Lin Chii Mei, Fu Haiqing, Martinovsky Maria, Bouhassira Eric, Aladjem Mirit I
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Curr Biol. 2003 Jun 17;13(12):1019-28. doi: 10.1016/s0960-9822(03)00382-8.
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.
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.
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.
真核生物基因组被划分为不同的复制时间结构域，这些结构域在S期以严格保守的顺序被激活。细胞分化可改变某些基因座的复制时间，但最近的实验在基因表达与复制时间的关系方面产生了相互矛盾的数据。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。
我们开发了一种哺乳动物实验系统，其中DNA复制时间可以以可控方式改变。该系统利用来自人类β-珠蛋白基因座的序列，当插入小鼠基因组时，这些序列表现出方向依赖性转录沉默。我们发现，在插入之前，小鼠靶位点在S期后期复制。插入后，复制时间取决于转基因的方向。在转录允许方向上，转基因和侧翼序列早期复制。在反向（易沉默）方向上，这些序列后期复制。早期复制与转基因染色质的组蛋白修饰相关，但在没有β-珠蛋白启动子的情况下也可观察到。重要的是，复制时间转换不需要转基因内的复制起点。
转基因插入哺乳动物异染色质可改变插入位点的DNA复制时间。这种不依赖于分化的复制时间转换不需要插入活性启动子或复制起点。这些观察结果表明，DNA复制时间可通过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。
