不同亚型内源性MLV表观遗传抑制网络的动态构建及分子机制

在表观遗传水平抑制内源性反转录病毒ERV的转录活性是高等真核细胞维护基因组稳定性的重要策略。近期组学研究揭示各类表观抑制因子即使在序列高度保守的不同ERV拷贝上的组织模式及功能发挥也呈现出明显异质性；我们的前期数据揭示表观抑制因子PRC1仅对小鼠白血病病毒MLV部分拷贝具靶向特异性。我们向小鼠ESC基因组定向编辑插入具初始转录活性的MLV，建成了一套能够全程检测其转录抑制过程的报告系统，再次验证了PRC1对MLV的选择性识别。为解析相关分子机制，本项目将运用该报告系统比较表观抑制因子在不同亚型MLV上动态装配及转录抑制的过程，并筛选关键招募序列；结合基因编辑、重组ZFP等技术解析各类抑制因子的功能及关联性；利用质谱方法或构建锌指结构域环肽库筛选MLV亚型特异性识别因子以澄清识别机制。解析MLV复杂多样的表观抑制机制的分子基础有助于阐明表观遗传防御体系随ERV序列变异适应性进化的相关规律。

Transcription repression of endogenous retroviruses (ERV) through epigenetic regulators is essential for the genomic stability in higher eukaryotes. Recent genome-wide mapping of the epigenetic regulators and corresponding repressive marks at ERV uncovered that the organization and functional involvement of each epigenetic regulator were not only very specific for distinct ERV families, but even for various ERV proviral copies within the same family. Our preliminary data also indicated that the components of polycomb repressive complex 1 (PRC1) selectively targeted to some particular copies of the Murine Leukemia Viruses (MLV) in mouse embryonic stem cells (mESC). Using CRISPR-facilitated homologous recombination, we successfully inserted the partial sequences of MLV provirus into transcriptionally active regions in the genome of mESC, then monitored their entire repression process in a reporter system, which led us observe the phenomenon of selective targeting of MLV by PRC1 again. To further address the underlying mechanisms, we propose to comprehensively apply the above reporter system to compare the dynamic assembly and repression processes of various epigenetic regulators at distinct subtypes of MLV, and to identify the key cis-element(s) within each subtype for epigenetic regulator recruitment. The combination of gene editing, recombinant ZFP strategies and other technologies will help us dissect the functional roles and inter-relationship of the epigenetic regulators involved. In addition, the specific recognition factor for each MLV subtype will be identified by mass spectrometric analyses or upon functional screening of a cyclic peptide library of zinc finger domains. Taken together, clarification of the molecular mechanisms of epigenetic regulatory network for each MLV subtype will improve our understanding for the evolution strategies of the epigenetic defending systems against the sequence variations of ERVs.