SUMM4 complex couples insulator function and DNA replication timing control

The asynchronous timing of replication of different chromosome domains is essential for eukaryotic genome stability, but the mechanisms establishing replication timing programs remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) domains in polytene chromosomes. SUUR negatively regulates DNA replication fork progression across IH; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. In vitro, Mod(Mdg4) stimulates the ATPase activity of SUUR, although neither SUUR nor SUMM4 can remodel nucleosomes. Mod(Mdg4)-67.2 and SUUR distribution patterns in vivo partially overlap, and Mod(Mdg4) is required for a normal spatiotemporal distribution of SUUR in chromosomes. SUUR and Mod(Mdg4)-67.2 mediate insulator activities of the gypsy mobile element that disrupt enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, mutations of SuUR or mod(mdg4) reverse the locus-specific UR. These findings reveal that DNA replication can be delayed by a chromatin barrier and thus, uncover a critical role for architectural proteins in replication timing control. They also provide a biochemical link between ATP-dependent motor factors and the activity of insulators in regulation of gene expression and chromatin partitioning. Overall design: DNA-sequencing of Drosophila salivary glands to measure under replication.

不同染色体结构域的复制异步时序对于真核基因组稳定性至关重要，但建立复制时序程序的分子机制仍未完全阐明。果蝇SNF2相关因子SUUR可介导多线染色体中晚复制插入型异染色质（intercalary heterochromatin, IH）结构域的复制不足（under-replication, UR）。SUUR可负调控跨异染色质的DNA复制叉行进，但其作用机制仍不明确。本研究开发了一种名为质谱辅助快速蛋白质复合物鉴定（MS-Enabled Rapid protein Complex Identification, MERCI）的新方法，成功分离得到由SUUR与染色质边界蛋白Mod(Mdg4)-67.2组成的稳定化学计量天然复合物SUMM4。体外实验表明，Mod(Mdg4)可激活SUUR的ATP酶活性，不过SUUR及SUMM4均无法重塑核小体。体内实验中，Mod(Mdg4)-67.2与SUUR的分布模式部分重叠，且Mod(Mdg4)对于SUUR在染色体上的正常时空分布不可或缺。SUUR与Mod(Mdg4)-67.2可介导吉普赛转座子（gypsy mobile element）的绝缘子活性，该活性可破坏增强子-启动子相互作用，并在基因组中建立常染色质-异染色质屏障。此外，SuUR或mod(mdg4)的突变可逆转位点特异性的复制不足。上述研究结果揭示，染色质屏障可延缓DNA复制，从而发现了架构蛋白在复制时序调控中的关键作用。同时，本研究也为ATP依赖的运动因子与绝缘子在基因表达调控及染色质分区中的活性之间建立了生化联系。实验整体设计：通过对果蝇唾液腺进行DNA测序，以检测复制不足现象。