Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.
Cell. 2019 Feb 7;176(4):816-830.e18. doi: 10.1016/j.cell.2018.11.036. Epub 2018 Dec 27.
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.
DNA 复制的时间顺序（复制时间[RT]）与基因组结构高度相关，但调节 RT 的顺式元件仍然难以捉摸。我们在小鼠胚胎干细胞中创建了一系列与多能性相关的拓扑关联结构域（TAD）的 CRISPR 介导缺失和反转。CTCF 相关的结构域边界对于 RT 并不必需。CTCF 蛋白耗竭削弱了大多数 TAD 边界，但对 RT 或 A/B 区室化没有影响。相比之下，删除三个 TAD 内不依赖 CTCF 的 3D 接触点会导致整个 TAD 的 RT 早期到晚期转移、A 到 B 区室的转换、TAD 结构的弱化以及转录的丧失。TAD 边界的非必需性和这些“早期复制调控元件”（ERCEs）的必要性通过在其他结构域中的缺失和反转得到了验证。我们的结果表明，离散的顺式调控元件协调了整个 TAD 的 RT、A/B 区室化、TAD 结构和转录，揭示了连接基因组结构和功能的基本原理。
