Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication

题目：鉴定顺式作用元件对哺乳动物 DNA 复制的时空调控

作者及单位：

Jiao Sima, Abhijit Chakraborty, Vishnu Dileep [ ... ] David M. Gilbert

Affiliations

Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA

发表刊物及时间：

Published: December 27, 2018DOI:https://doi.org/10.1016/j.cell.2018.11.036

摘要：

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[replication time, RT])与基因组结构高度耦合，但调节 DNA 复 制的顺式元件仍是难以捉摸的。我们在小鼠 ESCs 中创建了一系列 CRISPR 介导的缺失和反转 的多能相关拓扑关联域(topologically associating domain, TAD)。 CTCF 相关的结构域边界对于 RT 不是必需的。 CTCF 蛋白的缺失削弱了大多数 TAD 边界，但对 RT 或 A/B 的全基因组区域 没有影响。相比之下，三个 TAD 内的 CTCF 独立 3D 接触点的缺失导致了一个全域范围的先 后 RT 转移，是一种 A 到 B 的区域切换， 削弱了 TAD 结构和导致转录丢失。 TAD 边界的可分 性和这些“早期复制调控元件”(early replication control elements , ERCEs)的必要性通过在附加 域上的删除和反转得到验证。我们的结果表明，离散的顺式调控元件协调了全域 RT、 A/B 区域划分、 TAD 结构和转录，揭示了连接基因组结构和功能的基本原理。

图表选析：

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Figure 1Internal Segments Contribute Partially to Early Replication

(A) 3D structure and chromatin features of the Dppa2/4 domain. Presented in order are Hi-C heatmap (

Bonev et al., 2017) visualized using HiGlass, LaminB1 DamID (LaminB1), nuclear RNA (NucRNA), reference genes (Genes), CCCTC-binding factor ChIP (CTCF), SMC1 ChIP signal in ChIAPET datasets (SMC1), SMC1 ChIAPET identified loops (SMC1 loops, horizontal bars in black), and short nascent strand mapped replication origins (SNS origins) in mESCs. CTCF binding site orientation is indicated as pink triangles above the CTCF ChIP track. The grey vertical lines indicate the domain boundaries.

(B) Positions of the boundary deletions or inversions, boundary and internal deletions, and internal deletions.

(C–E) RT profiles of boundary deletion or inversions (C), boundary and internal deletions (D), and internal deletions (E), with mutated allele plotted in red lines and WT allele in black. The Dppa2/4 replication domain is highlighted in yellow; deleted regions are masked in white; breakpoints are indicated by grey dashed lines and inversions by red arrows.

(F) Flow cytometry demonstrating GFP-tagged CTCF depletion.

(G) RT profile of a 50-Mb region of chromosome 16 in untreated (black and grey lines) and CTCF depletion samples (red and pink lines). The Dppa2/4 domain is highlighted in yellow.

(A) Dppa2/4 区域的三维结构和染色质特征。按顺序呈现的是 Hi-C 热图(Bonev et al., 2017)。 可视化使用 HiGlass。LaminB1 DamID (LaminB1)， nuclear RNA (NucRNA)， 参考基因(genes)， CCCTC 结合因子芯片 (CTCF)， ChIAPET 数据集(SMC1)中的 SMC1 芯片信号， SMC1 ChIAPET 标识的循环(SMC1 循环，黑色横杠)。在 mESCs 中短新生链映射复制源(SNS origins)， CTCF 结合位点的方向表示为 CTCF 芯片轨迹上方的粉色三角形。 灰色竖线表示域边界。 (B) 边界缺失或逆序的位置，边界和内部缺失的位置，以及内部缺失的位置。 (C-E)边界删除或反转的 RT 剖面 (C) 边界和内部删除 (D) 内部删除 (E) 突变等位基因用红线标出， WT 等位基因用黑线标出。Dppa2/4 复制域以黄色突出显示， 删除区域用白色覆盖;断点用灰色虚线表示，用红色箭头表示反转。 (F) 流式细胞仪显示 gfp 标记的 CTCF 耗尽。 (G) 未处理(黑色和灰色线)和 CTCF 耗尽样本(红色和粉色线)中 16 号染色体的 50Mb 区域的 RT 剖面。 Dppa2/4 域用黄色突出显示。 还请参见图 S1、 S2 和 S3 以及表 S1、 S2 和 S3。

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Figure S2. Local 4C Interaction Pattern of the DppA2/4 Domain Are Not Affected after Deletion of the CTCF-Associated Domain Boundary, Related toFigures 1 and 3

图S2 4C DppA2/4局部交互模式在CTCF相关的领域边界删除后没有被影响，如图1和图3所示

Smoothed 4C contact counts from a bait within DppA2/4 domain (green triangle) are plotted for a 9Mb region in the 45kb or 335kb deletion (red). WT 46C mESCs is plotted in black as a control. Reads within 50kb of the bait are removed, and the corresponding RT profiles are overlaid as a blue histogram.

从DppA2/4域（绿色三角形）中一个“诱饵”进行的光滑的4C接触计数被绘制为9Mb区域中45kb或者335kb删除（红色）。 WT 46C mESCs作为一个控件被绘制在黑色中。50kb“诱饵”中的读取被移除，并且相应的RT轮廓作为蓝色柱状图叠加在一 起。

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Figure 5 ERCE-Containing Inversions

(A) WT capture Hi-C heatmap, DI, and domainograms are shown as in Figure 3, with inversion positions indicated with thick blue arrows. Sites a, b, and c are highlighted in red, yellow, and blue, respectively. The red stars indicate the TAD boundaries in this region.

(B and C) RT profile for the 680 kb (B) and 435 kb (C) inversions are presented with WT allele plotted in black, inverted allele in red in the actual linear distance, and the inverted allele in pink as it would appear with WT coordinates.

(D) Capture Hi-C of the 680 kb inversion presented according to the actual linear distance after inversion. Eigenvector and CTCF ChIP-seq peaks (with pink arrows indicating the orientation) are inverted from WT data. The blue box indicates the inverted region. The red stars and red dashed lines indicate newly formed TAD boundaries.

See also Figures S1 and S4 and Tables S1, S2, and S3

（A） 绘制的对照组(wild-type)Hi-C 热图、 方向性指数、 结构图如 图 3 所示， 其中反转位置用粗蓝色箭头表示。 点 a， b 和 c 分别以红 色， 黄色和蓝色突出显示。 红色星星表示该地区的 TAD 边界。 （B 和 C） 680kb（B） 和 435kb（C） 倒置反转后的 RT 曲线， 以黑色绘制显示的为 WT 等位基因，以实际线性距离显示的倒置等位 基因为红色， 出现在 WT 坐标上的倒置等位基因为粉红色。 （D）根据倒置后的实际线性距离，绘制了 680kb 倒置的 Hi-C。 特 征向量和 CCCTC binding factor(CTCF)的 ChIP-seq 峰（用指示方向的 粉红色箭头） 从 WT 数据反转。 蓝色框表示反转区域。 红色星形和 红色虚线表示新形成的 TAD 边界。

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Figure 7Model for ERCE Function

(A) ERCEs interact strongly with other predicted ERCEs outside Dppa2/4 domain. Virtual 4C profiles generated from capture Hi-C data are plotted from the viewpoints of sites a, b, c, or the predicted ERCE near the Nectin3/Pvrl3 promoter upstream of the Dppa2/4 domain. Bait regions are removed from the plots.

(B) The predicted ERCE is decorated with chromatin features similar to validated ERCEs in the Dppa2/4 domain.

(C) A co-regulation model illustrating the role of ERCEs in regulating RT, A/B compartmentalization, TAD architecture, and gene transcription.

图 7. ERCE 功能模型 (A)ERCEs 与 Dppa 2/4 域外的其他预测的 ERCEs 有很强的相互作用。由捕获 Hi-C 数据生成的 虚拟 4C 剖面是从 Dppa 2/4 结构域上游 Nectin 3/Pvr 3 启动子附近的位点 a、 b、 c 或预测的 ERCE 的角度绘制的。诱饵区域被从图上移除。 (B)预测的 ERCE 被与 Dppa 2/4 域确证的 ERCEs 相似的染色质特征所修饰。 (C)一个共同调节模型，说明 ERCEs 在调节 RT、 A/B 分割、 TAD 结 构和基因转录方面的作用

翻译小组：

黄敬潼、王俊豪、邓峻玮、陈凯星、陈志荣、郑凌伶