鄧瑋杭，李鑫輝

MNase-seq與核小體定占位研究

鄧瑋杭，李鑫輝

上海交通大學(xué)生物醫(yī)學(xué)工程學(xué)院，上海 200240

核小體是染色質(zhì)復(fù)雜三維結(jié)構(gòu)的基本單位，它在基因組上的定位及占位在DNA轉(zhuǎn)錄、復(fù)制和修復(fù)等基礎(chǔ)生物過(guò)程中發(fā)揮重要功能。在眾多核小體定占位研究技術(shù)中，微球菌核酸酶測(cè)序(micrococcal nuclease sequencing, MNase-seq)被認(rèn)為是目前最為高效的方法，因此應(yīng)用十分廣泛。研究人員利用該技術(shù)繪制了多種生物的核小體圖譜，并揭示了核小體組織特點(diǎn)的共性和差異。本文介紹了MNase-seq的技術(shù)原理以及在解析核小體組織及其功能中的應(yīng)用，總結(jié)了在染色質(zhì)構(gòu)象這一快速發(fā)展領(lǐng)域中的研究進(jìn)展，并展望了染色質(zhì)生物學(xué)的未來(lái)發(fā)展方向。由MNase-seq揭示的核小體組織結(jié)構(gòu)為基因表達(dá)和發(fā)育調(diào)控提供了新的見(jiàn)解，也有助于人們理解疾病的發(fā)生過(guò)程。

核小體；染色質(zhì)結(jié)構(gòu)；染色質(zhì)重塑；下一代測(cè)序技術(shù)；微球菌核酸酶

構(gòu)建人類細(xì)胞的一個(gè)關(guān)鍵步驟是將近2 m的DNA組裝成染色質(zhì)并包裝進(jìn)直徑10 μm的細(xì)胞核內(nèi)，而核小體作為染色質(zhì)組裝的基本單位，一般是由長(zhǎng)度為145~147 bp的DNA以左螺旋的方式纏繞組蛋白八聚體接近兩圈構(gòu)成的[1,2]。其中組蛋白八聚體由2個(gè)拷貝的4種核心組蛋白H2A、H2B、H3和H4組成，H1作為連接組蛋白。在基因組的大部分區(qū)域都有核小體覆蓋，約75%~90%的基因組DNA包裹在核小體中[3]。核小體定位、占位以及組蛋白修飾等影響著啟動(dòng)子、轉(zhuǎn)錄起始位點(diǎn)的建立以及染色質(zhì)高級(jí)結(jié)構(gòu)的組裝，參與基因轉(zhuǎn)錄、DNA復(fù)制和修復(fù)過(guò)程。核小體動(dòng)態(tài)組織通過(guò)改變?nèi)旧|(zhì)結(jié)構(gòu)的方式影響細(xì)胞功能，因此與生物體的基因調(diào)控、發(fā)育分化以及細(xì)胞應(yīng)激過(guò)程密切相關(guān)。

應(yīng)用MNase酶切配合下一代測(cè)序技術(shù)，人們加快了對(duì)核小體組織及染色質(zhì)結(jié)構(gòu)的研究，有望更深入了解基因調(diào)控模式以及染色質(zhì)功能。本文主要介紹了微球菌核酸酶測(cè)序(micrococcal nuclease seque-ncing, MNase-seq)的技術(shù)原理和相關(guān)衍生技術(shù)，總結(jié)了MNase-seq等技術(shù)在揭示基因組上核小體的組織特點(diǎn)中的作用，綜述了近年來(lái)多種生物細(xì)胞內(nèi)核小體圖譜的研究進(jìn)展，以期為今后以核小體組織為核心的染色質(zhì)構(gòu)象等研究提供參考。

1 MNase-seq技術(shù)原理及其發(fā)展

1.1 MNase-seq技術(shù)原理

利用微球菌核酸酶切割染色質(zhì)纖維，回收DNA并配合下一代測(cè)序技術(shù)來(lái)繪制核小體定位圖譜，稱作MNase-seq。盡管MNase-seq在近10年來(lái)才得以飛速發(fā)展，但早在20世紀(jì)70年代，研究人員就開(kāi)始利用MNase消化染色質(zhì)并研究其結(jié)構(gòu)[4,5]。MNase來(lái)源于金黃色葡萄球菌()，同時(shí)具有核酸內(nèi)切酶與外切酶活性。核小體間的連接DNA(linker DNA)對(duì)MNase的敏感性要比核小體DNA高25倍[6]，根據(jù)酶的這一特性，在對(duì)染色質(zhì)DNA進(jìn)行充分消化后，大部分核小體間的裸露DNA都被消化，由此可以富集到單核小體DNA片段。

MNase-seq的主要步驟包括：(1)提取細(xì)胞核；(2)用MNase對(duì)染色質(zhì)進(jìn)行酶切；(3)終止酶切、去除RNA及蛋白雜質(zhì)；(4)分離提純DNA片段；(5)文庫(kù)構(gòu)建；(6)上機(jī)測(cè)序。在MNase-seq的實(shí)驗(yàn)設(shè)計(jì)中，染色質(zhì)交聯(lián)程度以及MNase的酶切水平很關(guān)鍵。不少實(shí)驗(yàn)利用甲醛做交聯(lián)劑(crosslinking agent)，在細(xì)胞活體條件下對(duì)蛋白質(zhì)-DNA相互作用進(jìn)行固定，以免在染色質(zhì)制備過(guò)程中丟失這些相互作用及破壞核小體組織結(jié)構(gòu)[7]。也有研究者用MNase直接處理未固定的染色質(zhì)[8~10]，該方法減少了組蛋白以外的蛋白質(zhì)-DNA相互作用帶來(lái)的實(shí)驗(yàn)誤差。盡管該方法可能導(dǎo)致核小體結(jié)構(gòu)的改變，但研究發(fā)現(xiàn)是否使用甲醛固定對(duì)核小體的組織改變不大[11]。使用中等或較高水平的酶切使得酶切產(chǎn)物中80%~100%為單核小體片段，有利于減少建庫(kù)過(guò)程中片段篩選帶來(lái)的額外技術(shù)誤差。MNase酶切后進(jìn)行電泳分析，150 bp左右的條帶則是單個(gè)的完整核小體DNA。

MNase-seq的優(yōu)點(diǎn)在于技術(shù)難度較低，具有較高的分辨率，且數(shù)據(jù)處理相對(duì)簡(jiǎn)單。較高的分辨率得益于MNase的酶切特性，MNase處理染色質(zhì)可以高效去除連接DNA，得到的DNA片段末端正是包裹組蛋白的DNA兩端。相比用超聲打斷DNA的常規(guī)方法，MNase處理DNA可以獲得長(zhǎng)度較均一的DNA片段，從而得到核小體更為精確的位置坐標(biāo)。然而，由于不同DNA片段對(duì)MNase酶切的敏感性不同，這一酶切效率的差異使得MNase-seq測(cè)序片段的末端不能準(zhǔn)確反映核小體邊緣的位置，因此測(cè)序數(shù)據(jù)的處理分析尤為關(guān)鍵。MNase-seq數(shù)據(jù)處理過(guò)程主要為數(shù)據(jù)預(yù)處理及質(zhì)量控制、序列比對(duì)、核小體定位與占位分析以及數(shù)據(jù)的可視化[12]。在序列比對(duì)后，研究者們先后運(yùn)用多種算法(如iNPS[13]、DiNuP[14]等)分析MNase-seq測(cè)序數(shù)據(jù)，解析全基因組核小體定位精確圖譜或?qū)Σ町惢嘉缓诵◇w進(jìn)行分析。其中，Chen等[13]建立了iNPS算法，該算法在NPS（nucleosome positioning from sequencing）的基礎(chǔ)上增加了“核小體邊界信號(hào)調(diào)整”與“相鄰核小體合并或分離”步驟，它比通用的NPS算法識(shí)別核小體邊界信號(hào)的能力更強(qiáng)，因此可以多檢測(cè)到約60%的核小體。該算法具有更高的檢測(cè)準(zhǔn)確性和穩(wěn)健性，因此有利于下游數(shù)據(jù)分析。核小體定位的理論預(yù)測(cè)同樣具有較好的研究前景，研究者們利用核小體DNA/連接DNA的序列特性、堿基二聯(lián)體周期信號(hào)等建立數(shù)學(xué)模型(如Segal模型[3]、N-Score模型[15]等)對(duì)核小體定位進(jìn)行預(yù)測(cè)。近年來(lái)，MNase-seq測(cè)序數(shù)據(jù)分析方法的進(jìn)步使得該技術(shù)日趨成熟，模型的改進(jìn)與優(yōu)化使得人們對(duì)核小體定位預(yù)測(cè)的準(zhǔn)確性不斷提高，MNase-seq技術(shù)目前已廣泛應(yīng)用于各種研究場(chǎng)景。

MNase-seq具有的這些優(yōu)點(diǎn)使其成為檢測(cè)核小體分布的優(yōu)越的方法。目前，MNase-seq已被應(yīng)用于釀酒酵母()、果蠅()、人類()以及多種模式生物體內(nèi)的染色質(zhì)結(jié)構(gòu)研究。另一方面，通過(guò)MNase-seq測(cè)量染色質(zhì)被核小體及其他調(diào)控因子的占有水平，可以間接揭示染色質(zhì)可及性、發(fā)現(xiàn)潛在基因調(diào)控位點(diǎn)[7,12]。

1.2 MNase-seq衍生技術(shù)及相關(guān)技術(shù)

目前，以MNase-seq技術(shù)為基礎(chǔ)，發(fā)展出了一系列衍生技術(shù)(圖1)，例如(1) MNase-ChIP-seq[16]，被用于對(duì)特定調(diào)控因子、組蛋白修飾或變體的檢測(cè)；(2) MNase-Exo-seq，在酶切體系中加入核酸外切酶Ⅲ來(lái)彌補(bǔ)MNase的外切酶活性，從而更高程度地切割到核小體核心區(qū)域(core particle)，以獲得更精準(zhǔn)的核小體定位[17]；(3) MACC-seq (MNase accessibility sequencing)，同時(shí)對(duì)整個(gè)基因組上的核小體位置及

其可及性進(jìn)行測(cè)量，探究核小體占位與染色質(zhì)可及性的關(guān)系[18]；(4) MH-seq，識(shí)別MNase高敏位點(diǎn)(MNase hypersensitive sites, MHSs)，利用MNase可以檢測(cè)到DNase I或Tn5無(wú)法訪問(wèn)到的開(kāi)放染色質(zhì)區(qū)域[19]；(5) Array-seq，使用低濃度MNase酶切，用于檢測(cè)核小體陣列的規(guī)律性(regularity)及核小體間距(nucleosome spacing)[20]；(6) CUT&RUN (cleavage under targets and release using nuclease)技術(shù)，MNase被靶向作用于特定的組蛋白修飾位點(diǎn)或轉(zhuǎn)錄因子等蛋白結(jié)合位點(diǎn)[21]。

除MNase-seq及其衍生技術(shù)以外，染色質(zhì)免疫共沉淀測(cè)序技術(shù)(chromatin immunoprecipitation se-quencing, ChIP-seq)、染色質(zhì)開(kāi)放性測(cè)序技術(shù)(assay for transposase-accessible chromatin with high throu-ghput sequencing, ATAC-seq)、DNase I超敏感位點(diǎn)測(cè)序(DNase I hypersensitive site sequencing, DNase-seq)以及核小體占位及甲基化測(cè)序(nucleosome occupancy and methylome sequencing, NOMe-seq)等技術(shù)也在解析核小體定位及染色質(zhì)結(jié)構(gòu)及功能的研究中起到重要作用。表1對(duì)這些技術(shù)進(jìn)行了總結(jié)比較。

1.3 少量細(xì)胞及單細(xì)胞MNase-seq技術(shù)

多年來(lái)，大量細(xì)胞MNase-seq技術(shù)研究得較為充分并得到了廣泛應(yīng)用，但該方法存在兩個(gè)明顯的缺陷：首先，在研究臨床樣本等珍貴樣本時(shí)，收集足夠多的細(xì)胞存在困難；另外，該方法只能得到細(xì)胞群體中核小體定位的平均水平，而盡管在同源細(xì)胞群體中，細(xì)胞間的染色質(zhì)狀態(tài)仍存在明顯的異質(zhì)性，因此不能反映單個(gè)細(xì)胞內(nèi)真實(shí)的核小體定位狀態(tài)。隨著下一代測(cè)序技術(shù)的不斷進(jìn)步以及單細(xì)胞測(cè)序技術(shù)的發(fā)展，MNase-seq技術(shù)也更新迭代，少量細(xì)胞起始的MNase-seq及其衍生技術(shù)、單細(xì)胞MNase-seq技術(shù)[35](single-cell MNase-seq, scMNase- seq)日趨成熟，在近年來(lái)得到重要突破。少量細(xì)胞甚至單細(xì)胞起始的MNase-seq技術(shù)有助于研究胚胎發(fā)育或疾病發(fā)展中至關(guān)重要但稀少珍貴的細(xì)胞。

圖1 MNase-seq及其衍生技術(shù)

A: MNase-seq; B: Array-seq; C: MNase-ChIP-seq; D: CUT&RUN。

表1 研究核小體、染色質(zhì)結(jié)構(gòu)的常用技術(shù)

Mattia等[36]發(fā)展了少量細(xì)胞MACC-seq，最低50個(gè)細(xì)胞的投入量即可達(dá)到和大量細(xì)胞MACC相似的可信結(jié)果，擴(kuò)大了MACC技術(shù)的應(yīng)用范圍。2018年，Peter等[37]改進(jìn)了CUT&RUN技術(shù)，利用該技術(shù)方法對(duì)組蛋白修飾進(jìn)行測(cè)定，最低只需要投入約100個(gè)細(xì)胞。2019年，Sarah等[38]實(shí)現(xiàn)了在單個(gè)細(xì)胞中利用uliCUT&RUN (Ultra-low input CUT&RUN)技術(shù)對(duì)轉(zhuǎn)錄因子結(jié)合位點(diǎn)進(jìn)行分析，揭示了轉(zhuǎn)錄因子在胚胎干細(xì)胞中結(jié)合位點(diǎn)的多樣性。2018年，Lai等[39]開(kāi)發(fā)了scMNase-seq技術(shù)，實(shí)驗(yàn)流程主要包括：(1)流式分選收集單個(gè)細(xì)胞；(2)裂解細(xì)胞、MNase酶切消化染色質(zhì)；(3)分離提純DNA；(4)酶切末端補(bǔ)平、連接測(cè)序接頭、PCR擴(kuò)增約25個(gè)循環(huán)；(6)篩選合適長(zhǎng)度的片段(150~320 bp)上機(jī)測(cè)序。單個(gè)細(xì)胞含有的DNA量極少，因此scMNase-seq的技術(shù)要點(diǎn)在于最小化DNA損失。為達(dá)到這一目標(biāo)，實(shí)驗(yàn)細(xì)節(jié)的優(yōu)化包括在DNA提純過(guò)程中加入環(huán)狀質(zhì)粒使得DNA顆粒容易可見(jiàn)，以及在建庫(kù)流程中不預(yù)先分離單核小體長(zhǎng)度的DNA片段。他們利用該技術(shù)同時(shí)對(duì)NIH3T3細(xì)胞、小鼠（）胚胎干細(xì)胞及小鼠幼稚CD4+幼稚T細(xì)胞的全基因組核小體定位及染色質(zhì)可及性進(jìn)行研究。相比于大量細(xì)胞MNase-seq對(duì)核小體間距的研究只限于核小體定位精準(zhǔn)的區(qū)域(例如TSS附近)，scMNase-seq技術(shù)可以對(duì)單個(gè)細(xì)胞全基因組范圍的核小體間距模式進(jìn)行測(cè)量[40]。該研究表明，在異染色質(zhì)區(qū)域，相鄰核小體以約180 bp的間距規(guī)律排列，但在不同細(xì)胞中核小體的定位不一致；在活躍染色質(zhì)區(qū)域，相鄰核小體間的間隔存在差異，然而核小體的定位在不同細(xì)胞中趨于一致；在TH1增強(qiáng)子處，不同CD4+幼稚T細(xì)胞顯示不同的核小體缺失程度，在TH1增強(qiáng)子處缺失程度高的細(xì)胞具有更高TH1分化的潛能。

2 MNase-seq技術(shù)解析核小體組織特點(diǎn)及內(nèi)在功能

2.1 核小體組織特點(diǎn)及影響因素

核小體覆蓋了可測(cè)序基因組的大部分區(qū)域，整體而言，核小體重復(fù)地、有規(guī)律地出現(xiàn)在基因組上。釀酒酵母基因組中，核小體以約165 bp的間隔重復(fù)出現(xiàn)[41]，相鄰核小體由10~50 bp的連接DNA(linker DNA)相連[42~45]。然而，核小體在基因組上的組織具有異質(zhì)性，導(dǎo)致不同的染色質(zhì)折疊方式。核小體在異染色質(zhì)區(qū)域覆蓋率高，形成了緊湊封閉的染色質(zhì)結(jié)構(gòu)；然而在增強(qiáng)子、絕緣子等調(diào)控區(qū)域常表現(xiàn)為核小體缺失，形成開(kāi)放可及的染色質(zhì)[41]。并且，核小體在基因組上的組織不是固定不變的，具有定位和結(jié)構(gòu)上的動(dòng)態(tài)性[46]。核小體定位的動(dòng)態(tài)性體現(xiàn)在核小體可以在DNA上滑動(dòng)，且容易自發(fā)發(fā)生全部或部分解聚的動(dòng)態(tài)變化。在生理?xiàng)l件下，核小體易受染色質(zhì)重塑復(fù)合物(chromatin remodelers)、分子伴侶、聚合酶、轉(zhuǎn)錄因子等調(diào)控因子的影響而發(fā)生重定位；全局轉(zhuǎn)錄水平的變化以及外界環(huán)境刺激也會(huì)影響核小體的定位。核小體結(jié)構(gòu)的動(dòng)態(tài)性體現(xiàn)在組蛋白的翻譯后修飾(post-translational modifications, PTM)[47,48]，以及組蛋白變體[49,50]和非標(biāo)準(zhǔn)核小體的形成[51]等。

研究人員使用核小體定位(nucleosome position-ning)及占位(nucleosome occupancy)來(lái)描述核小體的組織狀態(tài)。核小體定位指核小體出現(xiàn)在基因組特定位置相對(duì)于其周邊的概率，反映核小體對(duì)特定DNA序列選擇的特性；核小體占位是指在基因組特定區(qū)域出現(xiàn)的核小體平均數(shù)目，體現(xiàn)了核小體密度。核小體的定位和占位具有堿基偏好性，通常，富含G、C堿基的DNA序列更有利于DNA與核小體的緊密結(jié)合，而高A、T含量DNA序列削弱了DNA與組蛋白的相互作用，連續(xù)出現(xiàn)的A堿基區(qū)域(AAAAA)在體內(nèi)和體外實(shí)驗(yàn)中都表現(xiàn)出最低的核小體占有率[52]。Albert等[53]利用MNase-ChIP-seq研究釀酒酵母核小體組織時(shí)發(fā)現(xiàn)并定義了DNA序列影響核小體定位的旋轉(zhuǎn)特性(rotational setting)和平移特性(translational setting)。其中旋轉(zhuǎn)特性與規(guī)律排列的二核苷酸有關(guān)，以10 bp為周期連續(xù)出現(xiàn)的AA/AT/ TA/TT二核苷酸以及相位相差5 bp的GG/CC/GC/ CG二核苷酸序列交錯(cuò)出現(xiàn)，塑造了DNA的急劇彎曲特性，有利于DNA對(duì)組蛋白八聚體的纏繞，從而具有強(qiáng)烈的核小體定位特性。

目前普遍認(rèn)為DNA序列對(duì)于核小體在基因組上的組織起到主要作用，僅通過(guò)核小體對(duì)DNA的序列偏好可以解釋體內(nèi)核小體組織形態(tài)的50%~60%[3]。Kaplan等[52]利用MNase-seq對(duì)培養(yǎng)于3種不同培養(yǎng)基的釀酒酵母體內(nèi)的核小體進(jìn)行測(cè)定，它們的核小體圖譜顯示出較高的相似性，表明盡管存在環(huán)境差異，DNA序列仍是影響核小體組織的最關(guān)鍵因素。根據(jù)核小體定位的序列偏好性，研究人員根據(jù)DNA序列及體外合成的核小體數(shù)據(jù)建立計(jì)算模型，對(duì)生理?xiàng)l件下核小體組織形式進(jìn)行預(yù)測(cè)[3,54]。在基因組上不同區(qū)域，DNA序列與核小體占位的相關(guān)性不同。在啟動(dòng)子區(qū)域，DNA序列與體內(nèi)核小體占位的相關(guān)系數(shù)較非啟動(dòng)子區(qū)域更低。由此說(shuō)明，除了DNA序列的作用，核小體在生物體內(nèi)的組織形態(tài)受到眾多細(xì)胞內(nèi)調(diào)控因子及轉(zhuǎn)錄水平的影響。

以染色質(zhì)重塑復(fù)合物為主的眾多染色質(zhì)調(diào)節(jié)因子也影響著核小體在基因組上的定位。染色質(zhì)重塑復(fù)合物是一種依賴ATP的酶類，它們可以越過(guò)核小體對(duì)DNA序列的內(nèi)在偏好性，利用ATP水解的能量移除、移動(dòng)或并入組蛋白來(lái)改變核小體的定位及構(gòu)象。在促進(jìn)轉(zhuǎn)錄因子與DNA的結(jié)合[55]、順式作用元件構(gòu)象建成及DNA復(fù)制、轉(zhuǎn)錄激活等過(guò)程中發(fā)揮重要作用[56]。SWI/SNF (switch/sucrose non- fermenting)復(fù)合物是研究得較為全面的染色質(zhì)重塑復(fù)合物，它們通常富集在轉(zhuǎn)錄起始位點(diǎn)、復(fù)制起點(diǎn)中核小體缺失位點(diǎn)(nucleosome-free region, NFR)的–1核小體處[57]，主要通過(guò)促進(jìn)核小體在DNA上的移動(dòng)或移除來(lái)發(fā)揮功能，參與釀酒酵母的應(yīng)激反應(yīng)[58]。近年來(lái)，SWI/SNF的作用機(jī)制被研究得更為透徹，研究表明，在黑色素細(xì)胞的分化中，SWI/SNF亞基BAF60A促進(jìn)色素基因及啟動(dòng)子對(duì)SWI/ SNF亞基BRG1的募集，從而促進(jìn)染色質(zhì)重塑與細(xì)胞分化[59]。ISWI(imitation SWI)復(fù)合物屬于SWI蛋白家族，它能協(xié)助染色質(zhì)的組裝和組織[60,61]，幫助具有規(guī)律間隔的核小體序列的建成[62]。CHD (chro-modomain-helicase-DNA-binding)也屬于SWI蛋白家族，它與ISWI共同決定了基因組上核小體間距(nucleosome spacing)的全局特征[63]。

2.2 核小體缺失位點(diǎn)的結(jié)構(gòu)和功能

在利用下一代測(cè)序技術(shù)研究核小體組織之前，研究者們利用MNase切割染色質(zhì)纖維并配合基因芯片技術(shù)(MNase-chip)，在釀酒酵母的核小體組織上有了開(kāi)創(chuàng)性的發(fā)現(xiàn)，即核小體在啟動(dòng)子區(qū)域普遍顯示出缺失特性[64~67]，這些區(qū)域被命名為核小體缺失位點(diǎn)。NFR也被發(fā)現(xiàn)存在于活躍增強(qiáng)子區(qū)域、復(fù)制起點(diǎn)以及轉(zhuǎn)錄因子結(jié)合位點(diǎn)[67]。核小體的缺失增加了該位點(diǎn)的染色質(zhì)可及性、具有更強(qiáng)的調(diào)控潛力，利于眾多反式作用因子正確發(fā)揮功能，包括染色質(zhì)調(diào)控因子、轉(zhuǎn)錄因子、復(fù)制和轉(zhuǎn)錄需要的酶類等[68]。

根據(jù)染色質(zhì)的可及性可將啟動(dòng)子分為兩種類型：開(kāi)放啟動(dòng)子(open promoter)和封閉啟動(dòng)子(covered promoter)[62]。開(kāi)放啟動(dòng)子具有開(kāi)放的染色質(zhì)狀態(tài)，在起始密碼子上游200 bp左右具有一段NFR，NFR內(nèi)具有暴露的轉(zhuǎn)錄激活因子結(jié)合位點(diǎn)；而封閉啟動(dòng)子具有較高的核小體占位，轉(zhuǎn)錄激活因子需要在染色質(zhì)重塑復(fù)合物的幫助下與核小體競(jìng)爭(zhēng)結(jié)合位點(diǎn)才能開(kāi)始轉(zhuǎn)錄。NFR區(qū)域上下游出現(xiàn)的第一個(gè)核小體被命名為–1/+1核小體，分別標(biāo)定了NFR的上游/下游邊界[46]。+1核小體定位在轉(zhuǎn)錄起始位點(diǎn)(trans-cription start site, TSS)下游的固定距離，它所結(jié)合的DNA序列具有很強(qiáng)的核小體定位特性。利用MNase- seq對(duì)不同物種啟動(dòng)子的核小體結(jié)構(gòu)進(jìn)行測(cè)量時(shí)發(fā)現(xiàn)，+1核小體的位置具有物種特異性。在酵母中，+1核小體與TSS在位置上存在重疊[69]，而在果蠅的基因啟動(dòng)子處，+1核小體通常位于TSS下游約135 bp處，因此具有比酵母更長(zhǎng)的NFR[42](圖2)。在表達(dá)基因TSS的+1核小體下游方向，形成了規(guī)律間隔、定位精準(zhǔn)且相位統(tǒng)一的核小體陣列(nucleosome phasing)，這一陣列大約向基因內(nèi)部延伸1000 bp左右，規(guī)律排列的特性隨著與TSS間距離的增大而減弱[46]。Dustin等[44]在人類CD4+T淋巴細(xì)胞中發(fā)現(xiàn)，+1核小體下游相位統(tǒng)一的核小體陣列在不表達(dá)的基因中不存在。并且，將啟動(dòng)子區(qū)域核小體排列與RNA聚合酶Ⅱ的ChIP-seq數(shù)據(jù)比對(duì)發(fā)現(xiàn)，啟動(dòng)子區(qū)域RNA聚合酶II水平越高，+1核小體及其下游的核小體定相現(xiàn)象越明顯；不同種類的RNA聚合酶II影響著+1核小體的位置。

圖2 酵母和果蠅+1/?1核小體的位置

A：釀酒酵母+1/?1核小體位置；B：果蠅+1/?1核小體位置。

啟動(dòng)子區(qū)域NFR的功能主要體現(xiàn)在兩個(gè)方面：首先，轉(zhuǎn)錄起始位點(diǎn)的NFR有助于轉(zhuǎn)錄起始前復(fù)合物的組裝從而開(kāi)始轉(zhuǎn)錄，而轉(zhuǎn)錄終止位點(diǎn)附近的NFR有助于轉(zhuǎn)錄復(fù)合物的解聚；另外，NFR下游+1核小體與DNA的解聚或結(jié)構(gòu)變化促進(jìn)了RNA聚合酶Ⅱ與DNA的結(jié)合，促進(jìn)了轉(zhuǎn)錄的進(jìn)行。+1核小體的位置在轉(zhuǎn)錄活動(dòng)中發(fā)揮重要作用，其位置向NFR上游偏移將會(huì)影響轉(zhuǎn)錄元件的組裝，降低轉(zhuǎn)錄效率[70,71]。在啟動(dòng)子區(qū)域以外，也發(fā)現(xiàn)有NFR及相位統(tǒng)一的核小體陣列的存在，例如絕緣子復(fù)合物及轉(zhuǎn)錄因子的結(jié)合位點(diǎn)。絕緣子CTCF(CCCTC-binding factor) 蛋白結(jié)合位點(diǎn)處核小體占位很低，表現(xiàn)出核小體缺失，并且在CTCF結(jié)合位點(diǎn)上下游發(fā)現(xiàn)具有對(duì)稱且規(guī)律的相位統(tǒng)一的核小體陣列[72]。與+1核小體類似，這些蛋白質(zhì)與DNA結(jié)合很緊密，具有強(qiáng)定位特性，但基因組上核小體定相排布的機(jī)制還未完全破解[41]。

2.3 核小體組織與基因表達(dá)調(diào)控

核小體對(duì)DNA的包裝，一方面壓縮了DNA，阻礙了眾多DNA結(jié)合蛋白與DNA的相互作用；另一方面，在這一過(guò)程中核小體能調(diào)整染色質(zhì)的包裝方式，促進(jìn)了細(xì)胞內(nèi)基因的正確表達(dá)[62]。核小體的定位與轉(zhuǎn)錄因子的結(jié)合以及基因的轉(zhuǎn)錄水平密切相關(guān)，且相互影響。

轉(zhuǎn)錄因子與核小體競(jìng)爭(zhēng)DNA結(jié)合位點(diǎn)會(huì)導(dǎo)致核小體組織的變化，進(jìn)而重塑該位點(diǎn)的染色質(zhì)可及性狀態(tài)從而調(diào)控基因表達(dá)。對(duì)于大多數(shù)轉(zhuǎn)錄因子而言，其對(duì)應(yīng)的結(jié)合位點(diǎn)上有核小體存在時(shí)，由于空間位阻、電荷相斥等影響，轉(zhuǎn)錄因子與DNA的親和性通常比裸露DNA低10倍以上。然而結(jié)構(gòu)蛋白(architectural proteins, AP)和染色質(zhì)重塑復(fù)合物能幫助轉(zhuǎn)錄因子與DNA的結(jié)合，這一過(guò)程通常促進(jìn)核小體從DNA上分離。Daniel等[10]利用ChIP-seq結(jié)合MNase-seq等技術(shù)對(duì)35種轉(zhuǎn)錄因子結(jié)合位點(diǎn)附近的核小體組織形態(tài)進(jìn)行分析，他們發(fā)現(xiàn)在轉(zhuǎn)錄因子結(jié)合位點(diǎn)峰值處通常形成NFR，且轉(zhuǎn)錄因子占位水平與其結(jié)合位點(diǎn)上下游核小體定位強(qiáng)度負(fù)相關(guān)。轉(zhuǎn)錄因子結(jié)合使得核小體解聚，進(jìn)一步促進(jìn)開(kāi)放染色質(zhì)的形成從而激活轉(zhuǎn)錄。釀酒酵母基因啟動(dòng)子是研究染色質(zhì)結(jié)構(gòu)對(duì)基因表達(dá)影響的重要模型。研究發(fā)現(xiàn)，在轉(zhuǎn)錄抑制狀態(tài)的啟動(dòng)子處，核小體呈規(guī)律排列形式，而轉(zhuǎn)錄因子Pho4p與UASp2位點(diǎn)的結(jié)合能導(dǎo)致核小體組織重構(gòu)，表現(xiàn)為–2和–3核小體解聚，核小體占位水平很低。這一核小體重構(gòu)導(dǎo)致了染色質(zhì)開(kāi)放位點(diǎn)的形成，進(jìn)而激活了PHO5的表達(dá)[73,74]。轉(zhuǎn)錄因子還可以通過(guò)改變局部染色質(zhì)的空間結(jié)構(gòu)來(lái)影響轉(zhuǎn)錄?；蚓哂袃蓚€(gè)重要的調(diào)控元件boxA與boxB，轉(zhuǎn)錄因子TFⅢC與這兩個(gè)調(diào)控元件的結(jié)合使boxA與boxB間的核小體向上游平移約40 bp。而后，轉(zhuǎn)錄因子TFⅢB的結(jié)合導(dǎo)致–1核小體重定位從而改變TATA上游染色質(zhì)空間結(jié)構(gòu)，即在TATA框處形成一段對(duì)核酸酶高敏的活躍染色質(zhì)，從而為轉(zhuǎn)錄起始前復(fù)合物(pre-initiation complex, PIC)的組裝和轉(zhuǎn)錄做好了準(zhǔn)備[75]。

轉(zhuǎn)錄水平同樣也會(huì)對(duì)核小體在基因組上的定位和占位水平產(chǎn)生影響。Sushma等[76]利用MNase-seq對(duì)熱激(heat shock)前后釀酒酵母全基因組核小體定位及占位進(jìn)行測(cè)量，發(fā)現(xiàn)細(xì)胞在經(jīng)歷轉(zhuǎn)錄干擾(transcriptional perturbation)前后，不發(fā)生全局范圍的核小體定位變化，大部分核小體的位置保持穩(wěn)定，染色質(zhì)重塑活動(dòng)通常只與基因啟動(dòng)子區(qū)域單個(gè)或兩個(gè)核小體的缺失或變換相關(guān)。在人CD4+T細(xì)胞中，Anton等[72]發(fā)現(xiàn)在不同轉(zhuǎn)錄頻率的基因啟動(dòng)子處，其N(xiāo)FR的缺失程度不同，高表達(dá)基因(>8 RPKM)較低表達(dá)基因(<1 RPKM)的核小體缺失程度更高，因此轉(zhuǎn)錄活性可能與NFR區(qū)域核小體移除相關(guān)。轉(zhuǎn)錄活動(dòng)還會(huì)影響核小體間隔，Lai等[35]利用單細(xì)胞MNase-seq技術(shù)，對(duì)小鼠CD4+T淋巴細(xì)胞和胚胎干細(xì)胞的核小體組織及染色質(zhì)可及性進(jìn)行研究，發(fā)現(xiàn)活躍轉(zhuǎn)錄基因內(nèi)部核小體間的間隔不均勻；而在沉默基因或異染色質(zhì)區(qū)域，由于沒(méi)有轉(zhuǎn)錄活動(dòng)的影響，核小體間隔高度均勻，連接DNA長(zhǎng)度變化不大。

核小體在基因組上的排列多起到抑制轉(zhuǎn)錄的作用，通常與影響RNA聚合酶II的延伸及PIC的組裝有關(guān)。轉(zhuǎn)錄過(guò)程中，核小體的存在成為了RNA聚合酶Ⅱ的延伸的物理屏障，在體內(nèi)和體外的實(shí)驗(yàn)中都發(fā)現(xiàn)了核小體參與RNA聚合酶II延伸暫停(pausing process)，從而影響轉(zhuǎn)錄效率[42,77]；基因啟動(dòng)子區(qū)域的核小體組織特別是+1核小體的定位直接決定了PIC是否可以成功組裝。Reja等[78]利用ChIP-exo技術(shù)研究酵母核糖體蛋白啟動(dòng)子時(shí)發(fā)現(xiàn)，經(jīng)熱激處理后，+1核小體向上游啟動(dòng)子區(qū)域移動(dòng)導(dǎo)致了轉(zhuǎn)錄抑制，因?yàn)?1核小體對(duì)上游DNA序列的占領(lǐng)阻礙了PIC與足夠DNA底物的結(jié)合。然而，核小體對(duì)轉(zhuǎn)錄的抑制作用可以被細(xì)胞內(nèi)多種調(diào)控因子逆轉(zhuǎn)，在人IFN-β基因啟動(dòng)子處，+1核小體對(duì)TATA框(TATA box)及TSS的占位阻礙了PIC的組裝，抑制基因轉(zhuǎn)錄。當(dāng)機(jī)體受到病毒感染，在增強(qiáng)體(enhanceosome)及其招募的SWI/SNF、GCN5乙酰轉(zhuǎn)移酶等多種復(fù)合物的共同作用下，+1核小體向下游移動(dòng)約36 bp，使得轉(zhuǎn)錄因子TFⅡD與核心啟動(dòng)子區(qū)域結(jié)合，從而起始轉(zhuǎn)錄過(guò)程[79~81]。

2.4 組蛋白修飾圖譜與表觀遺傳學(xué)

核小體不僅是染色質(zhì)組裝的基本單位，也是生物體表觀遺傳學(xué)修飾的主要載體。核小體的定位及組蛋白翻譯后修飾是表觀遺傳學(xué)在各領(lǐng)域研究中的重要內(nèi)容。其中，MNase-ChIP-seq技術(shù)發(fā)揮了重要功能，該技術(shù)目前已經(jīng)可以對(duì)全基因組范圍內(nèi)的組蛋白PTM進(jìn)行捕獲并測(cè)量。

組蛋白PTM被研究者們稱作“組蛋白密碼”(histone code)，因?yàn)镻TM可以直接改變?nèi)旧|(zhì)的物理結(jié)構(gòu)，或被細(xì)胞內(nèi)特定的蛋白質(zhì)識(shí)別，進(jìn)而激活或抑制下游染色質(zhì)功能[82]。組蛋白翻譯后修飾中較普遍的有賴氨酸的乙?；?、甲基化及泛素化，精氨酸的甲基化以及絲氨酸的磷酸化[83,84]，這些修飾多出現(xiàn)在轉(zhuǎn)錄調(diào)控元件如啟動(dòng)子、增強(qiáng)子處。作為一種常見(jiàn)的修飾形式，組蛋白乙酰化削弱了組蛋白-DNA的相互作用、提高核小體周轉(zhuǎn)率和染色質(zhì)可及性，利于轉(zhuǎn)錄因子的結(jié)合從而促進(jìn)基因表達(dá)，在高轉(zhuǎn)錄活性的基因啟動(dòng)子區(qū)域豐度較高[85~87]。研究表明，啟動(dòng)子區(qū)域H3K9ac修飾水平與基因表達(dá)水平正相關(guān)，且H3K9ac被證實(shí)在促進(jìn)人胚胎干細(xì)胞分化[88]、胰島β細(xì)胞增殖[89]等過(guò)程中起關(guān)鍵作用。相比于乙?；揎?，組蛋白甲基化修飾形式更加穩(wěn)定。一些甲基化形式能促進(jìn)轉(zhuǎn)錄，如H3K4me1/3、H3K36me3等，其中H3K4me3主要分布于活躍轉(zhuǎn)錄基因啟動(dòng)子處，它有助于啟動(dòng)子對(duì)轉(zhuǎn)錄因子TFⅡD、RNA聚合酶Ⅱ的招募；在增強(qiáng)子處，多出現(xiàn)明顯的H3K4me1修飾，而不出現(xiàn)H3K4me3修飾[90]。另一些組蛋白甲基化與基因沉默相關(guān)，H3K9me3、H3K27me3在哺乳動(dòng)物細(xì)胞中促進(jìn)染色質(zhì)凝集[91]。

早期的研究方向多集中于H2A.Z核小體變體定位[53]與組蛋白甲基化分布形式[92]。H2A.Z作為常見(jiàn)的核小體變體，H2A.Z參與轉(zhuǎn)錄調(diào)控、DNA修復(fù)等眾多生物過(guò)程[93]。H2A.Z通常出現(xiàn)在基因啟動(dòng)子處+1/?1核小體處，專一定位在+1核小體處的H2A.Z與轉(zhuǎn)錄起始相關(guān)[94]。H2A.Z的占位水平由它的并入水平與解離水平共同決定。其中，SWR1促進(jìn)了H2A.Z的并入，而RNA聚合酶II以及Kin28/Cdk7 激酶參與H2A.Z的解離，目前，已經(jīng)可以在活細(xì)胞中以單分子的分辨率追蹤核小體的變化過(guò)程[95]。另一方面，H2A.Z占位水平通常與DNA甲基化水平負(fù)相關(guān)，Murphy等[96]發(fā)現(xiàn)在斑馬魚(yú)（）的配子和胚胎發(fā)育時(shí)期，H2A.Z促使DNA甲基化模式“重編程”。其中，定位在編碼胚胎早期轉(zhuǎn)錄因子基因啟動(dòng)子區(qū)的H2A.Z使得啟動(dòng)子處于低甲基化水平從而促進(jìn)基因表達(dá)，這一過(guò)程在斑馬魚(yú)胚胎發(fā)育中發(fā)揮重要作用。

組蛋白修飾狀態(tài)的改變通常與基因功能改變、細(xì)胞惡性轉(zhuǎn)化相關(guān)。目前，研究人員可以利用下一代測(cè)序技術(shù)探究染色質(zhì)表觀遺傳改變，從而分析細(xì)胞健康狀態(tài)。癌細(xì)胞通常具有全局范圍的表觀遺傳異常，其中，特定的組蛋白修飾與癌癥的起始和增殖緊密相關(guān)[97]。Wang等[98]發(fā)現(xiàn)了H3乙?；c腫瘤細(xì)胞耐藥性的關(guān)系。他們通過(guò)抑制組蛋白去乙酰化酶HDAC3的表達(dá)，促進(jìn)了癌細(xì)胞基因啟動(dòng)子區(qū)域的H3乙酰化，從而增強(qiáng)基因的表達(dá)。而PD-L1有抑制T細(xì)胞活性的作用，使得癌細(xì)胞具有更強(qiáng)的耐藥性。這一表觀遺傳學(xué)機(jī)制的發(fā)現(xiàn)有利于發(fā)現(xiàn)治療腫瘤抗藥能力的潛在靶點(diǎn)。Aman等[99]發(fā)現(xiàn)在腎細(xì)胞癌中，低水平的H3K4甲基化通常與晚期癌癥和腫瘤轉(zhuǎn)移相關(guān)，其中H3K4me3 可能是一個(gè)較好的腫瘤轉(zhuǎn)移預(yù)測(cè)標(biāo)志物。Liana等[100]發(fā)現(xiàn)H3K9ac促進(jìn)了口腔粘膜癌細(xì)胞增殖的上皮間質(zhì)轉(zhuǎn)化(epithelial–mesenchymal transition, EMT)，因此H3K9ac可以作為口腔粘膜癌預(yù)后評(píng)估的關(guān)鍵標(biāo)志物。表觀遺傳通常是動(dòng)態(tài)可逆的，因此根據(jù)組蛋白表觀遺傳相關(guān)酶類設(shè)計(jì)藥物，可以恢復(fù)“正常的表觀狀態(tài)”從而起到治療效果，以組蛋白甲基轉(zhuǎn)移酶抑制劑、組蛋白去乙?；敢种苿橹鞯谋碛^遺傳學(xué)藥物發(fā)展前景較為樂(lè)觀[97]。

3 結(jié)語(yǔ)與展望

21世紀(jì)以來(lái)，下一代測(cè)序技術(shù)的高速發(fā)展加快了研究人員對(duì)染色質(zhì)結(jié)構(gòu)的研究，使人們?cè)跁r(shí)間和空間上對(duì)核小體組織有了更清晰的認(rèn)知。MNase-seq及其衍生技術(shù)揭示了全基因組范圍核小體的組織方式，即核小體在基因組上的分布是動(dòng)態(tài)且不均勻的，在特定區(qū)域會(huì)出現(xiàn)NFR。人們已經(jīng)認(rèn)識(shí)到核小體定位主要受到DNA序列的影響，染色質(zhì)重塑因子、轉(zhuǎn)錄因子、聚合酶等同樣參與了核小體組織及染色質(zhì)結(jié)構(gòu)的形成。染色質(zhì)構(gòu)象的建立及動(dòng)態(tài)平衡使得調(diào)控元件高效地對(duì)轉(zhuǎn)錄因子進(jìn)行招募、調(diào)控遠(yuǎn)端序列與靶基因的互作，因此與染色質(zhì)功能特別是基因表達(dá)調(diào)控密切相關(guān)。

近年來(lái)，對(duì)MNase-seq技術(shù)改良的重點(diǎn)在于減少必要的細(xì)胞投入量及降低背景信號(hào)，目前該技術(shù)已經(jīng)可以在單個(gè)細(xì)胞上實(shí)現(xiàn)[35]，有助于人們從單個(gè)細(xì)胞的核小體組織結(jié)構(gòu)層面理解細(xì)胞間的染色質(zhì)異質(zhì)性。在疾病研究中，將scMNase-seq與scDNase-seq、scChIP-seq以及多種表觀遺傳學(xué)分析方法相結(jié)合，有助于人們理解腫瘤異質(zhì)性、剖析腫瘤形成過(guò)程內(nèi)在的染色質(zhì)結(jié)構(gòu)基礎(chǔ)、分析不同疾病的表觀遺傳標(biāo)記，從而解決更多疾病機(jī)理問(wèn)題[101]。利用MNase-seq及相關(guān)技術(shù)對(duì)全基因組核小體定位和可及性的測(cè)量初步解析了染色質(zhì)結(jié)構(gòu)和基因調(diào)控機(jī)制，最近發(fā)現(xiàn)MNase-seq還可以預(yù)測(cè)高階染色質(zhì)結(jié)構(gòu)，然而其在染色質(zhì)三維相互作用的分析中存在困難。將MNase- seq技術(shù)與近年來(lái)發(fā)展迅速的Hi-C[102,103]等染色質(zhì)捕獲技術(shù)相結(jié)合，有助于人們更好地理解染色質(zhì)結(jié)構(gòu)與其功能的關(guān)系。隨著技術(shù)方法的不斷進(jìn)步以及多學(xué)科的交叉融合，人們將對(duì)復(fù)雜動(dòng)態(tài)的染色質(zhì)結(jié)構(gòu)及功能研究得更加深入。

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Resolving nucleosomal positioning and occupancy with MNase-seq

Weihang Deng, Xinhui Li

Nucleosomes are the basic unit of the three-dimensional structure of chromatin. It is now widely accepted that the positioning and occupancy of nucleosomes play important roles in fundamental genomic processes such as DNA transcription, replication and repair. Among the methods used to provide genome-wide nucleosomal positions and occupancy levels, MNase-seq has proven to be highly effective. Indeed, with this method, the nucleosomal landscapes of a variety of organisms have now been investigated, revealing both commonalities and differences. In this review, we first introduce the technical principles underlying MNase-seq, focusing on details essential to precisely resolve nucleosome positioning and occupancy. We then describe recent advances with this method, as well as future perspectives of its role in chromatin biology, with a particular focus of uncovering mechanistic insights of many disease process.

nucleosome; chromatin structure; chromatin remodeling; next-generation sequencing (NGS); micrococcal nuclease

2020-09-04;

2020-10-18

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：81972909)資助[Supported by the National Natural Science Foundation of China(No. 81972909)]

鄧瑋杭，在讀碩士研究生，專業(yè)方向：系統(tǒng)生物醫(yī)學(xué)。E-mail: weihangdeng@sjtu.edu.cn

李鑫輝，博士，助理研究員，研究方向：系統(tǒng)生物學(xué)與分子生物學(xué)。E-mail: xhli@sjtu.edu.cn

10.16288/j.yczz.20-178

2020/11/9 11:17:28

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20201106.1053.003.html

(責(zé)任編委: 李海濤)