寧椿游，何夢楠，唐茜子，朱慶，李明洲，李地艷

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基于Hi-C技術(shù)哺乳動物三維基因組研究進(jìn)展

寧椿游，何夢楠，唐茜子，朱慶，李明洲，李地艷

四川農(nóng)業(yè)大學(xué)動物科技學(xué)院，動物遺傳育種研究所，成都 611130

基因組DNA在細(xì)胞核中并不是呈線性的一字排列，而是以三維結(jié)構(gòu)高度折疊并濃縮成染色質(zhì)的方式儲存于核內(nèi)，具有特定的高級空間結(jié)構(gòu)和構(gòu)象。高通量染色體構(gòu)象捕獲(high-througnput chromosome conformation capture, Hi-C)技術(shù)于2009年首次被提出，目前已得到大規(guī)模運(yùn)用，使得人們對于三維基因組學(xué)有了更深刻的認(rèn)識。研究表明，哺乳動物基因組三維層級結(jié)構(gòu)單元由大到小依次為染色體疆域(chromosome territory, CT)、染色質(zhì)區(qū)室(chromatin compartment A/B)、拓?fù)潢P(guān)聯(lián)結(jié)構(gòu)域(topological associated domain, TAD)和染色質(zhì)環(huán)(chromatin loop)，這些層級結(jié)構(gòu)單元在基因轉(zhuǎn)錄和表達(dá)調(diào)控過程中發(fā)揮著重要作用。本文基于Hi-C技術(shù)從染色質(zhì)的三維層級結(jié)構(gòu)劃分、構(gòu)象單元作用以及三維基因組在發(fā)育、疾病等方面的應(yīng)用進(jìn)行闡述，旨在為更深入地了解哺乳動物三維基因組學(xué)研究提供參考。

三維基因組學(xué)；染色質(zhì)空間構(gòu)象；Hi-C技術(shù)；基因表達(dá)調(diào)控

染色質(zhì)是遺傳物質(zhì)的載體，其活性和功能由線性的基因組序列、序列之間的相互作用和動態(tài)變化的染色質(zhì)三維空間構(gòu)象共同決定。早期對基因的表達(dá)調(diào)控研究大都是基于一維(基因序列)和二維(不同序列的相互作用)的層面，將基因組作為線性分子模型去研究機(jī)體或細(xì)胞內(nèi)的各種調(diào)控機(jī)理。隨著更多的一維和二維基因組數(shù)據(jù)的產(chǎn)生，現(xiàn)有的線性模型不足以揭示這些離散的調(diào)控元件、結(jié)構(gòu)變異與基因功能的聯(lián)系。由此，基于染色質(zhì)空間構(gòu)象解釋基因表達(dá)調(diào)控機(jī)制的三維基因組學(xué)應(yīng)運(yùn)而生。

2009年，Lieberman-Aiden等[1]在上首次報(bào)道了以整個(gè)細(xì)胞核為研究對象，利用高通量測序技術(shù)，結(jié)合生物信息學(xué)分析方法，研究全基因組范圍內(nèi)DNA序列在空間位置上任意兩位點(diǎn)間互作關(guān)系的高通量染色體構(gòu)象捕獲(high-througnput chr-omosome confo-rmation capture, Hi-C)技術(shù)。Hi-C及其衍生技術(shù)的出現(xiàn)，使得人們能夠從技術(shù)上突破對于三維基因組學(xué)認(rèn)識的障礙。三維基因組學(xué)研究能夠解釋那些距離目標(biāo)基因幾kb甚至幾Mb的調(diào)控元件如何調(diào)控基因表達(dá)[2]，其研究重點(diǎn)在于解析細(xì)胞核內(nèi)染色質(zhì)的不同空間構(gòu)象及其結(jié)構(gòu)單元，探究不同類型的結(jié)構(gòu)單元如何介導(dǎo)轉(zhuǎn)錄調(diào)控元件與基因間的互作關(guān)系，從而闡明基因功能與轉(zhuǎn)錄調(diào)控的分子機(jī)制。本文通過目前已有的研究，對哺乳動物細(xì)胞核內(nèi)三維基因組結(jié)構(gòu)劃分、構(gòu)象單元作用以及目前Hi-C技術(shù)在三維基因組學(xué)應(yīng)用等方面進(jìn)行了介紹。

1 哺乳動物三維基因組的結(jié)構(gòu)單元

目前，部分哺乳動物和其他真核生物的細(xì)胞核內(nèi)染色質(zhì)三維折疊組裝的基本規(guī)律被揭示[3]。在哺乳動物細(xì)胞核內(nèi)，染色質(zhì)以嚴(yán)密的層級結(jié)構(gòu)折疊組裝成高級構(gòu)象，這些層級結(jié)構(gòu)單元由大到小依次為染色體疆域(chromosome territory, CT)、染色質(zhì)區(qū)室(chromatin compartment A/B)、拓?fù)潢P(guān)聯(lián)結(jié)構(gòu)域(topo-logical associated domain, TAD)和染色質(zhì)環(huán)(chromatin loop)[4,5](圖1)。其中，染色體疆域是普遍存在的基因組空間結(jié)構(gòu)，不同的染色體在細(xì)胞核內(nèi)占據(jù)不同的疆域；染色質(zhì)區(qū)室是由基因組表觀狀態(tài)所決定的較大的結(jié)構(gòu)單元，與染色質(zhì)活性密切相關(guān)；拓?fù)潢P(guān)聯(lián)結(jié)構(gòu)域是細(xì)胞核內(nèi)穩(wěn)定存在的空間結(jié)構(gòu)單元，在局部范圍內(nèi)介導(dǎo)基因的表達(dá)調(diào)控；染色質(zhì)環(huán)是直接調(diào)控基因表達(dá)的最精細(xì)的結(jié)構(gòu)和功能單元，通常由啟動子與遠(yuǎn)端增強(qiáng)子互作形成，在介導(dǎo)基因的轉(zhuǎn)錄激活中發(fā)揮著重要作用。

1.1 染色體疆域

早在20世紀(jì)，細(xì)胞學(xué)家Theodore Boveri研究蛔蟲()間期核中的染色質(zhì)時(shí)，發(fā)現(xiàn)染色質(zhì)在細(xì)胞核中并非隨機(jī)排列[6]。后來的研究發(fā)現(xiàn)，每條染色體在間期中都各自占據(jù)了一塊特定的不重疊的核區(qū)域，即染色體疆域(CT)[7]。不同染色體之間的重疊僅限于CTs的邊界[8]。CTs的定位與其基因密度有關(guān)，不同基因密度的CTs占據(jù)著不同的核位置[9]。在CTs中，每條染色體都被限制在各自具體的核空間中，僅僅一小部分延伸到鄰近的核空間中，因此染色體折疊形成CT被認(rèn)為是內(nèi)部核運(yùn)動的屏障[4,10]。CTs的定位還與細(xì)胞類型的特異性因素如復(fù)制時(shí)間和轉(zhuǎn)錄活性相關(guān)，早期復(fù)制位點(diǎn)和活性基因傾向定位于細(xì)胞核內(nèi)部，而晚期復(fù)制位點(diǎn)和抑制基因傾向于核邊緣[11,12]。Solovei等[13]發(fā)現(xiàn)，與白天活動的動物和大多數(shù)真核生物相比，夜行哺乳動物的視網(wǎng)膜桿狀細(xì)胞中的CT位置是倒置的。在夜行性視網(wǎng)膜桿狀細(xì)胞中，異染色質(zhì)定位于核中心，常染色質(zhì)位于核外圍。核組織的模型計(jì)算發(fā)現(xiàn)這種倒置的CT結(jié)構(gòu)形式能夠有效地引導(dǎo)光線，從而有助于夜行動物適應(yīng)夜晚的生活方式。

1.2 染色體區(qū)室

2009年，Lieberman-Aiden等[1]首次運(yùn)用Hi-C技術(shù)揭示了人淋巴母細(xì)胞(GM06990)的三維基因組結(jié)構(gòu)。該研究證實(shí)了之前通過3C (chromosome con-formation capture)技術(shù)和3D-FISH (3D fluores-cencehybridization)技術(shù)發(fā)現(xiàn)的CTs的存在，即那些較小的、基因富集(gene-rich)的染色體在空間上更接近。對同一條染色體內(nèi)部互作(interaction)進(jìn)行分析發(fā)現(xiàn)，染色質(zhì)間互作強(qiáng)度隨著基因組線性距離的增加而降低，且同一條染色體內(nèi)部互作強(qiáng)度高于不同染色體間的互作。該研究也首次提出了基因組空間結(jié)構(gòu)的另一重要特征，即染色質(zhì)是由com-partment A和B兩種基因組間隔區(qū)交叉分布構(gòu)成(圖2)。同一種compartment內(nèi)部具有更高的染色質(zhì)互作頻率，且在同一線性距離上，compartment B之間的互作頻率高于compartment A。其中，compartment A為開放(open)染色質(zhì)區(qū)室，多為常染色質(zhì)，是基因富集區(qū)域，GC含量高，基因高表達(dá)；而compartment B為封閉(close)染色質(zhì)區(qū)室，多為異染色質(zhì)區(qū)域，通常是基因沙漠(gene-desert)區(qū)域，GC含量低，基因表達(dá)量相對compartment A低。Compartment A和B的特征與其他基因組和表觀特征呈現(xiàn)高度相關(guān)，其中compartment A區(qū)域有激活的染色質(zhì)標(biāo)簽H3K36me3，具有更高的染色質(zhì)可接近性(DNAseⅠ高度敏感)，而compartment B與抑制性組蛋白標(biāo)簽H3K27me3高度相關(guān)。因此，compartment A是更加開放的、可接近的、轉(zhuǎn)錄激活的染色質(zhì)區(qū)域。

圖1 哺乳動物細(xì)胞核內(nèi)染色質(zhì)的層級結(jié)構(gòu)

高階染色質(zhì)結(jié)構(gòu)是基因表達(dá)的重要調(diào)節(jié)因子。雖然在基因組中已經(jīng)發(fā)現(xiàn)了動態(tài)染色質(zhì)結(jié)構(gòu)，但在哺乳動物發(fā)育和譜系規(guī)范中染色質(zhì)動態(tài)的完整范圍仍有待確定。美國加州大學(xué)Bing Ren 教授及其團(tuán)隊(duì)通過繪制人類ES細(xì)胞核4個(gè)ES細(xì)胞衍生譜系的全基因組染色質(zhì)相互作用圖譜，揭示了在譜系規(guī)范中廣泛的染色質(zhì)重組[14]。在胚胎干細(xì)胞分化成4種特定細(xì)胞系的過程中，至少有36%的基因組發(fā)生了空間可塑性重排(即compartment A/B switch)。這些重排與特定的細(xì)胞功能相關(guān)，B到A狀態(tài)改變的基因傾向于更高表達(dá)，而A到B狀態(tài)改變的基因傾向于更低的表達(dá)。這說明在一個(gè)全局范圍內(nèi)，com-partment A/B具有較高可塑性，并且與細(xì)胞特異性基因表達(dá)相關(guān)，并不起決定性的作用。

圖2 Hi-C數(shù)據(jù)顯示每條染色體兩種類型的compartments

A：Hi-C原始互作矩陣；B：相關(guān)系數(shù)矩陣。根據(jù)本課題組對豬第18號染色體的Hi-C測序結(jié)果(數(shù)據(jù)未發(fā)表)繪制。

在更高分辨率的Hi-C互作圖譜中，compartment A/B還能被分成更小的subcompartments，即A1、A2和B1、B2、B3，并且每一個(gè)subcompartment都與部分特異性的組蛋白修飾模式相關(guān)聯(lián)[15]。在果蠅()細(xì)胞中同樣存在這5種主要的subcom-partments染色質(zhì)類型(2個(gè)激活性，3個(gè)抑制性)[16]，表明這種相似的compartments染色質(zhì)結(jié)構(gòu)在后生動物中高度保守。

1.3 拓?fù)潢P(guān)聯(lián)結(jié)構(gòu)域

2012年5月，同時(shí)報(bào)道了美國麻省大學(xué)醫(yī)學(xué)院分子遺傳學(xué)家Job Dekker以及美國加州大學(xué)Ludwig癌癥研究所Bing Ren教授的研究成果，他們均發(fā)現(xiàn)了哺乳動物細(xì)胞內(nèi)染色質(zhì)折疊的二級結(jié)構(gòu)單元——TAD[17,18]。研究發(fā)現(xiàn)，將Hi-C互作圖譜的分辨率提高到40 kb或更高時(shí)，高度自我相關(guān)的染色質(zhì)區(qū)域在互作熱圖上表現(xiàn)為間隔的三角形，即拓?fù)潢P(guān)聯(lián)結(jié)構(gòu)域(TAD) (圖3)。其中，Bing Ren教授及其團(tuán)隊(duì)研究了小鼠()的胚胎干細(xì)胞(mESCs)、大腦皮層(cortex)以及人的胚胎干細(xì)胞(hESCs)和肺成纖維細(xì)胞(IMR90)的Hi-C數(shù)據(jù)，在小鼠胚胎干細(xì)胞的Hi-C數(shù)據(jù)分析中找到了約2200個(gè)平均大小為0.88 Mb、約占基因組91%區(qū)域的TAD結(jié)構(gòu)，且在這些TAD內(nèi)部的互作顯著高于TAD間的互作[17]。此外，非哺乳動物如果蠅[19]、斑馬魚()[20]、線蟲()[21]以及酵母()[22,23]等基因組也具有這種相似的TAD結(jié)構(gòu)，而在擬南芥()的Hi-C結(jié)果中并未發(fā)現(xiàn)類似TAD樣的結(jié)構(gòu)[24,25]。但最近的研究發(fā)現(xiàn)，水稻(L.)中同樣存在非典型的TAD結(jié)構(gòu)，并且平均分布在水稻的12條染色體中，表明TAD結(jié)構(gòu)在植物中可能并不保守[26,27]。

圖3 Hi-C測序數(shù)據(jù)中鑒定得到的拓?fù)浣Y(jié)構(gòu)域(TAD)

A：500 kb Hi-C互作矩陣；B：20 kb Hi-C互作矩陣。根據(jù)本課題組對豬第18號染色體的Hi-C測序結(jié)果(數(shù)據(jù)未發(fā)表)繪制。

越來越多的證據(jù)表明，TAD作為基因組折疊的功能單元，在不同的動物細(xì)胞中穩(wěn)定存在[28,29]。首先，TAD在不同細(xì)胞間的位置相對穩(wěn)定，并且其似乎并不與組織特異性的基因表達(dá)或組蛋白修飾相關(guān)；其次，TAD的定位也具有保守性，在人和小鼠的ES細(xì)胞中，共有的TAD邊界達(dá)到50%~70%[14]，并且這種保守性還體現(xiàn)在果蠅等昆蟲上[30]，表明TAD是動物基因組的固有特性。此外，研究還發(fā)現(xiàn)，TAD邊界與復(fù)制域(replication domain)邊界存在著大量的重合，說明TAD是復(fù)制時(shí)間調(diào)節(jié)的穩(wěn)定單位[31]。

TAD作為基因組三維結(jié)構(gòu)單元具有重要特征，其具體形成機(jī)制正在被不斷揭示。研究顯示，TAD邊界富集著大量的標(biāo)記因子，包括H3K4me3和H3K36me3組蛋白修飾位點(diǎn)、轉(zhuǎn)錄起始位點(diǎn)(trans-cription start site, TSS)、看家基因、tRNA、短散在元件(SINE)以及阻遏子CTCF和黏連蛋白復(fù)合物(cohesin complex)，暗示這些因子在建立TAD的過程中存在著重要作用。在小鼠ES細(xì)胞中，分別有75%和33%的TAD邊界在CTCF結(jié)合位點(diǎn)、看家基因位置的20 kb以內(nèi)[17]；而在基因表達(dá)時(shí)，CTCF能夠與黏連蛋白協(xié)同合作，使得線性距離較遠(yuǎn)的增強(qiáng)子與基因的啟動子相結(jié)合，激活轉(zhuǎn)錄表達(dá)，由此說明CTCF的結(jié)合和高表達(dá)水平的轉(zhuǎn)錄活性可能與TAD的形成有關(guān)。為了揭示CTCF和cohesin在TAD形成中的作用，Bing Ren教授團(tuán)隊(duì)分別對CTCF和cohesin進(jìn)行了精確敲除，并結(jié)合4C（chromo-some conformation capture-on-chip）、Hi-C和3D- FISH技術(shù)檢測了染色質(zhì)組裝的變化及其對基因表達(dá)的影響[32]。研究發(fā)現(xiàn)，CTCF和cohesin對于TAD的形成具有不同的作用，cohesin主要參與TAD內(nèi)部的染色質(zhì)互作，而CTCF主要參與它們之間的空間隔離。CTCF穩(wěn)定地綁定在染色質(zhì)上，并且決定了cohesin的定位從而維持邊界的穩(wěn)定。如果沒有CTCF，cohesin將不能準(zhǔn)確定位，會形成跨越邊界的非特異性互作。當(dāng)cohesin被降解后，所有的loop域(同一條染色體上的兩個(gè)位點(diǎn)之間具有CTCF和cohesin蛋白綁定的區(qū)域)都消失了，但com-partment域(具有相似組蛋白修飾的間隔區(qū)域)或組蛋白標(biāo)簽并不會受到影響[33]。Loop域的缺失并不會導(dǎo)致廣泛的基因異常表達(dá)，但確實(shí)會顯著影響小部分基因的表達(dá)活性。Schwarzer等[34]也發(fā)現(xiàn)，TAD的形成依賴于cohesin，而compartment域卻不受影響。在對染色質(zhì)結(jié)構(gòu)的進(jìn)化分析中發(fā)現(xiàn)，CTCF和cohesin對于驅(qū)動染色質(zhì)結(jié)構(gòu)的改變也起著直接的作用[35]。也有部分研究發(fā)現(xiàn)，將CTCF或coh-esin進(jìn)行功能性敲除或敲低雖然能夠引起局部互作的缺失和基因表達(dá)紊亂，但compartment或TAD在敲除之后仍然得以保留[36,37]。Barutcu等[38]也發(fā)現(xiàn)，敲除或者插入(X染色體中存在15個(gè)CTCF位點(diǎn)的保守區(qū)域)序列片段都不足以以特定性別或等位基因的方式改變活性X染色體中的TAD邊界。這可能是由于在這些實(shí)驗(yàn)中對CTCF或cohesin的敲除并不完全或所用細(xì)胞類型的差異所致，或者是除了CTCF/cohesin結(jié)合外，可能還存在著其他的機(jī)制調(diào)控TAD的形成。

最近對果蠅的研究發(fā)現(xiàn)，TAD并不是由CTCF和cohesin所定義。通過超高深度Hi-C技術(shù)方法，研究人員發(fā)現(xiàn)果蠅基因組中TAD的實(shí)際數(shù)目是現(xiàn)有注釋的10倍，而且整個(gè)基因組全部為TAD所覆蓋，并且果蠅染色質(zhì)中絕大多數(shù)的TAD邊界都是由特異性的絕緣子蛋白復(fù)合物BEAF-32/CP190或BEAF-32/ Chromator所定義，而不是與人同源的CTCF/coh-esin[39]?，F(xiàn)有證據(jù)表明，BEAF-32是果蠅中特異性結(jié)合DNA的絕緣蛋白之一，而CP190/Chromator恰好可與BEAF-32結(jié)合并介導(dǎo)遠(yuǎn)距離相互作用，類似于哺乳動物細(xì)胞中的cohesin。這些結(jié)果表明，雖然CTCF/cohesin在果蠅中并不參與TAD的形成，但是，與其功能相似但不同源的蛋白復(fù)合物起到了哺乳動物細(xì)胞中CTCF/cohesin相同的作用。此外，除了CTCF和cohesin，DNA超螺旋在TAD的形成可能也發(fā)揮著作用[40]，而超螺旋結(jié)構(gòu)域的邊界在位置上與TAD邊界確實(shí)有著部分的重疊區(qū)域[41]，但這種因素是否真正影響了TAD結(jié)構(gòu)的形成，則需要進(jìn)一步的實(shí)驗(yàn)驗(yàn)證。

TAD是基因組的基本特性，其結(jié)構(gòu)的完整性是基因調(diào)控所必須的。刪除TAD的邊界片段會使基因調(diào)控陷入混亂，原本沉默的基因開始表達(dá)，而原本表達(dá)的基因被沉默。研究發(fā)現(xiàn)，在癌癥病人中，TAD邊界區(qū)域往往與大量的超級增強(qiáng)子的位點(diǎn)相重合，說明其穩(wěn)定性與癌癥的發(fā)生密切相關(guān)[42]。2015年，首次報(bào)道了TAD與遺傳學(xué)疾病的關(guān)聯(lián)[43]。德國馬克斯普朗克分子遺傳學(xué)研究所和柏林夏洛蒂醫(yī)科大學(xué)的科學(xué)家運(yùn)用最新的基因組編輯技術(shù)CRISPR/ Cas成功將調(diào)控3種人類罕見疾病(短指癥、多趾畸形和并趾)的基因所在的TAD邊界破壞，使得小鼠模型產(chǎn)生相應(yīng)遺傳疾病表型。在小鼠的肢體組織和患者的成纖維細(xì)胞中，與疾病有關(guān)的染色質(zhì)結(jié)構(gòu)改變使啟動子和非編碼DNA出現(xiàn)異?；プ?。在野生型小鼠中，基因的增強(qiáng)子正常激活其自身表達(dá)，而在3種患病的小鼠模型中，由于DNA結(jié)構(gòu)變異，的增強(qiáng)子分別錯誤地激活了以及基因，使其發(fā)生異位表達(dá)，從而產(chǎn)生短指、多趾畸形和并趾的疾病表型；進(jìn)一步研究表明，只有在CTCF相關(guān)的TAD邊界區(qū)域被破壞時(shí), 才會出現(xiàn)這種問題。該研究證實(shí)了TAD結(jié)構(gòu)的破壞會導(dǎo)致遠(yuǎn)距離調(diào)控元件的重排，使得增強(qiáng)子會作用于錯誤的靶基因而引起異位表達(dá)，導(dǎo)致致病表型。這項(xiàng)研究證明了TAD功能的重要性，人們可以在此基礎(chǔ)上預(yù)測人類結(jié)構(gòu)變異的致病性，尤其是在基因組的非編碼區(qū)域。該研究對基因組變化引起疾病的機(jī)制提出了新的見解。

1.4 染色質(zhì)環(huán)

在哺乳動物細(xì)胞核內(nèi)，由于染色質(zhì)濃縮聚合的性質(zhì)，導(dǎo)致基因纖維上兩個(gè)遠(yuǎn)端位點(diǎn)會產(chǎn)生隨機(jī)碰撞而以較低的頻率相互作用。然而，在某些確定的基因位點(diǎn)間，這種遠(yuǎn)距離互作的頻率卻顯著高于預(yù)測值。在sub-Mb分辨率的Hi-C互作圖譜中，這些遠(yuǎn)距離互作的位點(diǎn)大量存在，它們構(gòu)成了染色質(zhì)穩(wěn)定結(jié)構(gòu)的基礎(chǔ)，或直接參與轉(zhuǎn)錄等調(diào)控過程。因此，由基因位點(diǎn)的遠(yuǎn)距離互作而介導(dǎo)染色質(zhì)纖維折疊形成的環(huán)狀結(jié)構(gòu)，稱之為“Chromatin loop”，即染色質(zhì)環(huán)。2014年，美國Broad研究院Aiden教授團(tuán)隊(duì)通過超高分辨率的原位Hi-C方法(Hi-C, 1 kb)，詳細(xì)地展示了長達(dá)2米的人類基因組在直徑約10微米的細(xì)胞核內(nèi)的全部折疊方式[15]，獲得了人的類淋巴母細(xì)胞(GM12878)49億個(gè)染色質(zhì)互作信息，并首次列出了整個(gè)人類基因組上形成的9448個(gè)染色質(zhì)環(huán)(loop)。研究發(fā)現(xiàn)，這些loop的兩端通常連接著已知基因的啟動子和增強(qiáng)子，并且這些loop相關(guān)的啟動子所在基因具有更高的表達(dá)水平和更強(qiáng)的細(xì)胞特異性。因此，這些loop確實(shí)是啟動子-增強(qiáng)子的長距離互作所形成，并直接調(diào)控基因的表達(dá)。隨著技術(shù)的發(fā)展，越來越多的超高分辨率的Hi-C結(jié)果都先后揭示出不同細(xì)胞間由于長距離互作而形成的loop結(jié)構(gòu)[44~47]。盡管這些研究對于鑒定不同細(xì)胞的長距離互作的算法各有差異，但是都發(fā)現(xiàn)了一些loop互作共有的規(guī)律特征。首先，這些長距離互作通常發(fā)生在同一個(gè)TAD或者sub-TAD內(nèi)部，排除一些特異性的基因區(qū)域(如基因)外，基因組中發(fā)生極長距離的互作相對較少[48]；其次，活性的啟動子、增強(qiáng)子以及CTCF結(jié)合位點(diǎn)通常與長距離互作密切相關(guān)[46,49]。此外，除啟動子-增強(qiáng)子互作形成的loop結(jié)構(gòu)外，啟動子-啟動子以及增強(qiáng)子-增強(qiáng)子的互作也能形成復(fù)雜的loop網(wǎng)絡(luò)結(jié)構(gòu)[49,50]。

研究發(fā)現(xiàn)，38%的 loop與contact domain具有一致性，即形成loop的錨點(diǎn)通常位于domain的邊界區(qū)域，65%的loop的出現(xiàn)通常伴隨著domain的出現(xiàn)，因此這些domain被稱為loop domain。并且，這些臨近的loop通常具有傳遞性(transitivity)，即形成相鄰兩個(gè)loop的互作位點(diǎn)L1-L2和L2-L3，往往在L1-L3之間也會有l(wèi)oop的產(chǎn)生，說明這3個(gè)位點(diǎn)具有同一個(gè)空間位置。進(jìn)一步研究loop形成機(jī)制時(shí)發(fā)現(xiàn)，大部分的loop (peak)所涉及的兩個(gè)peak loci具有顯著的絕緣蛋白CTCF (86%)和cohesin的兩個(gè)亞基—RAD21 (86%)和SMC3 (87%)的富集，說明CTCF和cohesin參與loop結(jié)構(gòu)的形成[15]。隨后，Aiden教授團(tuán)隊(duì)對CTCF和cohesin參與loop結(jié)構(gòu)形成的機(jī)制進(jìn)行研究，并提出了CTCF/cohesin介導(dǎo)的環(huán)擠壓模型(Loop Extrusion Model)[51]。在這個(gè)模型中(圖4)，cohesin環(huán)在NIPBL裝載蛋白作用下形成cohesin復(fù)合物并結(jié)合到染色質(zhì)上，延DNA序列向相反的方向滑動，擠壓染色質(zhì)形成loop環(huán)，直到遇到綁定在CTCF motif序列的阻遏子CTCF蛋白，擠壓過程即被終止[51~53]。在環(huán)擠壓過程中，染色體結(jié)構(gòu)維持(stuctural maintenance of chromosomes, SMC)蛋白家族中的SMC1、SMC3以及RAD21參與形成cohesin的亞基結(jié)構(gòu)[54]。此外，部分cohesin環(huán)能夠在WAPL和PDS5蛋白作用下從擠壓過程中釋放[54]。研究還發(fā)現(xiàn)，由CTCF和cohesin介導(dǎo)形成的loop結(jié)構(gòu)在不同的細(xì)胞間具有穩(wěn)定的保守性，并以此劃分TAD以及sub-TAD[15,55,56]。此外，這些CTCF結(jié)合位點(diǎn)都具有收斂的CTCF模體序列，因而能夠解釋在所有的CTCF結(jié)合位點(diǎn)中只有一小部分參與domain邊界的界定[15,47,57]。

2 Hi-C技術(shù)在三維基因組學(xué)中的應(yīng)用

隨著Hi-C技術(shù)的不斷發(fā)展，Single cell Hi-C、Hi-C、Dnase Hi-C等一系列衍生技術(shù)相繼出現(xiàn)[22,58~61]。這些技術(shù)不僅能夠用來揭示哺乳動物細(xì)胞核內(nèi)染色質(zhì)空間構(gòu)象方式，闡明其折疊規(guī)律及其作用機(jī)制，在三維基因組學(xué)應(yīng)用方面也發(fā)揮著重要作用。因此，Hi-C及其衍生技術(shù)能夠用來輔助組裝基因組，構(gòu)建哺乳動物全基因組單倍型，比較不同細(xì)胞/物種間染色質(zhì)互作的差異及其介導(dǎo)的基因表達(dá)差異，探究機(jī)體發(fā)育規(guī)律以及復(fù)雜疾病的發(fā)病機(jī)制等。

圖4 CTCF/cohesin介導(dǎo)的環(huán)擠壓模型

2.1 輔助基因組組裝

Hi-C技術(shù)用于輔助基因組組裝是目前提高基因組組裝質(zhì)量的一種必要手段，具體是指在已經(jīng)完成基本組裝的基因組草圖(Draft genome)序列(Scaffolds/ Contigs)和染色體數(shù)目已知的前提下，利用Hi-C測序數(shù)據(jù)將Draft genome序列進(jìn)行不同染色體的群組劃分，并確定各序列在染色體上的順序和方向，使基因組組裝水平提升到染色體水平。其主要原理是染色體內(nèi)互作強(qiáng)度高于染色體間的互作，同一染色體上近距離互作強(qiáng)于遠(yuǎn)距離互作[62]。Hi-C輔助基因組組裝主要分為3步：(1) Cluster：將contigs或scaf-folds聚類到不同的染色體組；(2) Order：在每個(gè)染色體組中按順序排列contigs或scaffolds；(3) Orient：為每一個(gè)排好順序的相鄰的contigs或scaffolds確定方向。自2013年Burton等[62]首次利用Hi-C技術(shù)輔助組裝了人、小鼠及果蠅的基因組后，近年來，研究者相繼對擬南芥[63]、山羊()[64]、藜麥()[65]、埃及伊蚊()[66]、大麥(L)[67]以及甘蔗(L)[68]等動植物的基因組進(jìn)行Hi-C輔助組裝，為進(jìn)行更深入的基因組學(xué)研究奠定了基礎(chǔ)。

2.2 構(gòu)建全基因組單倍型

單倍型是存在于染色單體內(nèi)具有統(tǒng)計(jì)學(xué)關(guān)聯(lián)性的一類單核苷酸多態(tài)性(single nucleotide polymorp-hisms, SNPs)，這些進(jìn)行共同遺傳的多個(gè)基因座上等位基因的組合信息對人類遺傳、疾病風(fēng)險(xiǎn)預(yù)測以及農(nóng)業(yè)動植物經(jīng)濟(jì)性狀連鎖標(biāo)記等方面研究具有重要價(jià)值[69]。相比于傳統(tǒng)的單倍型分析技術(shù)對于DNA片段分析長度的限制，Hi-C技術(shù)能夠使其在全基因范圍內(nèi)進(jìn)行單倍型組裝，且檢測效率以及分析的準(zhǔn)確性都較高。早2013年，Bing Ren教授團(tuán)隊(duì)首次利用Hi-C技術(shù)對人細(xì)胞進(jìn)行了全基因組單倍型組裝，構(gòu)建了準(zhǔn)確率達(dá)98%的人的單倍型群體[70]。此后，越來越多的研究報(bào)道了Hi-C技術(shù)用于構(gòu)建基因組單倍型[14,71,72]。另外，研究者還開發(fā)了直接針對Hi-C測序數(shù)據(jù)的單倍型分析工具HapCUT2[73]。這些研究結(jié)果都說明Hi-C技術(shù)具有革命性的優(yōu)勢，能夠廣泛用于哺乳動物群體的單倍型構(gòu)建。

2.3 基因的表達(dá)調(diào)控

Hi-C技術(shù)除了可以進(jìn)行輔助組裝基因組分析外，還可對基因的表達(dá)調(diào)控以及基因功能進(jìn)行研究。染色質(zhì)互作的形成和功能對于細(xì)胞的命運(yùn)決定和分化等過程至關(guān)重要，在基因特異性表達(dá)調(diào)控中發(fā)揮重要作用。之前的研究表明，染色質(zhì)環(huán)(chromatin loop)的兩端通常連接著基因的啟動子和增強(qiáng)子，線性距離較遠(yuǎn)的增強(qiáng)子能夠通過loop結(jié)構(gòu)被募集到已知基因的啟動子區(qū)域，從而激活基因的轉(zhuǎn)錄。Mifsud等[74]通過高分辨捕獲Hi-C (Capture Hi-C, CHi-C)技術(shù)，構(gòu)建了兩種人類血細(xì)胞(GM12878和CD34+)中超過22 000個(gè)長距離的啟動子互作圖譜，鑒定了超過11 600 000個(gè)兩種細(xì)胞類型共有的互作，它們跨越啟動子和遠(yuǎn)端位點(diǎn)之間的數(shù)百個(gè)堿基；研究還發(fā)現(xiàn)，與疾病相關(guān)的SNPs位點(diǎn)明顯富集在基因的互作區(qū)域，暗示著遠(yuǎn)距離突變可能會破壞相關(guān)基因的表達(dá)調(diào)控而導(dǎo)致疾病的發(fā)生。Rubin等[75]利用CHi-C聯(lián)合ChIP-seq技術(shù)，在全基因組范圍內(nèi)研究了分離培養(yǎng)的人原代角質(zhì)細(xì)胞分化過程中增強(qiáng)子和啟動子的互作模式，確認(rèn)了兩種類型的啟動子-增強(qiáng)子互作：獲得型(gained)互作，在分化過程中增強(qiáng)，并與enhancer獲得H3K27ac活化標(biāo)記一致；穩(wěn)定型(stable)互作，在未分化細(xì)胞中已預(yù)先建立，enhancer有H3K27ac的標(biāo)記，并與黏連蛋白cohesin相關(guān)。但這兩種互作均未在多能性細(xì)胞中檢測到，表明這種譜系特異的染色質(zhì)構(gòu)象在組織的前體細(xì)胞中形成，并且在終末分化中重塑。Bonev等[76]對小鼠神經(jīng)細(xì)胞分化過程中的染色質(zhì)結(jié)構(gòu)進(jìn)行了超高分辨率的解析，發(fā)現(xiàn)基因的轉(zhuǎn)錄活動與染色質(zhì)的絕緣以及遠(yuǎn)距離互作相關(guān)，但dCas9介導(dǎo)的激活不足以重新形成TAD邊界；此外，在所有的細(xì)胞類型中，長距離互作主要發(fā)生在外顯子富集的gene body與激活基因間，且在神經(jīng)細(xì)胞分化過程中，活性TADs之間的互作變得不明顯，而非活動TADs之間的互作則越來越強(qiáng)，說明由分化引起的基因轉(zhuǎn)錄激活使得TADs的構(gòu)象發(fā)生變化。X染色體失活(X-chromosome inactivation, XCI)會引起X染色體結(jié)構(gòu)重塑，轉(zhuǎn)變成沉默的異染色質(zhì)[77]。在雌性哺乳動物發(fā)育中，X染色體失活由兩條X染色體中一條的非編碼RNA發(fā)生上調(diào)引起[78~80]。Giorgetti等[81]利用Hi-C技術(shù)解析了小鼠失活X染色體的結(jié)構(gòu)特征以及基因表達(dá)情況：在小鼠神經(jīng)前體細(xì)胞(NPCs)和胚胎干細(xì)胞中，失活的X染色體結(jié)構(gòu)重塑中和含有邊界發(fā)揮著重要作用，并且在失活的X染色體中，除了“逃脫”沉默的基因附近，其他位置失去了有活性和失活的compartment A/B以及TADs。

2.4 機(jī)體與細(xì)胞發(fā)育

2015年，Battulin等[82]對小鼠精細(xì)胞和胚胎成纖維細(xì)胞進(jìn)行Hi-C結(jié)果的比較，在1 Mb分辨率下，精細(xì)胞的compartment A/B與胚胎成纖維細(xì)胞具有高度相似性，這與之前報(bào)道的小鼠胚胎干細(xì)胞的三維基因組結(jié)構(gòu)相一致[17]。而當(dāng)研究人員將分辨率提高到40 kb時(shí)，彼此之間的TAD邊界出現(xiàn)差異，且在特定的基因座位點(diǎn)上，兩種細(xì)胞染色質(zhì)的互作差異顯著。與成纖維細(xì)胞相比，精子細(xì)胞的間期細(xì)胞核小10倍左右，其基因組高度濃縮的包裝形式導(dǎo)致了精子細(xì)胞染色質(zhì)遠(yuǎn)距離互作的富集，由此說明配子細(xì)胞的染色質(zhì)構(gòu)象與體細(xì)胞存在差異。目前的研究認(rèn)為，染色質(zhì)不同層級的構(gòu)象(如compartment A/B和TAD)在體細(xì)胞中是穩(wěn)定存在的保守結(jié)構(gòu)單元，但這種構(gòu)象是與生俱來還是從配子轉(zhuǎn)變?yōu)楹献拥脑缙谂咛グl(fā)育時(shí)期形成的，值得人們關(guān)注。之前由于細(xì)胞數(shù)量和實(shí)驗(yàn)手段的限制，染色體三維結(jié)構(gòu)在哺乳動物早期胚胎發(fā)育過程中的動態(tài)變化鮮為人知。近年來，隨著單細(xì)胞Hi-C (single-cell Hi-C)技術(shù)的運(yùn)用，研究者不僅能從普通Hi-C大量群體細(xì)胞中獲得平均數(shù)據(jù)評估染色質(zhì)折疊和潛在的互作，還能利用單細(xì)胞Hi-C技術(shù)分辨單個(gè)染色體的構(gòu)象模型，精確調(diào)控細(xì)胞的狀態(tài)和功能[58,83]。2017年，和“背靠背”發(fā)表研究論文，研究者們都發(fā)現(xiàn)哺乳動物染色體三維結(jié)構(gòu)在著床前胚胎發(fā)育過程中的動態(tài)重組過程[84,85]。研究結(jié)果顯示，精子保留經(jīng)典的染色質(zhì)高級結(jié)構(gòu)，包括TADs和compartments；相反，處于MⅡ期的卵子染色體呈現(xiàn)出一種均一性結(jié)構(gòu)，缺乏TADs和compartments結(jié)構(gòu)。染色體三維結(jié)構(gòu)在受精后首先呈現(xiàn)出一種極其松散的狀態(tài)，兩套親本基因組在空間上部分分離且染色體compartments不同，差異持續(xù)到8細(xì)胞期。在隨后的胚胎早期發(fā)育過程中，染色質(zhì)高級結(jié)構(gòu)逐步以親本特異的方式建立和成熟，并且不完全依賴于合子基因組的轉(zhuǎn)錄激活。Flyamer等[86]也發(fā)現(xiàn)，受精完成后，父源和母源染色質(zhì)需要在受精卵中進(jìn)行空間排布重組，并且此過程中父源和母源染色質(zhì)的重組方式不同。此外，Kaaij等[87]研究發(fā)現(xiàn)，在斑馬魚的早期胚胎發(fā)育過程中，因?yàn)槿狈献拥霓D(zhuǎn)錄活動，其基因組高度結(jié)構(gòu)化；當(dāng)合子基因組被激活后，斑馬魚染色體失去結(jié)構(gòu)特征，并且這些特征在隨后的發(fā)育過程中被重新建立。

染色質(zhì)重塑是調(diào)控基因時(shí)序性表達(dá)的重要環(huán)節(jié)，往往發(fā)生在衰老細(xì)胞中，并且衰老細(xì)胞核中會形成衰老相關(guān)異染色質(zhì)聚集(senescence-associated heter-ochromatic foci, SAHF)。Chandra等[88]利用Hi-C技術(shù)對衰老細(xì)胞和正常ES細(xì)胞的染色質(zhì)空間構(gòu)象進(jìn)行探究，發(fā)現(xiàn)與正常細(xì)胞相比，在衰老細(xì)胞的異染色質(zhì)中存在大量依賴于序列和核纖層蛋白的局部互作缺失，且衰老細(xì)胞中出現(xiàn)特有的異染色質(zhì)聚集，這可能是SAHF形成的中間產(chǎn)物。此外，另一項(xiàng)研究也發(fā)現(xiàn)，當(dāng)衰老發(fā)生時(shí)，染色質(zhì)重塑是由于CTCF簇的形成，導(dǎo)致loop的重組，并且HMGB2蛋白參與此過程[89]。

2.5 在疾病研究中的應(yīng)用

許多復(fù)雜疾病的發(fā)生往往與其組織細(xì)胞的三維基因組構(gòu)象改變密切相關(guān)。Won等[90]通過Hi-C技術(shù)構(gòu)建了人大腦皮質(zhì)的高分辨率3D圖譜，分析鑒定了數(shù)百個(gè)在人類譜系中已知的啟動子-增強(qiáng)子互作基因，并且將染色質(zhì)互作與GWAS研究中確定的精神分裂癥相關(guān)非編碼變異相結(jié)合，突出了多個(gè)候選精神分裂癥風(fēng)險(xiǎn)基因和相關(guān)通路，其中一個(gè)遠(yuǎn)端的精神分裂癥變異位點(diǎn)能夠調(diào)控風(fēng)險(xiǎn)基因的表達(dá)，支持其作為精神分裂癥易感基因的潛在作用。心力衰竭主要是由心肌細(xì)胞的生物化學(xué)變化引起，已有研究表明這一復(fù)雜的細(xì)胞功能障礙是基因表達(dá)改變的結(jié)果，受轉(zhuǎn)錄因子和染色質(zhì)重塑酶的影響[91~94]。Rosa-Garrido等[95]利用5 kb分辨率的全基因組捕獲Hi-C與DNA測序結(jié)合，對心臟特異性敲除CTCF的小鼠心肌細(xì)胞進(jìn)行了研究，發(fā)現(xiàn)CTCF元件缺失和心肌壓力超負(fù)荷能大幅減少loop結(jié)構(gòu)，重塑loop內(nèi)部互作，導(dǎo)致功能元件與啟動子區(qū)域的互作明顯減弱，從而擾亂基因的轉(zhuǎn)錄調(diào)控。Anene Nzelu等[96]也發(fā)現(xiàn)，患病小鼠與正常小鼠心室肌細(xì)胞的染色質(zhì)構(gòu)象單元compartments A/B的變化與基因的表達(dá)變化模式相關(guān)，通過對H3K27ac標(biāo)記的富集區(qū)域進(jìn)行分析，確定了細(xì)胞特異性基因表達(dá)的調(diào)控元件，并通過CRISPR敲除和基因座上游的一個(gè)調(diào)控區(qū)域，導(dǎo)致與其互作基因的表達(dá)下調(diào)。Loviglio等[97]通過Hi-C、FISH以及4C-seq技術(shù)確認(rèn)了染色體16p11.2上兩個(gè)拷貝數(shù)變異(copy number variants，CNV)傾向的區(qū)域：16p11.2遠(yuǎn)端BP2-BP3間220 kb區(qū)域和16p11.2近端BP4-BP5間600 kb區(qū)域影響染色質(zhì)的成環(huán)作用，并影響成環(huán)區(qū)域間所含基因的協(xié)調(diào)表達(dá)和調(diào)控，提示染色質(zhì)互作異常與孤獨(dú)癥譜系障礙(autism spectrum disorders)、肥胖/體重不足及巨頭/小頭畸形的表型相關(guān)，并且確認(rèn)了在基因組其他區(qū)域類似表型相關(guān)的順式及反式染色體互作，表明染色體互作圖譜可以揭示功能和臨床診斷相關(guān)的疾病易感基因。此外，也有研究報(bào)道利用Hi-C技術(shù)對類風(fēng)濕關(guān)節(jié)炎、Crohn氏病等自身免疫疾病的發(fā)病機(jī)制進(jìn)行探究，發(fā)現(xiàn)啟動子互作區(qū)域的異常與這些疾病的調(diào)控機(jī)制密切相關(guān)[44]。

與正常細(xì)胞相比，癌細(xì)胞由于遺傳以及表觀遺傳的改變，使得基因表達(dá)紊亂[98~101]；并且癌癥是一種以細(xì)胞核的主要形態(tài)變化為特征的疾病[102,103]。因此，染色質(zhì)空間構(gòu)象的變化與癌癥的發(fā)生密切相關(guān)(圖5)。Barutcu等[104]通過Hi-C技術(shù)對乳腺上皮細(xì)胞(MCF-10A)和乳腺癌細(xì)胞系(MCF-7)進(jìn)行分析，發(fā)現(xiàn)與MCF-10A細(xì)胞相比，MCF-7細(xì)胞中小而基因富集的16~22號染色體間的互作頻率更低，且兩種細(xì)胞的染色體內(nèi)部互作也區(qū)別明顯；此外，MCF-10A細(xì)胞在端粒區(qū)以及亞端粒區(qū)的互作要強(qiáng)于MCF-7細(xì)胞。Taberlay等[105]利用Hi-C技術(shù)對前列腺癌中包括拷貝數(shù)變異、遠(yuǎn)距離染色質(zhì)互作重塑以及非典型基因表達(dá)的染色體三維結(jié)構(gòu)破壞進(jìn)行了研究，發(fā)現(xiàn)癌細(xì)胞保留了將其基因組分割成Mb級別的TAD的能力，但由于附加的TAD邊界的建立，這些TAD比正常細(xì)胞中的TAD小，且很大一部分新的與癌癥相關(guān)的特異性TAD邊界發(fā)生在CNV變化的區(qū)域。此外，前列腺癌患者17p13.1的一個(gè)常見缺失導(dǎo)致了單個(gè)TAD分為兩個(gè)明顯更小的TAD，而TAD的改變伴隨著TAD內(nèi)新的腫瘤特異性染色質(zhì)互作的形成，并在啟動子、增強(qiáng)子以及絕緣子等調(diào)控元件上富集，引發(fā)基因表達(dá)的改變。此外，研究者還利用Hi-C技術(shù)探究了神經(jīng)母細(xì)胞瘤[106]、膠質(zhì)瘤[107]以及急性T淋巴細(xì)胞白血病[108]等惡性腫瘤的發(fā)病機(jī)制，旨在從三維基因組角度解析染色體空間構(gòu)象重組對于癌癥發(fā)生的重要作用。

圖5 癌癥中染色質(zhì)三維結(jié)構(gòu)的破壞導(dǎo)致基因異常表達(dá)

癌癥的發(fā)生往往伴隨著基因組變異。癌癥基因組的測序提供的第一個(gè)直接信息是體細(xì)胞基因組變異率如何在正常細(xì)胞與癌細(xì)胞之間變化[109~115]。研究發(fā)現(xiàn)，癌癥基因組中的突變率與染色質(zhì)折疊密切相關(guān)，在Mb尺度下，異染色質(zhì)相關(guān)的組蛋白修飾標(biāo)志物H3K9me3的單一特征水平可占到變異率的40%以上[116]。Litchfield等[117]聯(lián)合利用Hi-C與GWAS對睪丸生殖細(xì)胞腫瘤(testicular germ cell tumors, TGCTs)中的SNPs進(jìn)行鑒定，確認(rèn)了19個(gè)新的風(fēng)險(xiǎn)位點(diǎn)，并結(jié)合之前已發(fā)現(xiàn)的25個(gè)位點(diǎn)[118,119]，將總共44個(gè)風(fēng)險(xiǎn)位點(diǎn)與候選的致病基因進(jìn)行互作網(wǎng)絡(luò)分析，發(fā)現(xiàn)TGCTs的易感基礎(chǔ)是發(fā)育調(diào)控因子的大范圍紊亂導(dǎo)致的。Romanel等[120]利用Hi-C技術(shù)對引起前列腺癌的早發(fā)性體細(xì)胞變異的非編碼多態(tài)性調(diào)節(jié)元件7p14.3與其調(diào)控基因的結(jié)構(gòu)進(jìn)行了分析，表明7p14.3位點(diǎn)的多態(tài)性可能通過雄性激素依賴的DNA損失修復(fù)功能影響前列腺癌的致病傾向。此外，還有研究利用捕獲Hi-C技術(shù)對乳腺癌[121,122]、結(jié)直腸癌[123]等癌癥的風(fēng)險(xiǎn)位點(diǎn)進(jìn)行了鑒定，揭示出基因座中的重要遠(yuǎn)距離染色質(zhì)互作參與癌癥發(fā)生的致病機(jī)制。

Hi-C技術(shù)還能通過檢測癌癥病人原發(fā)性腫瘤樣本組織中平衡和非平衡的染色質(zhì)重塑，包括易位和反轉(zhuǎn)，以及獲得性CNV，預(yù)測癌癥的發(fā)生及預(yù)后，這將極大降低癌癥的檢測成本[124]。此外，Hi-C技術(shù)還用于鑒定經(jīng)福爾馬林固定的石蠟包埋腫瘤樣本的結(jié)構(gòu)變異，這種與Hi-C技術(shù)類似的高通量構(gòu)象捕獲技術(shù)被稱為“Fix-C”技術(shù)，能夠識別未被其他方法檢測到的新結(jié)構(gòu)變異，這種方法能夠在癌癥進(jìn)展期間從FFPE樣本中詳細(xì)解析全基因組重塑事件，為患者護(hù)理的目標(biāo)分子診斷提供信息[125]。

越來越多的研究表明，染色體的三維空間構(gòu)象紊亂正在成為癌癥等疾病發(fā)生過程中一種新的致病機(jī)制，以Hi-C技術(shù)為代表的三維基因組技術(shù)將極大地促進(jìn)復(fù)雜疾病以及癌癥的相關(guān)研究，通過解析病發(fā)細(xì)胞中基因與其調(diào)控元件之間互作的改變，找到新的致病位點(diǎn)，并為開發(fā)新的靶向治療藥物提供線索。

3 結(jié)語與展望

隨著時(shí)代的進(jìn)步以及技術(shù)的革新，基因組學(xué)的發(fā)展取得了長足的進(jìn)步，從“人類基因組計(jì)劃”(HGP)[126]到“人類基因組百科全書計(jì)劃”(ENCODE)[127]的順利完成，科研人員可以深入分析、解讀和注釋基因組序列信息和功能。但是，基因組DNA并不是在染色體上呈線性排列，其三維空間構(gòu)象對DNA復(fù)制、基因轉(zhuǎn)錄調(diào)控、染色質(zhì)濃縮和分離等基本生物學(xué)過程都有著不可或缺的重要作用。Hi-C技術(shù)的提出以及其大規(guī)模的運(yùn)用，使得人們可以從空間層面去揭示這些不同調(diào)控元件的互作關(guān)系，認(rèn)識染色質(zhì)構(gòu)象對基因表達(dá)調(diào)控的機(jī)制及作用。2015年，科學(xué)家開始正式實(shí)施一個(gè)全新的全球合作項(xiàng)目——“4D核體計(jì)劃”[128]，計(jì)劃用5年或者更長的時(shí)間從空間(三維)和時(shí)間(四維)角度來研究細(xì)胞核結(jié)構(gòu)形成原理，探索細(xì)胞核結(jié)構(gòu)對基因表達(dá)、細(xì)胞功能，以及對發(fā)育和疾病發(fā)生、發(fā)展的影響。因此，Hi-C技術(shù)以及三維基因組的全面發(fā)展，必然會為全面解讀人類基因組信息、攻克復(fù)雜疾病和促進(jìn)人類醫(yī)學(xué)進(jìn)步提供有力的支持。

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Advances in mammalian three-dimensional genome by using Hi-C technology approach

Chunyou Ning, Mengnan He, Qianzi Tang, Qing Zhu, Mingzhou Li, Diyan Li

Mammalian genomic DNA in the cell nucleus doesn’t exist in linear form but is highly folded and condensed into chromatin with a three-dimensional (3D) structure possessing a specific spatial structure and conformation. Hi-C, the high-throughput chromosome conformation capture technology, was first published in 2009, and it provides an in-depth view of 3D genomics. According to the size of DNA unit, the 3D hierarchical units of mammalian genome can be categorizedsequentially as chromosome territory (CT), chromatin compartment A/B, topological associated domain (TAD), and chromatin loop. These hierarchical structural units play vital roles in gene transcription and regulation. In this review, we summarize the 3D hierarchical division of chromosomes, the effects of hierarchical units and the applications of Hi-C technology in development and disease. This review is intended to provide insights for the further study of 3D genomics in mammals.

three dimensional (3D) genomics; chromatin spatial organization; Hi-C technology; gene transcriptional regulation

2018-11-21;

2019-01-23

國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(編號：2018YFD0500403)和國家自然科學(xué)基金項(xiàng)目(編號：31772576)資助[Supported by the National Key R&D Program of China (No. 2018YFD0500403) and the National Natural Science Foundation of China (No. 31772576)]

寧椿游，博士研究生，研究方向：動物遺傳育種與繁殖。E-mail: ningchunyou@hotmail.com

李地艷，博士，研究員，研究方向：功能基因組學(xué)研究。E-mail: diyanli@sicau.edu.cn

10.16288/j.yczz.18-317

2019/2/28 16:39:29

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190228.1639.002.html

(責(zé)任編委: 趙方慶)