Role of Heterochromatin protein 1 Beta in Genome Maintenance and Oncogenesis

描述(申请人提供)：DNA损伤反应(DDR)介导DNA双链断裂(DSB)修复，保护细胞免受损伤诱导的转化或死亡。细胞DNA被组织成蛋白质DNA复合体(染色质)，以控制蛋白质对DNA的访问和调节DNA依赖功能。在真核生物中，有两种主要类型的染色质：异染色质(基因贫乏)和常染色质(基因丰富)，其区别在于特定的组蛋白尾部修饰和非组蛋白染色质蛋白组成的差异。在非组蛋白染色质蛋白中，异染色质蛋白1(HP1)是研究最深入的例子。在哺乳动物中，有三种HP1亚型(HP1a、HP1B和HP1g)，它们的结构特征都是由两个由铰链区隔开的保守结构域：N-端染色体结构域(CD)和C-端染色体结构域(CSD)。我们先前证明，在人类细胞中过表达HP1a或HP1B会增加基因组的不稳定性和对电离辐射(IR)诱导的细胞杀伤的敏感性。此外，耗尽了 Cbx1(小鼠HP1B)在小鼠细胞中增加了基因组的不稳定性，自发的ATM(共济失调-毛细血管扩张突变)自磷酸化，减少了IR诱导的g-H_2AX焦点形成的频率，增加了IR诱导的细胞杀伤和致癌转化。其他研究人员最近的研究支持我们的结果，即HP1B对DNA DSB修复既有积极的也有消极的影响，并表明功能HP1B的准确水平是IR敏感性的关键决定因素。由于HP1b如何与修复相关蛋白相互作用来调节DNA DSB修复的大多数机制细节尚不清楚，我们将确定不同的结构域如何与染色质/修复蛋白组分相互作用来调节DNA DSB修复。我们假设HP1B的负面作用是通过CD结构域与H3K9Me结合介导的，因为这个结构域的缺失可以提高细胞的存活率，而积极的作用可能是由于HP1B CSD与乙酰化组蛋白H4K16(H4K16ac)的相互作用，这是一种独特的组蛋白修饰，可以防止高阶染色质堆积，这可能会阻碍蛋白质对DNA的访问以及与参与DDR的蛋白质的相互作用。DNA损伤修复缺陷与致癌转化和肿瘤发生有关，因此，我们将确定(I)在ATM存在和不存在的情况下Cbx1+/-小鼠和(Ii)Cbx1条件性基因敲除小鼠中Cbx1降低对肿瘤发展的影响。这些研究将确定HP1B调节细胞IR反应和肿瘤发生的机制。我们的假设--非组蛋白修饰因子HP1B调节染色质结构，并通过与DDR成分的相互作用，促进肿瘤发生--是一个需要深入研究的新想法。进一步了解正常组织和肿瘤组织染色质结构之间的生化差异的机制基础，将有助于开发新的策略来调整IR反应和改善临床放射治疗。

DESCRIPTION (provided by applicant): The DNA damage response (DDR) mediates DNA double strand break (DSB) repair and protects cells from damage induced transformation or death. Cellular DNA is organized into protein DNA complexes (chromatin) in order to control DNA access by proteins and regulated DNA dependent functions. In eukaryotes, there are two major types of chromatin: heterochromatin (gene poor) and euchromatin (gene rich) that are distinguished by specific histone tail modifications and differences in nonhistone chromatin protein constituents. Among nonhistone chromatin proteins, heterochromatin protein 1 (HP1) is the best- studied example. In mammals, there are three HP1 isoforms (HP1a, HP1b and HP1g) all structurally characterized by two conserved domains separated by a hinge region: an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD). We previously demonstrated that overexpression of HP1a or HP1b in human cells increases genomic instability and sensitivity to ionizing radiation (IR)-induced cell killing. Moreover, depletion of Cbx1 (mouse HP1b) in mouse cells increased genomic instability, spontaneous ATM (ataxia-telangiectasia mutated) autophosphorylation, reduced the frequency of IR-induced g-H2AX foci formation, increased IR-induced cell killing and oncogenic transformation. Recent studies by other investigators support our results indicating HP1b has both negative as well as positive effects on DNA DSB repair and suggest that the precise level of functional HP1b is a critical determinant to IR sensitivity. Since most mechanistic details as to how HP1 b interacts with repair associated proteins to modulate DNA DSB repair are unexplored, we will determine how different domains interact with chromatin/repair protein components to regulate DNA DSB repair. We hypothesize that the negative effect of HP1b is mediated through CD domain binding to H3K9me, since deletion of this domain can improve cell survival and the positive effect could be due to HP1b CSD interactions with acetylated histone H4K16 (H4K16ac) a unique histone modification that prevents higher order chromatin packing, which can impede protein access to DNA and with proteins involved in the DDR. Defective DNA damage repair is linked with oncogenic transformation and tumorigenesis, therefore, we will determine the impact of decreased Cbx1 on tumor development in (i) Cbx1+/- mice in the presence and absence of Atm and (ii) Cbx1 conditional knockout mice. These studies will define the mechanism by which HP1b regulates the cellular IR response and tumorigenesis. Our hypothesis-that the non-histone modifying factor HP1b regulates chromatin structure and, through interactions with DDR components, contributes to oncogenesis-is a novel idea requiring in depth studies. Further understanding about the mechanistic basis for biochemical differences between normal and tumor tissue chromatin structure will facilitate the development of new strategies for modifying IR response and improving clinical radiation therapy.