Molecular functions of DNA methylation
DNA甲基化與組蛋白修飾及非組蛋白共同決定了染色質(zhì)結(jié)構(gòu)和開(kāi)放程度。因此，DNA甲基化有助于調(diào)控基因表達(dá)、轉(zhuǎn)座子沉默、染色質(zhì)互作（圖4）以及性狀遺傳（Box 1）。
Gene regulation
植物中基因相關(guān)的DNA甲基化發(fā)生在啟動(dòng)子區(qū)或者轉(zhuǎn)錄基因區(qū)。盡管某些情況下DNA甲基化會(huì)促進(jìn)基因的表達(dá)，比如說(shuō)擬南芥中ROS1基因以及番茄中數(shù)百個(gè)抑制果實(shí)成熟的基因，但多數(shù)情況下啟動(dòng)子DNA甲基化會(huì)抑制基因的轉(zhuǎn)錄。啟動(dòng)子DNA甲基化通過(guò)阻止轉(zhuǎn)錄激活子的結(jié)合或促進(jìn)轉(zhuǎn)錄抑制子的結(jié)合直接抑制基因的表達(dá)，或者通過(guò)促進(jìn)H3K9me2等抑制性組蛋白修飾和抑制組蛋白乙?；却龠M(jìn)性組蛋白修飾來(lái)間接抑制基因的表達(dá)（圖4a）。啟動(dòng)子甲基化如何激活基因的轉(zhuǎn)錄還不是很清楚。想來(lái)，DNA甲基化可能會(huì)增強(qiáng)某些轉(zhuǎn)錄激活子的結(jié)合或是抑制某些轉(zhuǎn)錄抑制子的結(jié)合。啟動(dòng)子區(qū)的DNA甲基化通常是來(lái)自附近轉(zhuǎn)座子或其他重復(fù)序列上甲基化擴(kuò)散的結(jié)果?；蜞徑霓D(zhuǎn)座子和重復(fù)序列同樣受到主動(dòng)DNA去甲基化的作用以保護(hù)基因免受轉(zhuǎn)錄沉默。在啟動(dòng)子DNA甲基化激活基因表達(dá)的情況中，主動(dòng)去甲基化會(huì)導(dǎo)致基因的轉(zhuǎn)錄沉默。
DNA甲基化控制
基因表達(dá)，轉(zhuǎn)座子沉默和染色體相互作用?；騿?dòng)子中的| DNA甲基化(m)通常會(huì)抑制轉(zhuǎn)錄，但在某些情況下，它可以增加轉(zhuǎn)錄3,8,12,106,107?；蝮w中b| DNA甲基化主要存在于CG context12,13,120,121，其功能尚不清楚。一些異染色質(zhì)內(nèi)含子中| DNA甲基化吸引了抗沉默1 (ASI1) - ASI1免疫沉淀蛋白1 (AIPP1) -增強(qiáng)霜霉病2 (EDM2)復(fù)合物，以促進(jìn)mRNA 132 - 136過(guò)程中選擇性遠(yuǎn)端聚腺苷化位點(diǎn)(紅色星號(hào))的選擇。ASI1與染色質(zhì)結(jié)合并結(jié)合RNA;EDM2在異染色質(zhì)中識(shí)別二甲基組蛋白H3賴氨酸9 (H3K9me2)。d | DNA甲基化對(duì)于沉默轉(zhuǎn)座子和其他DNA重復(fù)序列也很重要，這些重復(fù)序列主要位于內(nèi)包熵外異染色質(zhì)12120中。| DNA甲基化參與了內(nèi)包熵區(qū)域之間的染色體相互作用和一些相互作用的異染色質(zhì)島，這些異染色質(zhì)島是染色質(zhì)抑制區(qū)域，位于其他純色染色體臂上，以豐富的轉(zhuǎn)座子和小rnas145146為特征。在所有的面板中，轉(zhuǎn)座子和其他重復(fù)序列是黃色的，基因是藍(lán)色的。黑色和黃色的染色體區(qū)域分別代表著著絲粒和包內(nèi)熵區(qū)域。POL II, RNA聚合酶II 。
In A. thaliana, only approximately 5% of the genes are methylated in promoter regions. As a result, DNA methylation does not regulate the transcription of many genes, and most mutants with decreased or increased DNA methylation do not have severely impaired growth or development11. By contrast, crop plants with larger genomes can have a higher transposon content and more transposons that are close to genes; consequently, thereare more genes with promoter methylation3. Therefore, DNA methylation has more important roles in gene regulation in several crop plants than in A. thaliana, and DNA methylation mutants in these crop plants are generally either lethal or have severe growth and developmental defects3,116-119.
在擬南芥中，僅僅約有5%的基因的啟動(dòng)子區(qū)存在甲基化。因此，DNA甲基化對(duì)于大多數(shù)的基因來(lái)說(shuō)并不調(diào)控它們的轉(zhuǎn)錄，所以大多數(shù)增加或降低DNA甲基化水平的突變并不會(huì)導(dǎo)致嚴(yán)重的生長(zhǎng)和發(fā)育缺陷。相反，作物擁有更大的基因組，因此其含有更高的轉(zhuǎn)座子含量，進(jìn)而會(huì)有更多的臨近基因的轉(zhuǎn)座子，結(jié)果導(dǎo)致了作物中基因啟動(dòng)子區(qū)的甲基化水平會(huì)增高。因此，DNA甲基化在某些作物中作用于基因表達(dá)調(diào)控要比在擬南芥中更加重要，并且在這些作物中DNA甲基化突變要么致死，要么會(huì)導(dǎo)致嚴(yán)重的生長(zhǎng)和發(fā)育缺陷。
The gene bodies of over one-third of A. thaliana genes are methylated12. In contrast to transposons and repeats, which are usually heavily methylated in all three cytosine contexts, DNA methylation in gene bodies has very little non-CG methylation12,13,120,121 (Fig. 4b). Gene body methylation (gbM) preferentially occurs at exons and is absent from the transcription start and stop sites121. As a conserved feature in most angiosperms, genes with gbM tend to be longer than unmethylated genes and are generally constitutively expressed12,121,122. In two angiosperms, Eutrema salsugineumand Conringia planisiliqua, genome-wide loss of gbM was attributed to the loss of CMT3123,124. Levels of gbM decreased in A. thaliana with reduced histone H3.3 levels, and this correlated with increased density of the linker histone H1, suggesting that gbM is facilitated by histone H3.3, which inhibits histone H1-dependent chromatin folding and consequently increases chromatin accessibility to DNA methylases125.
三分之一的擬南芥基因的基因區(qū)存在甲基化。與轉(zhuǎn)座子和重復(fù)序列中存在三種不同類型的胞嘧啶甲基化，基因區(qū)的DNA甲基化存在很少的non-CG類型甲基化（圖4b）。基因區(qū)甲基化會(huì)優(yōu)先發(fā)生在外顯子上，但在起始和終止位點(diǎn)上不存在甲基化。大多數(shù)被子植物比較保守的一個(gè)特征就是，能夠發(fā)生DNA甲基化的基因要比沒(méi)有DNA甲基化的基因長(zhǎng)度更長(zhǎng)，并且通常是組成型表達(dá)的。在山崳菜和線果芥中，由于缺失了CMT3而導(dǎo)致了全基因組范圍上不存在基因區(qū)的DNA甲基化。在擬南芥中，基因區(qū)DNA甲基化水平會(huì)隨著組蛋白H3.3水平的降低而降低，并且與組蛋白H1的密度增加相關(guān)，說(shuō)明組蛋白H3.3通過(guò)抑制組蛋白H1依賴性的染色質(zhì)折疊，進(jìn)而增加染色質(zhì)對(duì)DNA甲基化酶的開(kāi)放程度來(lái)促進(jìn)基因區(qū)的DNA甲基化。
Gene body CG methylation is almost completely absent in the A. thaliana met1-3 mutant, in which steady-state mRNA levels of gbM genes do not appear to be globally increased relative to unmethylated genes12. Additionally, natural variation in gbM does not correlate with global gene expression levels in A. thaliana populations126. On the other hand, a comparison between the grass Brachypodium distachyon and rice (Oryza sativa Japonica group) showed that gbM is strongly conserved among orthologues of the two species and affects a biased subset of long, slowly evolving genes121. Thus, the biological importance of gbM in angiosperms seems to be species dependent. Considering that enrichment of the histone variant H2A.Z in gene bodies correlates with gene responsiveness to environmental and developmental stimuli and that the genomic distributions of H2A.Z and DNA methylation in A. thaliana are anti-correlative, gbM was proposed to reduce gene expression variability by excluding H2A.Z from nucleosomes127. In addition, gbM in plants may prevent aberrant transcription from internal cryptic promoters128. Indeed, in mouse cells, intragenic DNA methylation protects the gene body from spurious Pol II entry and cryptic transcription initiation129. It was also suggested that gbM increases pre-mRNA splicing efficiency in plants127, which is consistent with the observation that a small portion of alternative exon-intron junctions are affected by the global loss of CG methylation in the O. sativa met1-2 mutant130.
擬南芥met1-3突變體中基因區(qū)基本沒(méi)有CG甲基化，在該突變體中，基因區(qū)存在甲基化的基因的mRNA穩(wěn)定程度相對(duì)于基因區(qū)不存在甲基化基因并沒(méi)有顯著的提升。另外，擬南芥群體中基因區(qū)甲基化的自然變異并不于基因的表達(dá)水平相關(guān)。另一方面，短柄草與水稻的比較分析顯示在這兩個(gè)物種同源基因之間基因區(qū)甲基化十分保守，并且會(huì)偏向于影響一些長(zhǎng)的、演化比較慢的基因。因此，被子植物中基因區(qū)甲基化的生物學(xué)重要性似乎各個(gè)物種各有不同?？紤]到組蛋白變體H2A.Z在基因區(qū)的富集與響應(yīng)于環(huán)境和發(fā)育刺激的基因相關(guān)，并且擬南芥中H2A.Z和DNA甲基化的分布呈負(fù)相關(guān)模式，所以可以認(rèn)為基因區(qū)甲基化通過(guò)將H2A.Z從核小體上排除開(kāi)來(lái)進(jìn)而降低基因的表達(dá)變異。另外，植物中的基因區(qū)甲基化可能起到防止基因在啟動(dòng)子區(qū)潛在的起始密碼子處開(kāi)始轉(zhuǎn)錄的作用。確實(shí)，在小鼠細(xì)胞中，基因內(nèi)的DNA甲基化會(huì)保護(hù)基因區(qū)免受錯(cuò)誤的Pol II 結(jié)合及隱藏性轉(zhuǎn)錄起始。同時(shí)，基因區(qū)甲基化增加植物初級(jí)mRNA的剪切效率，這與在水稻met1-2突變體中所觀察到的缺失CG甲基化會(huì)影響一小部分可變外顯子-內(nèi)含子連接點(diǎn)的現(xiàn)象一致。
Some gene introns harbour transposons or other repeats, which are heavily methylated in all cytosine sequences and regulate mRNA processing, for example, alternative polyadenylation. Loss of DNA methylation in a long interspersed nuclear element retrotransposon in the intron of the homeotic gene DEFICIENS causes alternative splicing and premature termination and consequently the generation of the unproductive mantled somaclonal variant of oil palm131. An intron of the A. thaliana INCREASE IN BONSAI METHYLATION 1 (IBM1; also known as JMJ25) gene, which encodes a histone H3K9 demethylase, contains a heterochromatic repeat element that is recognized by a newly discovered protein complex that promotes distal polyadenylation of IBM1 transcripts132-136 (Fig. 4c). This protein complex consists of ANTI-SILENCING 1 (ASI1),ENHANCED DOWNY MILDEW 2 (EDM2) and ASI1-IMMUNOPRECIPITATED PROTEIN 1 (AIPP1). ASI1 is an RNA-binding protein that contains a BAH domain that may mediate its chromatin association with the heterochromatin region within the IBM1 intron132,133. EDM2 contains a composite plant homeodomain (PHD) that recognizes both the transcription- repressing H3K9me2 and transcription-activating H3K4me3 modifications, which together characterize introns that contain heterochromatin repeats134. AIPP1 interacts with both ASI1 and EDM2, thereby promoting the formation of the complex, which also promotes distal polyadenylation of many other genes that similarly harbour intronic heterochromatin135, although the mechanism by which the complex promotes alternative polyadenylation is unknown. Mutation of ASI1, EDM2 or AIPP1 indirectly causes gene silencing owing to the loss of full-length, functional transcripts of IBM1. ASI1 also associates with AIPP2, which has a PHD domain, AIPP3, which has a BAH domain, and the POL II carboxy-terminal domain phosphatase CARBOXY-TERMINAL PHOSPHATASELIKE 2135. Intriguingly, mutations in the three proteins had opposing effects on gene regulation compared with mutations in the ASI1-AIPP1-EDM2 complex135.
一些基因內(nèi)含子會(huì)包含轉(zhuǎn)座子或者重復(fù)序列，所以可能會(huì)存在不同類型的胞嘧啶甲基化，并調(diào)控mRNA的加工，比如說(shuō)可變聚腺苷酸化。在油棕的同源異型基因DEFICIENS的內(nèi)含子中的LINE逆轉(zhuǎn)座子上DNA甲基化的缺失會(huì)導(dǎo)致可變剪切和提前終止，并最終導(dǎo)致產(chǎn)生非生殖性體細(xì)胞無(wú)性系變體。擬南芥編碼組蛋白H3K9去甲基化酶的IBM1的內(nèi)含子含有一個(gè)異染色質(zhì)重復(fù)序列，能夠被一個(gè)新鑒定的蛋白復(fù)合物所識(shí)別，促進(jìn)IBM1轉(zhuǎn)錄本的遠(yuǎn)端多聚腺苷酸化（圖4c）。該蛋白復(fù)合物由ASI1、EDM2和AIPP1構(gòu)成。ASI1是一個(gè)RNA結(jié)合蛋白，包含一個(gè)BAH結(jié)構(gòu)域，可能介導(dǎo)其染色質(zhì)與IBM1內(nèi)含子區(qū)內(nèi)異染色質(zhì)之間的關(guān)聯(lián)。EDM2含有一個(gè)植物同源結(jié)構(gòu)域PHD，能夠同時(shí)識(shí)別轉(zhuǎn)錄抑制H3K9me2和轉(zhuǎn)錄激活H3K4me3兩種修飾，兩者同時(shí)構(gòu)成了含異染色質(zhì)重復(fù)序列的內(nèi)含子特征。AIPP1同時(shí)與ASI1和EDM2互作，因而促進(jìn)該復(fù)合物的形成，同時(shí)促進(jìn)許多其它有著類似內(nèi)含子的異染色質(zhì)的基因的遠(yuǎn)端多聚腺苷酸化，盡管目前對(duì)于該復(fù)合物是如何促進(jìn)可變多聚腺苷酸化的具體分子機(jī)制還不是很清楚。ASI1、EDM2或者AIPP1的突變因?yàn)槿L(zhǎng)、功能性IBM1轉(zhuǎn)錄本的缺失間接導(dǎo)致基因沉默。ASI1同時(shí)還與含有一個(gè)PHD結(jié)構(gòu)域的AIPP2、含有一個(gè)BAH結(jié)構(gòu)域的AIPP3以及POL II羧基末端結(jié)構(gòu)域磷酸酶CARBOXY-TERMINAL PHOSPHATASELIKE 2相關(guān)。有趣的是，這三個(gè)蛋白的突變與ASI1-AIPP1-EDM2復(fù)合物的突變相比存在相反的基因調(diào)控影響模式。
