Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
Genome Res. 2012 Jan;22(1):64-75. doi: 10.1101/gr.126003.111. Epub 2011 Nov 16.
Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.
精确的 DNA 复制对于基因组的维护至关重要，但在未转化的分化后生动物细胞中，从全基因组水平上研究这一过程具有内在的困难。为了确定后生动物 DNA 复制如何受到抑制，我们检查了在果蝇多线唾液腺中选择性复制不足的区域，发现这些区域转录沉默，并富含抑制性的 H3K27me3 标记。在对分化后生动物组织中起始识别复合物（ORC）结合的全基因组首次分析中，我们发现 ORC 结合在这些大区域内显著减少，表明起始减少是导致复制不足的一种机制。染色质蛋白 SUUR 抑制复制叉的延伸也是减少拷贝数的另一种抑制机制。尽管当 SUUR 发生突变并恢复拷贝数时，抑制性组蛋白标记被去除，但转录和 ORC 结合都没有恢复。将 SUUR 蛋白固定在特定位置不足以阻止复制，然而。这些结果表明，在分化过程中，DNA 复制的发育控制（在起始和延伸阶段）是改变基因拷贝数的一种机制。
