裂殖酵母组蛋白H2B保守致癌突变影响DNA复制压力应答的机制研究

Glioblastoma multiforme or lymphoma has been recently found that histone H2B contains recurrent and hotspot mutations such as G53D or P104L, respectively. However, the molecular mechanism has not been reported. 46 copies of human H2B genes and 19 protein variants also make study complex and difficult. Previously, we studied the fission yeast Schizosaccharomyces pombe that contains the sole copy of H2B gene; and revealed the mechanism of H2B acetylation-mimic mutation H2BK33Q compromising cellular response to DNA replication stress. Our preliminary experiments also showed that H2B onco-mutations H2BG52D or H2BP102L are conserved in S. pombe and they are sensitive to high temperature and drugs causing DNA replication stress. H2BG52D and H2BP102L mutants exhibit increased spontaneous or replication stress induced DNA damage, as well as delayed damage repair. These onco-mutations do not affect checkpoint activation but decrease the efficiency of homologous recombination (HR). Therefore we propose that H2BG52D and H2BP102L defect in DNA replication stress response through changing nucleosome or chromatin structure, reducing HR end resection efficiency and decreasing the ability of stalled replication fork restart. To this end, we plan firstly to test the effect of H2BG52D and H2BP102L on the velocity or frequency of stalling or collapsed forks, and on each response pathway to DNA replication stress. Then we will study the nucleosome stability and removal during HR end resection in these mutants. Finally we will consider to what extent the changes of gene expression or H3K4 methylation in mutants involved in above mechanism. These studies will elucidate the detailed molecular mechanism of H2B onco-mutations, enlarge the understanding of oncohistone pathology, and provide the novel clinical target and idea to the adult glioblastoma multiforme or lymphoma.

成人神经胶质瘤含有组蛋白H2BG53D热点突变，淋巴瘤含有H2BP104L突变。但H2B突变致癌机制尚无研究，并且人H2B的46个基因和19个蛋白变体也使研究复杂化。我们前期用仅含一个H2B基因的裂殖酵母揭示了H2B乙酰化突变K33Q减弱DNA复制压力应答的机制，并且预实验发现裂殖酵母H2BG52D和P102L保守致癌突变体对温度和复制压力药物敏感，自发或MMS诱导的DNA损伤增加且修复延迟，checkpoint正常激活但同源重组修复效率降低。我们推测H2BG52D和P102L有DNA复制压力应答缺陷：改变了核小体或染色质结构，降低同源重组末端切割效率，减弱停顿复制叉重起始能力。为此我们将检测突变对断裂复制叉重起始速率、垮塌频率与压力应答各通路的影响，研究核小体稳定和末端切割时核小体移除变化，探讨突变对基因表达或H3K4甲基化的改变及其影响，以阐明H2B突变致成人神经胶质瘤或淋巴瘤的机制。

项目背景：儿童罕见脑癌或骨癌中存在高频的组蛋白H3.3基因K27M，G34R/V/W/L，K36M突变，其主要的致癌机制是通过影响H3K27或K36的甲基化水平从而改变了与癌症发生相关的基因表达。近年来也发现，在多数的成人常见肿瘤中广泛存在相对低频的4种组蛋白的基因突变，且主要发生在基因的内部和非翻译后修饰的位点。这些非经典组蛋白突变的致癌机制还不清楚。另外，非经典组蛋白致癌突变以及相关癌症的种类呈现多样性，难以揭示致癌突变的普遍规律。.研究内容：裂殖酵母具有单拷贝的H2B基因，我们利用其作为模式生物，构建了16个H2B保守且高频的致癌突变菌株。我们研究了它们的基因组不稳定表型，深入探究了H2BG52D或H2BP102L突变影响基因组不稳定的分子机制。.重要结果：裂殖酵母H2BG52D或H2BP102L保守致癌突变菌株对温度及DNA复制或损伤药物敏感；自发或MMS诱导的DNA损伤增加且修复延迟；停顿或垮塌复制叉的重起始合成速率降低；细胞周期检验点蛋白激酶正常激活但同源重组修复的效率降低；同源重组起始过程呈现过度的末端切割，降低Rad51的募集；其机制可能是H2BG52D或H2BP102L突变导致H2BK119位的单泛素化水平降低，因为通过失活H2B去泛素化酶活性从而增强H2B泛素化后基本上能挽救其基因组不稳定表型。.关键数据：H2BG52D或H2BP102L致癌突变通过降低H2BK119单泛素化水平抑制了DNA损伤断裂后的同源重组修复效率进而导致基因组不稳定。.科学意义：本文揭示了H2B致癌突变影响基因组稳定的机制可基本分为H2B泛素化降低依赖型和非依赖型两种。对于H2B泛素化降低类型的致癌突变，H2B去泛素化酶USP22的抑制剂可能治疗其相关的癌症。