PRC1.6復(fù)合體表觀遺傳調(diào)控生殖譜系特異性基因的時(shí)空表達(dá)

孫曉偉，李宏陽，王健，程博,2

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PRC1.6復(fù)合體表觀遺傳調(diào)控生殖譜系特異性基因的時(shí)空表達(dá)

孫曉偉1，李宏陽1，王健1，程博1,2

1. 蘭州大學(xué)生命科學(xué)學(xué)院，蘭州 730000 2. 蘭州大學(xué)，教育部細(xì)胞活動(dòng)及逆境適應(yīng)重點(diǎn)實(shí)驗(yàn)室，蘭州 730000

多梳抑制復(fù)合體1 (polycomb repressive complex 1, PRC1)是一類通過催化和識(shí)別染色質(zhì)表觀遺傳修飾進(jìn)而調(diào)控基因表達(dá)的蛋白復(fù)合體，主要參與干細(xì)胞干性維持、細(xì)胞分化以及細(xì)胞周期調(diào)控等生理過程，該復(fù)合體功能異常影響機(jī)體發(fā)育或?qū)е掳┌Y發(fā)生。在哺乳動(dòng)物細(xì)胞內(nèi)，PRC1根據(jù)組成差異被進(jìn)一步細(xì)分為6種不同的亞型PRC1.1~PRC1.6，它們?cè)诎谢蛉鹤R(shí)別、表觀調(diào)控機(jī)制及生物學(xué)功能上存在明顯特化。近年來研究發(fā)現(xiàn)，PRC1.6復(fù)合體在胚胎干細(xì)胞及體細(xì)胞中對(duì)于穩(wěn)定抑制生殖譜系特異性基因的表達(dá)至關(guān)重要，同時(shí)對(duì)于生殖干細(xì)胞的干性維持以及精子發(fā)生過程中生殖譜系特異性基因的精密調(diào)控承擔(dān)著重要作用。本文在介紹PRC1.6復(fù)合體的發(fā)現(xiàn)、各組分的分子功能及其參與的生化反應(yīng)途徑的基礎(chǔ)上，系統(tǒng)闡述了該復(fù)合體在胚胎發(fā)育、性腺發(fā)育、精子發(fā)生等過程中對(duì)生殖譜系特異性靶基因群的時(shí)空表達(dá)發(fā)揮的重要調(diào)控功能，并探討了PRC1.6與已知表觀遺傳調(diào)控網(wǎng)絡(luò)的相互作用，以期為進(jìn)一步探索生殖譜系基因表達(dá)、精子發(fā)生的表觀遺傳調(diào)控機(jī)制以及男性不育的致病機(jī)制提供參考。

多梳抑制復(fù)合體1；PRC1.6；轉(zhuǎn)錄抑制；生殖譜系特異性基因；時(shí)空表達(dá)

表觀遺傳調(diào)控是實(shí)現(xiàn)真核生物基因選擇性表達(dá)的主要途徑。多梳蛋白家族(polycomb groups，PcGs)是一類重要的表觀遺傳調(diào)控因子，與多種干細(xì)胞的干性維持、細(xì)胞分化、細(xì)胞周期的調(diào)控、細(xì)胞衰老、X染色體失活等一系列細(xì)胞生理活動(dòng)密切相關(guān)[1,2]。它主要通過兩類蛋白復(fù)合體發(fā)揮功能——多梳抑制復(fù)合體Ⅰ和Ⅱ (polycomb repressive complex 1/2, PRC1/2)。近年來在高等動(dòng)物細(xì)胞中不斷鑒定出組分不同的PRC1亞型，且它們的生物學(xué)功能在靶基因群的選擇及調(diào)控基因表達(dá)的作用機(jī)制中有所差異。PRC1.6復(fù)合體屬于其中一類PRC1亞型，主要包含RING1B、PCGF6 (polycomb group ring finger protein 6)、MAXMyc-associated factor X)、MGA (MAX gene associated)、E2F6和L3MBTL2 (lethal (3) malignant brain tumour like 2)等組分。近年來通過對(duì)PRC1.6復(fù)合體不同核心組分在生物化學(xué)、分子生物學(xué)、細(xì)胞生物學(xué)及發(fā)育生物學(xué)等方面的研究，發(fā)現(xiàn)該復(fù)合體對(duì)于哺乳動(dòng)物發(fā)育過程中多種相關(guān)細(xì)胞譜系的建成及維持是必需的。本文在介紹該復(fù)合體的發(fā)現(xiàn)、核心組分的分子生物學(xué)功能的基礎(chǔ)上，對(duì)該復(fù)合體在高等動(dòng)物胚胎發(fā)育、性腺發(fā)育、精子發(fā)生、胚胎干細(xì)胞(embryonic stem cells, ESCs)及生殖干細(xì)胞(germ line stem cells, GSCs)維持等過程中發(fā)揮的生物學(xué)功能展開了系統(tǒng)論述。

1 多梳蛋白家族PcGs

PcGs是一大類通過催化和識(shí)別組蛋白表觀遺傳修飾來改變?nèi)旧|(zhì)構(gòu)象、對(duì)靶基因進(jìn)行轉(zhuǎn)錄調(diào)控的蛋白。第一個(gè)多梳蛋白編碼基因()是Pamela Lewis于1947年在黑腹果蠅()中發(fā)現(xiàn)的，1978年Edward Lewis發(fā)現(xiàn)突變導(dǎo)致果蠅發(fā)生同源異型轉(zhuǎn)化(如雄性果蠅產(chǎn)生多對(duì)性梳的異常發(fā)育表型)，進(jìn)一步研究發(fā)現(xiàn)多梳蛋白是通過抑制同源異型基因的表達(dá)來調(diào)控果蠅的體節(jié)發(fā)育[1,3,4]，其功能與發(fā)揮基因激活作用的三胸蛋白家族TrxGs(trithorax groups)相拮抗[5,6]。但是近幾年也有關(guān)于PcGs蛋白激活基因表達(dá)的報(bào)道[7~9]，表明PcGs蛋白在執(zhí)行轉(zhuǎn)錄調(diào)控方面的機(jī)制可能是非常復(fù)雜且多效的。PcGs蛋白主要通過PRC1和PRC2兩類蛋白復(fù)合體形式發(fā)揮作用。其中，EZH2是PRC2中具有催化活性的核心組分，可以催化組蛋白H3第27位賴氨酸的三甲基化(H3K27me3)[10]。其他的PRC2組分參與復(fù)合體組裝及活性維持[11]。PRC1具有E3泛素連接酶活性，該活性由RING1A/ RING1B及PCGF的環(huán)指結(jié)構(gòu)域(ring finger domain)的部分親水性表面構(gòu)成[12,13]，催化組蛋白H2A第119位賴氨酸的單泛素化(H2AK119ub1)[14]。PRC1和PRC2 調(diào)控的靶基因群存在部分重疊，在多數(shù)情況下二 者存在相互招募的關(guān)系，互助實(shí)現(xiàn)對(duì)靶基因的轉(zhuǎn)錄 調(diào)控[15]。

高等動(dòng)物中PRC1的組分較果蠅要復(fù)雜的多，除了對(duì)應(yīng)果蠅PRC1的每個(gè)組分都衍生出多個(gè)同源蛋白之外，還增添了很多其他組分，且組分之間的組合方式非常復(fù)雜多樣[16]。目前在哺乳動(dòng)物中發(fā)現(xiàn)PRC1亞型有6類，稱為PRC1.1~PRC1.6，其中PRC1.2和PRC1.4因包含有經(jīng)典的Pc蛋白同系物CBX蛋白而被稱作經(jīng)典PRC1 (canonical PRC1)，其余幾種亞型因缺乏CBX蛋白而被列為非典型PRC1 (non-canonical PRC1)。本文著重討論了PRC1.6亞型，關(guān)于其他亞型的組成及功能均有較多報(bào)道，請(qǐng)讀者參考相關(guān)綜述[17,18]。

2 PRC1.6復(fù)合體的發(fā)現(xiàn)、核心組分及其分子功能

2.1 PRC1.6復(fù)合體的發(fā)現(xiàn)

PRC1.6復(fù)合體先后被不同的研究團(tuán)隊(duì)發(fā)現(xiàn)，并被賦予了不同的名稱(表1)。2002年，Ogawa等[19]在HeLa及成纖維細(xì)胞中利用免疫沉降技術(shù)、甘油密度梯度離心并結(jié)合質(zhì)譜檢測(cè)發(fā)現(xiàn)了與E2F6-標(biāo)簽融合蛋白結(jié)合的蛋白復(fù)合體—E2F6復(fù)合物(E2F6.com)。該復(fù)合體中除了包含轉(zhuǎn)錄因子E2F6/DP-1，還有另一對(duì)轉(zhuǎn)錄因子異二聚體MAX/MGA；同時(shí)發(fā)現(xiàn)該復(fù)合體在甘油密度梯度分離中與組蛋白甲基轉(zhuǎn)移酶G9a、Eu-HMTase1 (GLP)部分重疊，具有H3K9的甲基轉(zhuǎn)移酶活性[19]。2011年，美國紐約大學(xué)醫(yī)學(xué)院Danny Reinberg課題組的Trojer等[20]研究L3MBTL2的功能時(shí)再次發(fā)現(xiàn)了該復(fù)合體，將其命名為PRC1L4 (PRC1-like4)復(fù)合體；2012年，同課題組的Gao等[21]通過免疫沉降/質(zhì)譜實(shí)驗(yàn)發(fā)現(xiàn)了人源細(xì)胞中包含不同PCGF因子(PCGF1~PCGF6)的PRC1復(fù)合體，并根據(jù)包含PCGF因子的編號(hào)將PRC1復(fù)合體細(xì)分為PRC1.1~PRC1.6復(fù)合體亞型，各亞型除了共同組分是PRC1復(fù)合體的酶學(xué)核心組分RING1B (RING2)外其余組分差異較大。其中PRC1.6組分包括PCGF6、RING1B、RYBP、MAX、MGA、L3MBTL2、E2F6、DP-1/2、HP1γ和HDAC1/2等。2017年，Endoh等[22]在小鼠ESCs中利用帶標(biāo)簽的PCGF6蛋白也分離到了類似的復(fù)合體并將其稱為PCGF6-PRC1。以上4種復(fù)合體雖然名稱各異，但是核心組分都含有PCGF6、E2F6、RING1B、L3MBTL2和HP1γ等因子(表1)，本質(zhì)上屬于同一復(fù)合體或十分相近的同類復(fù)合體。為了敘述簡(jiǎn)潔，本文在后續(xù)介紹中將其統(tǒng)稱為PRC1.6復(fù)合體。

表1 PRC1.6復(fù)合體的發(fā)現(xiàn)及組分

2.2 PRC1.6復(fù)合體各組分的分子功能

如上所述，PRC1.6復(fù)合體是一個(gè)多組分蛋白復(fù)合體，其組分大致可以分成與催化組蛋白修飾相關(guān)的因子和與復(fù)合體裝配或靶向定位相關(guān)的因子。同其他PRC1亞型類似，PRC1.6復(fù)合體最主要的酶學(xué)活性是由RING1B提供的E3泛素連接酶活性，催化產(chǎn)生H2AK119ub1；而RYBP、PCGF6等因子對(duì)于RING1B的活性發(fā)揮起到重要的促進(jìn)作用[14,21,23]。在該復(fù)合體中包含異染色質(zhì)結(jié)合蛋白HP1γ，說明其可能介導(dǎo)PRC1.6與甲基化的H3K9之間存在功能互作；除此之外，Gao等[21]還在PRC1.6復(fù)合體中檢測(cè)到了組蛋白去乙?；窰DAC1/2，說明該復(fù)合體在某些情況下可能與組蛋白去乙?；竻f(xié)同作用抑制靶基因的轉(zhuǎn)錄。

PRC1.6蛋白復(fù)合體各組分的已知分子功能見表2。在ESCs中通過免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)PCGF6與L3MBTL2、MAX、RING1B和RYBP具有相互作用，敲除時(shí)阻斷了L3MBTL2與復(fù)合體中RING1B、RYBP和MAX等組分的互作，這說明整個(gè)復(fù)合體的完整性在一定程度上依賴于PCGF6[24]。另有研究表明PCGF6可通過招募RING1B到靶基因上催化H2AK119位點(diǎn)的泛素化并抑制其表達(dá)[22]。MGA對(duì)于PRC1.6復(fù)合體的裝配和穩(wěn)定性發(fā)揮著更加關(guān)鍵的作用，在HEK293T細(xì)胞中敲除發(fā)現(xiàn)不僅該復(fù)合體各特異性組分在靶基因上的富集程度均受到明顯抑制，且其中幾個(gè)核心特異組分的蛋白總量明顯下降，如PCGF6、L3MBTL2和E2F6[25]。從L3MBTL2的RNA干擾實(shí)驗(yàn)可知，部分靶基因的上調(diào)與其基因上富集的H2K119ub1水平的明顯下降相伴發(fā)生，說明該因子對(duì)于PRC1.6復(fù)合體在靶基因區(qū)域組蛋白H2AK119位點(diǎn)的泛素化是必需的。同時(shí)，體外實(shí)驗(yàn)還發(fā)現(xiàn)L3MBTL2能夠誘發(fā)染色質(zhì)凝縮[20]。

目前，關(guān)于PRC1.6復(fù)合體的靶向定位的研究結(jié)果顯示該復(fù)合體的定位機(jī)制較為復(fù)雜，由多個(gè)組分協(xié)同執(zhí)行，包括MAX/MGA和E2F6/DP-1/2這兩對(duì)轉(zhuǎn)錄因子形成的異二聚體以及L3MBTL2[25](表2)。MAX和MGA通過各自的bHLHZip (basic Helix- Loop-Helix-Zipper)結(jié)構(gòu)域結(jié)合形成異二聚體，并特異性識(shí)別E-boxes序列CACGTG；此外MGA的氮端還包含另一個(gè)DNA結(jié)合結(jié)構(gòu)域，可識(shí)別T-box序列AGGC/TGC/TGA，該異二聚體在PRC1.6復(fù)合體的組裝、穩(wěn)定性及靶基因識(shí)別上都發(fā)揮著重要作用[25,26]。另一對(duì)轉(zhuǎn)錄因子是E2F6和DP-1或DP-2，二者結(jié)合共同識(shí)別E2F家族識(shí)別序列GCGGGA[27]。與E2F家族的其他轉(zhuǎn)錄激活因子不同，E2F6通常對(duì)其結(jié)合的序列調(diào)控的靶基因產(chǎn)生轉(zhuǎn)錄抑制效應(yīng)。L3MBTL2包含4個(gè)MBT結(jié)構(gòu)域，結(jié)合組蛋白H3和H4，但該結(jié)合是否依賴于組蛋白的甲基化狀態(tài)目前還有爭(zhēng)議[28,29]。

表2 PRC1.6復(fù)合體中各組分的主要生化及分子功能

3 PRC1.6復(fù)合體在胚胎發(fā)育中抑制生殖譜系相關(guān)基因的表達(dá)

近年來不斷積累的針對(duì)PRC1.6組分的RNA干擾或基因敲除實(shí)驗(yàn)已經(jīng)充分證實(shí)PRC1.6復(fù)合體在維持ESCs的自我更新及調(diào)控其分化能力方面，尤其在抑制生殖譜系相關(guān)基因表達(dá)中發(fā)揮重要作用。在小鼠中，該復(fù)合體組分的敲除往往造成胚層及/或胚外譜系不同程度的發(fā)育異常，多數(shù)情況下產(chǎn)生胚胎致死表型(表3)，由此可見，PRC 1.6復(fù)合體在ESCs以及胚胎發(fā)育過程中發(fā)揮至關(guān)重要的調(diào)控功能。

表3 PRC1.6組分下調(diào)對(duì)ESCs及小鼠胚胎發(fā)育或性腺發(fā)育的影響

3.1 PRC1.6復(fù)合體參與ESCs干性維持及調(diào)控ESCs正常分化

PRC1.6復(fù)合體的組分RING1B、MAX、MGA、HP1γ、PCGF6和L3MBTL2等都已被報(bào)道對(duì)于ESCs的干性維持是必需的，這些組分在被敲除后均會(huì)引起ESCs分化異常(表3)。另有一些因子如RYBP雖然對(duì)于ESCs的自我更新是非必需的，但對(duì)于ESCs的正常分化卻是不可缺少的[38]。2013年，Maeda等[39]在小鼠ESCs中通過siRNA文庫從864個(gè)候選基因中篩選能夠抑制生殖細(xì)胞分化路徑的重要基因，篩選到多個(gè)PRC1.6復(fù)合體組分，包括、和，其中敲低效應(yīng)最為顯著，可誘發(fā)ESCs進(jìn)入類似減數(shù)分裂的狀態(tài)。Suzuki等[40]在ESCs中誘導(dǎo)性敲除后也發(fā)現(xiàn)表達(dá)上調(diào)的基因主要包括減數(shù)分裂和精子發(fā)生過程中的相關(guān)基因，如、和等。通過細(xì)胞免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)在Dox誘導(dǎo)敲除10天后，ESCs發(fā)生類似減數(shù)分裂細(xì)胞的形態(tài)學(xué)變化，出現(xiàn)減數(shù)分裂前期(細(xì)線期和偶線期)相關(guān)蛋白SYCP3的表達(dá)，因此PRC1.6復(fù)合體功能異常導(dǎo)致減數(shù)分裂相關(guān)基因的異常高表達(dá)，使ESCs越過原始生殖細(xì)胞(primordial germ cells, PGCs)直接形成類似減數(shù)分裂前期的細(xì)胞，說明MAX對(duì)于調(diào)控減數(shù)分裂的起始至關(guān)重要[40]。

在小鼠ESCs中PRC1.6復(fù)合體不同組分的ChIP-Seq數(shù)據(jù)顯示，該復(fù)合體在全基因組中靶向上萬個(gè)位點(diǎn)(由PCGF6、L3MBTL2和MGA共同靶向)；當(dāng)逐個(gè)敲除不同組分時(shí)，這些PRC1.6復(fù)合體靶基因中轉(zhuǎn)錄本水平發(fā)生上調(diào)的基因數(shù)目一般為數(shù)百個(gè)，如敲除導(dǎo)致882個(gè)基因上調(diào)[25,26]；敲除導(dǎo)致421個(gè)[25]或167個(gè)[41]基因上調(diào)。這類對(duì)PRC1.6復(fù)合體調(diào)控最敏感的基因中包含不同類型的發(fā)育相關(guān)基因(如神經(jīng)發(fā)育基因)，但最顯著的是特異性地包含生殖譜系及減數(shù)分裂關(guān)鍵基因[24,25,40,41]，這是其區(qū)分于其他PRC1復(fù)合體亞型所獨(dú)具的功能。在敲除的ESCs中表達(dá)顯著上調(diào)(>10倍)的基因中有49個(gè)都是在精子發(fā)生過程中必不可少的基因[22]。在ESCs中敲除后發(fā)現(xiàn)與減數(shù)分裂相 關(guān)的基因、、和表達(dá)量上調(diào)[25]。同樣，敲除的ESCs也呈現(xiàn)出生殖譜系相關(guān)基因特異性上調(diào)的現(xiàn)象[38]。另有研究表明，PRC1.6復(fù)合體同時(shí)抑制體細(xì)胞中生殖譜系相關(guān)基因的表達(dá)，如敲除基因的小鼠呈現(xiàn)出生殖譜系相關(guān)基因(和等)在體細(xì)胞中的異常表達(dá)[42,43]。這些實(shí)驗(yàn)證據(jù)都支持PRC1.6復(fù)合體在ESCs等其他非生殖細(xì)胞類型中發(fā)揮穩(wěn)定抑制生殖譜系特異性基因表達(dá)的作用，維持這些細(xì)胞類型的身份，限制其向生殖細(xì)胞譜系異常分化。

3.2 PRC1.6復(fù)合體對(duì)于正常胚胎發(fā)育必不可少

PRC1.6復(fù)合體對(duì)于哺乳動(dòng)物胚胎發(fā)育的正常進(jìn)行是必需的，但每個(gè)組分被敲除后小鼠所呈現(xiàn)的胚胎發(fā)育異常狀況卻有所區(qū)別，這一定程度上反映出每個(gè)組分在該復(fù)合體功能發(fā)揮中的權(quán)重不同以及部分組分可能因?yàn)閰⑴c多種復(fù)合體而具備更為多樣的發(fā)育調(diào)控功能(表3)。有研究表明，敲除小鼠在E5.5~6.5時(shí)表現(xiàn)出胚胎及胚外組織發(fā)育阻滯，且不具備明顯的胚胎特征或胚胎與胚外組織分界不清晰[44]。的表達(dá)在小鼠E3.5時(shí)的內(nèi)細(xì)胞團(tuán)中以及E4.5~E6.5時(shí)具全能性的上胚層(epiblast)中均可檢測(cè)到，當(dāng)使用Mga/Mga純合敲除，內(nèi)細(xì)胞團(tuán)在E4.5后隨即發(fā)育停滯，細(xì)胞增殖無明顯變化但細(xì)胞凋亡增加，上胚層不能正常形成[45]。由Mga衍生出來的Mga等位基因呈現(xiàn)出低表達(dá)表型，約一半Mga/Mga純合小鼠在出生及哺乳期死亡，其余能存活并可育[45]。早期通過原位雜交實(shí)驗(yàn)發(fā)現(xiàn)在胚胎發(fā)育E9.5~10.5間廣譜表達(dá)，在肢芽、腮弓及尾部區(qū)域高表達(dá)[26]。為進(jìn)一步澄清其在E4.5之后的發(fā)育調(diào)控功能，近期研究發(fā)現(xiàn)表達(dá)低劑量的Mga/Mga雜合轉(zhuǎn)基因小鼠在E7.5~E12.5之間死亡，主要由于E7.5之前上胚層全能性細(xì)胞發(fā)育停滯導(dǎo)致[46]，所以Mga對(duì)于上胚層的正常分化是必不可少的。缺失會(huì)導(dǎo)致多能性ESCs增殖及分化異常，在E7.5左右胚胎因不能正常形成原腸胚而致死，致死原因尚不明確[41]。

4 PRC1.6復(fù)合體調(diào)控雄性性腺發(fā)育及精子生成過程中生殖譜系基因的順次激活

PRC1.6復(fù)合體不僅可以影響胚胎發(fā)育中生殖譜系相關(guān)基因的表達(dá)，還可以影響精子生成和雄性性腺發(fā)育。從表3中可知，當(dāng)分別敲除和時(shí)，小鼠都可存活，但會(huì)表現(xiàn)出不同程度的睪丸發(fā)育不良及精子生成障礙。特異性敲除生殖細(xì)胞中的小鼠呈現(xiàn)出睪丸發(fā)育異常和不育表型，GSCs數(shù)目明顯下降并且異常分化[53]。最新研究表明，L3MBTL2在小鼠精原細(xì)胞減數(shù)分裂前期高表達(dá)，特異性敲除生殖細(xì)胞中的雄鼠雖然可育但表現(xiàn)出睪丸重量減輕，附睪中的精子數(shù)目減少及異常精子增多，并出現(xiàn)睪丸早衰現(xiàn)象[69]。進(jìn)一步研究發(fā)現(xiàn)這些雄鼠的精原細(xì)胞在減數(shù)分裂前期出現(xiàn)染色體異常聯(lián)會(huì)，且影響延長(zhǎng)型精子中組蛋白乙?；胶途映墒爝^程中組蛋白向魚精蛋白的轉(zhuǎn)變，說明參與精子生成過程進(jìn)而影響性腺發(fā)育。這些研究結(jié)果證明了PRC1.6復(fù)合體確實(shí)在精子發(fā)生及性腺發(fā)育過程中具有重要的作用。

近期關(guān)于PRC1.6復(fù)合體對(duì)生殖細(xì)胞分化過程中生殖譜系基因的表達(dá)調(diào)控也有相應(yīng)的報(bào)道。Maezawa等[53]構(gòu)建了生殖細(xì)胞特異性敲除的小鼠，并從出生7天的雄鼠中分離出未分化的精原細(xì)胞(Thy1+標(biāo)記)和已初步分化的精原細(xì)胞(c-Kit+標(biāo)記)。RNA-Seq檢測(cè)結(jié)果揭示，與野生型相比Ring1b小鼠的Thy1+細(xì)胞中分別有116個(gè)基因表達(dá)下調(diào)和69個(gè)基因表達(dá)上調(diào)，c-Kit+細(xì)胞中有1381基因表達(dá)下調(diào)和269個(gè)基因表達(dá)上調(diào)。GO分析發(fā)現(xiàn)大多數(shù)上調(diào)基因的功能與精子發(fā)生沒有直接的關(guān)聯(lián)，而下調(diào)基因中富含調(diào)控精子分化的重要功能基因。Thy1+和c-Kit+的細(xì)胞中因敲除表達(dá)下調(diào)的基因中有78個(gè)重疊，包含許多精子發(fā)生過程中必要的調(diào)控基因(如、和)，其中下調(diào)最顯著的是，后續(xù)ChIP實(shí)驗(yàn)證實(shí)是RING1B的直接靶基因。c-Kit+細(xì)胞中因敲除特異性下調(diào)的基因主要用于調(diào)節(jié)精原細(xì)胞分化(如、和)，減數(shù)分裂的開啟(如和)和piRNA的調(diào)控(如、和)；在正常精子發(fā)生中這些基因會(huì)在Thy1+精原細(xì)胞向c-Kit+精原細(xì)胞分化的過程中被高度激活，且進(jìn)一步研究證實(shí)RING1B與SALL4可以共同靶向并上調(diào)這群基因的表達(dá)。該研究表明RING1B可與SALL4協(xié)同調(diào)控生殖譜系基因的順次激活，這對(duì)生殖譜系細(xì)胞的正常分化及進(jìn)入減數(shù)分裂等過程至關(guān)重要。

5 PRC1.6復(fù)合體是精子發(fā)生表觀遺傳調(diào)控網(wǎng)絡(luò)的重要組成部分

精子發(fā)生是一個(gè)受到多因素調(diào)控的復(fù)雜發(fā)育過程。表4簡(jiǎn)要概括了精子發(fā)生過程中已知的表觀遺傳調(diào)控機(jī)制的大致類型。參與甲基化的各類DNA甲基轉(zhuǎn)移酶(DNMTs)的表達(dá)及活性在生殖細(xì)胞發(fā)育過程中呈現(xiàn)明顯的動(dòng)態(tài)變化，這些DNMTs的異常往往直接導(dǎo)致生殖細(xì)胞發(fā)育異常以及個(gè)體雄性不育[70]。胚胎發(fā)育及生殖細(xì)胞發(fā)育過程中呈現(xiàn)動(dòng)態(tài)變化的組蛋白甲基化、乙?；刃揎棇?duì)于建立細(xì)胞譜系的特異性身份至關(guān)重要[71,72]。其他組蛋白修飾，如泛素化、磷酸化以及近幾年新發(fā)現(xiàn)的組蛋白巴豆酰化等對(duì)精子發(fā)育也十分重要[73,74]。在精子發(fā)育過程中，組裝核小體的經(jīng)典組蛋白在特定發(fā)育階段被不同睪丸特異性組蛋白變體所替換，逐漸減弱DNA和組蛋白之間的作用，為大多數(shù)組蛋白在單倍體精子成熟的過程中被魚精蛋白所替代做準(zhǔn)備[75,76]。非編碼RNA如piRNA介導(dǎo)的基因沉默機(jī)制是精子發(fā)育及其他情況下抑制可轉(zhuǎn)座元件活性的重要途徑，對(duì)于精子的正常發(fā)育必不可少[77]；同時(shí)生殖細(xì)胞特異性表達(dá)的lncRNAs或miRNAs等在精子發(fā)育過程中也都起到重要的調(diào)控作用[78~81]。另外，近幾年發(fā)現(xiàn)RNA的修飾尤其是甲基化修飾可參與RNA的轉(zhuǎn)錄后加工、核輸出、翻譯及RNA穩(wěn)定性調(diào)節(jié)等，在造血干細(xì)胞分化、精子發(fā)生、神經(jīng)發(fā)育等發(fā)育過程中發(fā)揮重要作用[82,83]。

表4 精子發(fā)育過程中的表觀遺傳調(diào)控方式

如表4所示，生殖譜系基因的表達(dá)受到非常復(fù)雜精密的表觀遺傳調(diào)控網(wǎng)絡(luò)的調(diào)控，PRC1.6復(fù)合體作為該表觀遺傳調(diào)控網(wǎng)絡(luò)的一部分，與該調(diào)控網(wǎng)絡(luò)的其他“版塊”之間存在著緊密的交互作用。目前已知PRC1.6復(fù)合體與DNA甲基化、組蛋白H3K9me3和H3K27me3等重要的表觀遺傳修飾存在協(xié)同作用，且這些協(xié)同互作呈現(xiàn)出明顯的動(dòng)態(tài)變化(圖1)。例如，在胚胎發(fā)育早期E2F6對(duì)于減數(shù)分裂相關(guān)基因的轉(zhuǎn)錄抑制主要通過與PRC2協(xié)同完成，但不依賴于DNA甲基轉(zhuǎn)移酶DNMT3B[98]；然而，在小鼠體細(xì)胞中，E2F6則更多地通過招募DNMT3B對(duì)靶基因啟動(dòng)子進(jìn)行甲基化來實(shí)現(xiàn)對(duì)這些生殖譜系特異性基因表達(dá)的有效抑制[99]。有趣的是，雖然小鼠ESCs中E2F6與DNMT3B之間沒有協(xié)同關(guān)系，但MAX與DNA甲基轉(zhuǎn)移酶DNMT1，DNMT3A，DNMT3B之間存在明顯的協(xié)同作用并抑制生殖譜系相關(guān)基因的轉(zhuǎn)錄[100]。除了PRC1.6復(fù)合體自身包含HP1γ，可實(shí)現(xiàn)對(duì)轉(zhuǎn)錄抑制標(biāo)記H3K9me3的結(jié)合之外，最近有研究揭示MAX與H3K9甲基轉(zhuǎn)移酶SETDB1 可協(xié)同介導(dǎo)生殖譜系相關(guān)基因的轉(zhuǎn)錄抑制，但該機(jī)制并不依賴于PRC1.6復(fù)合體[100]。另外，在PCGF6 敲除的小鼠ESCs中，piRNA互作蛋白PIWIL1和PIWIL2的表達(dá)量均明顯上升，意味著PRC1.6復(fù)合體通過抑制piRNA調(diào)控途徑中重要RNA結(jié)合蛋白的表達(dá)水平來控制其活性[24]。除此之外，PRC1.6復(fù)合體組分自身的表達(dá)水平受到嚴(yán)密調(diào)控，目前這方面的報(bào)道相對(duì)較少，已知RYBP[101]和E2F6[102]等均可以受到miRNA的調(diào)控。

圖1 PRC1.6復(fù)合體與其他表觀遺傳調(diào)控機(jī)制的相互作用

圖2 PRC1.6復(fù)合體的組成、分子功能及活性異常產(chǎn)生的發(fā)育缺陷

標(biāo)注紅色的組分為具有酶活性或輔助酶活性的因子；標(biāo)注藍(lán)色的組分為具有DNA結(jié)合能力的轉(zhuǎn)錄因子；標(biāo)注綠色的組分為具有染色質(zhì)結(jié)合能力的因子。

6 結(jié)語與展望

本文總結(jié)了多梳蛋白PRC1.6復(fù)合體主要組分的分子功能及其在胚胎發(fā)育和精子發(fā)生中所扮演的重要角色(圖2)。胚胎發(fā)育過程中PRC1.6復(fù)合體通過抑制生殖譜系相關(guān)基因及其他發(fā)育調(diào)控基因的表達(dá)確保了胚胎的正常分化。PRC1.6復(fù)合體也參與精子發(fā)生的表觀遺傳調(diào)控，順次激活其不同階段生殖譜系相關(guān)基因的表達(dá)，是正常精子發(fā)生及性腺發(fā)育的必要條件。盡管在過去的幾年中有關(guān)PRC1.6復(fù)合體的組成及作用機(jī)制等方面取得了可喜的進(jìn)展，但是對(duì)于該復(fù)合體具體的工作機(jī)制包括組分之間及整個(gè)復(fù)合體與其他表觀調(diào)控途徑之間的協(xié)調(diào)關(guān)系等的研究還有待深入。如PRC1.6各組分在調(diào)節(jié)減數(shù)分裂及精子形成過程中的具體分工還不夠明確，以及該復(fù)合體如何實(shí)現(xiàn)對(duì)體細(xì)胞及不同分化階段的生殖細(xì)胞中相關(guān)特異性基因的差異調(diào)控等。隨著基因編輯技術(shù)以及現(xiàn)代分子生物學(xué)的進(jìn)一步深入發(fā)展，PRC1.6各組分的具體功能以及它們之間的相互作用機(jī)制一定會(huì)研究得更加清晰，并將為與PRC1.6復(fù)合體組分突變或功能缺陷相關(guān)的男性不育的診斷與治療帶來新的思路。

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Controlling the spatiotemporal expression of germ line specific genes by PRC1.6 complex

Xiaowei Sun1, Hongyang Li1, Jian Wang1, Bo Cheng1,2

Polycomb repressive complex 1 (PRC1) is a class of epigenetic regulatory complexes that normally represses gene expression by catalyzing and/or recognizing chromatin modifications. PRC1 mainly functions in stem cell maintenance, cell differentiation, cell cycle regulation and related processes. PRC1 also have aberrant functions which has been implicated in many types of developmental diseases and cancers. Mammalian PRC1 complexes are divided into six subtypes based on their composition and function; subtypes include PRC1.1 to PRC1.6. Each PRC1 subtype regulates a unique collection of target genes. The PRC1.6 complex subtype plays key roles in specifically repressing transcription of genes controlling germ cell development in embryonic stem cells and other somatic cell types. Recent research demonstrates that the PRC1.6 complex is also crucial for the timely activation of the germ line of specific genes during spermatogenesis, which is essential for proper gonad development. In this review, we summarize the identification of molecular functions of each core component of the PRC1.6 complex including how it recognizes and represses germ line specific genes. We also update the biological roles of this complex in regulating the spatiotemporal expression of germ line specific genes during embryonic development, gonad development, and spermatogenesis. Lastly, the crosstalk between the PRC1.6 complex and the other main epigenetic regulatory mechanisms involved in controlling spermatogenesis is discussed. Our discussion of the PRC1.6 complex in regulating germ line specific genes informs the studies of molecular processes of spermatogenesis and contributes to the understanding of the pathogenic mechanisms of male infertility.

polycomb repressive complex 1 (PRC1); PRC1.6; transcriptional repression; germ line specific genes; spatiotemporal expression

2019-01-27;

2019-03-14

國家自然科學(xué)基金項(xiàng)目(編號(hào)：31471233, 31771447)，中央高校基本科研業(yè)務(wù)費(fèi)(編號(hào)：lzujbky-2017-it51, lzujbky-2018-k05)和教育部細(xì)胞活動(dòng)與逆境適應(yīng)重點(diǎn)實(shí)驗(yàn)室開放基金項(xiàng)目(編號(hào)：lzujbky-2017-kb05, lzujbky-2018-kb05)資助[Supported by the National Natural Science Foundation of China (Nos. 31471233, 31771447), the Fundamental Research Funds for the Central Universities (Nos. lzujbky-2017-it51, lzujbky-2018-k05) and the Foundation of the Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations (Nos. lzujbky-2017-kb05, lzujbky-2018-kb05)]

孫曉偉，在讀博士研究生，專業(yè)方向：細(xì)胞生物學(xué)。E-mail: sunxw18@lzu.edu.cn李宏陽，在讀碩士研究生，專業(yè)方向：細(xì)胞生物學(xué)。E-mail: hyli17@lzu.edu.cn孫曉偉和李宏陽并列第一作者。

程博，博士，教授，研究方向：真核基因表達(dá)調(diào)控。E-mail: bocheng@lzu.edu.cn

10.16288/j.yczz.18-332

2019/4/1 9:34:53

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190401.0934.001.html

(責(zé)任編委: 苗龍)