Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication
題目：鑒定順式作用元件對哺乳動物 DNA 復制的時空調(diào)控
作者及單位：
Jiao Sima, Abhijit Chakraborty, Vishnu Dileep [ ... ] David M. Gilbert
Affiliations
Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
發(fā)表刊物及時間：
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 復制的時間順序(復制時間[replication time, RT])與基因組結構高度耦合，但調(diào)節(jié) DNA 復 制的順式元件仍是難以捉摸的。我們在小鼠 ESCs 中創(chuàng)建了一系列 CRISPR 介導的缺失和反轉 的多能相關拓撲關聯(lián)域(topologically associating domain, TAD)。 CTCF 相關的結構域邊界對于 RT 不是必需的。 CTCF 蛋白的缺失削弱了大多數(shù) TAD 邊界，但對 RT 或 A/B 的全基因組區(qū)域 沒有影響。相比之下，三個 TAD 內(nèi)的 CTCF 獨立 3D 接觸點的缺失導致了一個全域范圍的先 后 RT 轉移，是一種 A 到 B 的區(qū)域切換， 削弱了 TAD 結構和導致轉錄丟失。 TAD 邊界的可分 性和這些“早期復制調(diào)控元件”(early replication control elements , ERCEs)的必要性通過在附加 域上的刪除和反轉得到驗證。我們的結果表明，離散的順式調(diào)控元件協(xié)調(diào)了全域 RT、 A/B 區(qū)域劃分、 TAD 結構和轉錄，揭示了連接基因組結構和功能的基本原理。
圖表選析：
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 區(qū)域的三維結構和染色質(zhì)特征。按順序呈現(xiàn)的是 Hi-C 熱圖(Bonev et al., 2017)。 可視化使用 HiGlass。LaminB1 DamID (LaminB1)， nuclear RNA (NucRNA)， 參考基因(genes)， CCCTC 結合因子芯片 (CTCF)， ChIAPET 數(shù)據(jù)集(SMC1)中的 SMC1 芯片信號， SMC1 ChIAPET 標識的循環(huán)(SMC1 循環(huán)，黑色橫杠)。在 mESCs 中短新生鏈映射復制源(SNS origins)， CTCF 結合位點的方向表示為 CTCF 芯片軌跡上方的粉色三角形。 灰色豎線表示域邊界。 (B) 邊界缺失或逆序的位置，邊界和內(nèi)部缺失的位置，以及內(nèi)部缺失的位置。 (C-E)邊界刪除或反轉的 RT 剖面 (C) 邊界和內(nèi)部刪除 (D) 內(nèi)部刪除 (E) 突變等位基因用紅線標出， WT 等位基因用黑線標出。Dppa2/4 復制域以黃色突出顯示， 刪除區(qū)域用白色覆蓋;斷點用灰色虛線表示，用紅色箭頭表示反轉。 (F) 流式細胞儀顯示 gfp 標記的 CTCF 耗盡。 (G) 未處理(黑色和灰色線)和 CTCF 耗盡樣本(紅色和粉色線)中 16 號染色體的 50Mb 區(qū)域的 RT 剖面。 Dppa2/4 域用黃色突出顯示。 還請參見圖 S1、 S2 和 S3 以及表 S1、 S2 和 S3。
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
圖S2 4C DppA2/4局部交互模式在CTCF相關的領域邊界刪除后沒有被影響，如圖1和圖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.
從DppA2/4域（綠色三角形）中一個“誘餌”進行的光滑的4C接觸計數(shù)被繪制為9Mb區(qū)域中45kb或者335kb刪除（紅色）。 WT 46C mESCs作為一個控件被繪制在黑色中。50kb“誘餌”中的讀取被移除，并且相應的RT輪廓作為藍色柱狀圖疊加在一 起。
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） 繪制的對照組(wild-type)Hi-C 熱圖、 方向性指數(shù)、 結構圖如 圖 3 所示， 其中反轉位置用粗藍色箭頭表示。 點 a， b 和 c 分別以紅 色， 黃色和藍色突出顯示。 紅色星星表示該地區(qū)的 TAD 邊界。 （B 和 C） 680kb（B） 和 435kb（C） 倒置反轉后的 RT 曲線， 以黑色繪制顯示的為 WT 等位基因，以實際線性距離顯示的倒置等位 基因為紅色， 出現(xiàn)在 WT 坐標上的倒置等位基因為粉紅色。 （D）根據(jù)倒置后的實際線性距離，繪制了 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 粉紅色箭頭） 從 WT 數(shù)據(jù)反轉。 藍色框表示反轉區(qū)域。 紅色星形和 紅色虛線表示新形成的 TAD 邊界。
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.
圖 7. ERCE 功能模型 (A)ERCEs 與 Dppa 2/4 域外的其他預測的 ERCEs 有很強的相互作用。由捕獲 Hi-C 數(shù)據(jù)生成的 虛擬 4C 剖面是從 Dppa 2/4 結構域上游 Nectin 3/Pvr 3 啟動子附近的位點 a、 b、 c 或預測的 ERCE 的角度繪制的。誘餌區(qū)域被從圖上移除。 (B)預測的 ERCE 被與 Dppa 2/4 域確證的 ERCEs 相似的染色質(zhì)特征所修飾。 (C)一個共同調(diào)節(jié)模型，說明 ERCEs 在調(diào)節(jié) RT、 A/B 分割、 TAD 結 構和基因轉錄方面的作用
翻譯小組：
黃敬潼、王俊豪、鄧峻瑋、陳凱星、陳志榮、鄭凌伶
