DAB2IP调控DNA损伤诱发的染色质重塑机制研究

DNA double-strand breaks (DSBs) are the most severe form of DNA damage induced by ionizing radiation. To detect and repair DSBs, DNA damage response regulators need to overcome the barrier of condensed chromatin to access lesion sites. The posttranslational modification of histones and non-histone proteins is one of the fundamental mechanisms to promote the formation of open, relaxed of chromatin structure at DSBs and that allow the DNA damage repair machinery to access the spatially confined region surrounding the DSBs. How the DNA DSBs repair couple the chromatin-remodeling signal has not been fully elucidated. The member of Ras-GAP protein family DAB2IP, is able to suppress PI3K-Akt pro-survival pathway, and interacts with the apoptosis-stimulated kinas (ASK1)-JNK pathway to enhance cells death. Our previous studies showed that loss of DAB2IP acquired radioresistence in different cell lines. However, the exact function of DAB2IP in DNA damage response has not yet been clarified. Here, our preliminary data indicated that DAB2IP could present in nucleus bind with chromatin. More importantly, loss of DAB2IP impairs the activation of DNA damage regulators, including the phosphorylation of DNA-PKcs and ATM. Overexpression of DAB2IP promotes IR induced G1 checkpoint and apoptosis. To further analyze its role in DNA damage repair pathway, we identified that DAB2IP can interact with SAFB1, PARP-1 and RBMX using mass spec. The SAFB1 is a nonenzymatic architectural component of chromatin and co-regulates gene expression through mediating the modification of histones around promoter. Recent work revealed that SAFB1 cooperates with histone acetylation to allow the remodeling of chromatin and efficient spreading of DNA damage signal. RBMX is one of interaction proteins of SAFB1, which was also involved in DSBs homologous recombination (HR) repair. Both the function of SAFB1 and RBMX in DNA damage response are regulated by PARP-1 mediated poly(ADP-ribosyl)ation. On the basis of these results and our preliminary data, we will further explore the role of DAB2IP-SAFB1-PARP-1-RBMX signal pathway in irradiation induced chromatin remodeling, including: 1) Whether DAB2IP plays an important role in DNA damage-induced chromatin remodeling through regulating SAFB1-mediated histones and nonhitone proteins’ posttranslation modification; 2) we will further identify if the DAB2IP affects the DSBs repair pathway choice via RBMX (If SAFB1 participates in this progression); 3) And whether PARP-1 mediated poly(ADP-ribosyl)ation contributes to DAB2IP’s function in DNA damage induced chromatin remodeling and repair pathway choice; 4) Whether DAB2IP-SAFB1-PARP-1-RBMX pathway affects irradiation-induced large scale heterochromatin relaxation. This investigation may be of value in the development of radioprotective compounds against DAB2IP-SAFB1-PARP-1-RBMX signal pathway.

DNA损伤修复信号如何协调染色质重塑目前尚未完全阐明。Ras-GAP蛋白家族成员DAB2IP促进细胞辐射敏感性，DAB2IP缺失削弱辐射诱发的DNA损伤信号，如抑制DNA-PKcs和ATM磷酸化。质谱鉴定DAB2IP与SAFB1、PARP-1、RBMX相互作用。SAFB1是染色质非酶类结构组分，通过介导启动子区域的组蛋白修饰调控基因表达，调节染色质重塑并促进DNA损伤信号传递。RBMX是SAFB1相互作用蛋白，参与DNA DSBs同源重组修复。此项目探索DAB2IP-SAFB1-PARP-1-RBMX信号通路在电离辐射诱发的染色质重塑中的功能，1）DAB2IP是否通过SAFB1介导的组蛋白和非组蛋白翻译后修饰调控DNA损伤诱导的染色质重塑；2）DAB2IP是否通过RBMX调控DNA DSBs修复途径选择（SAFB1是否参与）；3）PARP-1在此过程中的作用。

本研究揭示了DAB2IP调控DNA损伤信号转导和细胞周期进程的新功能：.(1) FANCD2是范可尼贫血（FA）信号通路的核心分子，在维持细胞基因组稳定性过程中起重要作用。我们发现DAB2IP与FANCD2相互作用，促进DNA损伤发生后FANCD2向染色质募集。缺失DAB2IP削弱FANCD2在DNA损伤形成聚焦点的能力。与这些发现相一致，缺失DAB2IP细胞放射敏感性显著增加。除此之外，DAB2IP缺失细胞在HU处理后呈现更剧烈的DNA复制叉应激。.（2）有丝分裂期动粒-微管（KT-MT）粘附异常和纺锤体装配监测点缺陷是最易造成细胞染色体不稳定增加的原因。我们发现缺失DAB2IP削弱KT-MT粘附并造成大量的有丝分裂异常现象。DAB2IP可与Plk1相互作用促进其激酶活性，这种作用依赖于有丝分裂期CDK1介导的DAB2IP在T531和T546位点磷酸化。DAB2IP两个磷酸化位点突变诱发APC/C-MCC复合物稳定性降低。除此之外，DAB2IP促进MPS1蛋白稳定性，缺失DAB2IP细胞对MPS1抑制剂更敏感。在DAB2IP缺失肿瘤细胞中重建DAB2IP增加细胞对紫杉醇类药物和Plk1激酶抑制BI2536敏感性。我们的工作发现了DAB2IP通过Plk1-MPS1-APC/C-MCC复合物调控细胞周期的新机制。.（3）敲除DAB2IP基因小鼠在受到大剂量射线照射后肠组织损伤程度加重，再生隐窝数量明显下降，难以恢复，提示DAB2IP在放射性肠损伤中具有重要作用。.（4）小分子物质丁香醛促进照后TRX2在隐窝细胞中表达，修复放射性肠损伤。