Dynamic alterations of replication timing in mammalian cells
Dynamic alterations of replication timing in mammalian cells
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DOI：
10.1016/s0960-9822(03)00382-8
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发表时间：
2003-06-17
期刊：
影响因子：
9.2
通讯作者：
Aladjem, MI
中科院分区：
文献类型：
--
作者：
Lin, CM;Fu, HQ;Aladjem, MI
真核生物基因组被划分为不同的复制时序域，它们在S期以严格保守的顺序被激活。细胞分化可以改变一些基因座的复制时间，但最近的实验得出了关于基因表达和复制时间之间关系的矛盾数据。哺乳动物细胞中复制时间的遗传和表观遗传决定因素尚未阐明。结果：我们开发了一种哺乳动物实验系统，其中DNA复制的时间可以以受控的方式改变。该系统利用来自人类β -珠蛋白位点的序列，当插入小鼠基因组时，这些序列表现出取向依赖的转录沉默。我们发现，在插入之前，小鼠靶位点在S期复制较晚。插入后，复制时间取决于转基因的方向。在允许转录的方向上，转基因和侧翼序列复制得较早。在相反（易于沉默）的方向上，这些序列复制较晚。早期复制与转基因染色质的组蛋白修饰相关，但可以在缺乏-珠蛋白启动子的情况下观察到。重要的是，复制定时开关不需要转基因内的复制起源。结论：转基因插入哺乳动物异染色质可以改变插入位点DNA复制的时间。这种与差异无关的复制定时开关不需要插入主动启动子或复制起点。这些观察结果表明，DNA复制的时间可以通过DNA序列的变化来操纵，但复制时间的决定因素与指定复制起始位点的序列不同。
Background: The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated.Results: We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human beta-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the beta-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene.Conclusions: Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.