I- sce - I系统来检查造血早期祖细胞和髓细胞谱系中的DSB修复和重组，以及这种类型的损伤促进非法重组的可能性。方法将：(1)确定修复dsb和染色体间重组的潜力，以促进特定谱系内的基因组重排；(2)确定MLL和AF-4基因断点簇区域内dsb修复的潜力，从而导致t（4;11）易位，类似于在临床环境中观察到的情况；(3)使用基于基因组学微阵列的方法来表征造血发育阶段或损伤剂对细胞DNA损伤反应特异性的影响。这些研究将为DSB在造血细胞亚群中重新连接的生物学，以及负责正常抑制基因组重排和最终肿瘤发生的因素提供重要的见解。揭示易位的病因和后果可能会导致新的治疗和预防方法。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the influence of hematopoietic-specific developmental programs on the repair of one type of DNA damage--a double strand break (DSB)--and the initial molecular events that lead to translocations, which are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas. Chromosomal DSBs are formed during normal metabolic processes including DNA replication and antigen receptor rearrangement in developing lymphoid cells, as well as following exposure to DNA damaging agents. There is an association between certain chemotherapeutic agents and secondary leukemia, e.g. treatment with topoisomerase II inhibitors and rearrangements involving 1lq23, and sequence analysis of breakpoints suggests that DNA damage is involved in the translocation process. However, the mechanisms by which specific translocations occur within developing hematopoietic subpopulations in the initial formation of rearrangements remain unclear. These questions can be addressed by adaptation of a genetic system developed in murine ES cells based on the rare-cutting yeast endonuclease I-Sce I to introduce DSBs at defined genomic loci and analyze recombinant repair events at the molecular level. This system also scores for translocations, duplications, and deletions that may result. This proposal will adapt the I-Sce I system to examine DSB repair and recombination in hematopoietic early progenitor and myeloid cell lineages and the potential for this type of damage to promote illegitimate recombination. Approaches will: (1) determine the potential for repair of DSBs and interchromosomal recombination to promote genome rearrangements within specific lineages; (2) determine the potential for repair of DSBs within the breakpoint cluster regions of the MLL and AF-4 genes to result in t(4;11) translocations similar to those observed in the clinical setting; and (3) use a genomics microarray-based approach to characterize the influence of the stage of hematopoietic development or the damaging agent on the specificity of a cell's DNA damage response. These studies will provide important insight into the biology of DSB rejoining in hematopoietic cell subpopulations, and the factors responsible for the normal suppression of genome rearrangements and, ultimately, tumorigenesis. Unraveling the etiology and consequences of translocations may lead to new approaches to therapy and prevention.