首頁資訊科學(xué)網(wǎng)—連載：大牛朱健康最新植物DNA甲基化綜述（2）

科學(xué)網(wǎng)—連載：大牛朱健康最新植物DNA甲基化綜述（2）

來源：泰然健康網(wǎng) 時(shí)間：2024年12月03日 20:35

2018年5月21日，Nature Reviews Molecular Cell Biology在線發(fā)表了中國科學(xué)院上海植物逆境生物學(xué)研究中心朱健康研究員、張惠明研究員與郎曌博研究員共同完成的題為“Dynamics and function of DNA methylation in plants”的綜述文章。本博客將持續(xù)解讀該文章，本文為連載第二期，包括第一章節(jié)DNA甲基化動(dòng)態(tài)的第一部分內(nèi)容：RdDM介導(dǎo)的DNA甲基化建立。

Dynamics and function of DNA methylation in plants

First author: Huiming Zhang; Affiliations: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences (中國科學(xué)院上海植物逆境生物學(xué)研究中心): Shanghai, China

Corresponding author: Jian- Kang Zhu

DNA methylation dynamics

A specific DNA methylation state reflects the outcome of the dynamic regulation of establishment, maintenance and active-removal activities. These activities are catalysed by various enzymes that are targeted to specific genomic regions by distinct pathways. Plant DNA methylation occurs in all cytosine sequence contexts: CG, CHG and CHH (H represents A, T or C)12,13. In A. thaliana, genome-wide DNA methylation is characterized by heavy methylation in heterochromatin, which is enriched with transposable elements (transposons) and other repetitive DNA sequences12,14. Interspersed transposon-associated DNA methylation also exists in euchromatic chromosome arms12.

一個(gè)特異性DNA甲基化狀態(tài)反映了甲基化建立、維持和主動(dòng)去除的動(dòng)態(tài)調(diào)控的結(jié)果。這些過程受到多個(gè)酶的催化，并且由不同通路靶向到特異性基因組區(qū)域。植物DNA甲基化發(fā)生在所有的胞嘧啶序列上，包括三種類型，即CG、CHG和CHH，其中H代表了A、T或C。在擬南芥中，全基因組范圍的DNA甲基化特征主要是異染色質(zhì)中的高度甲基化，主要富集在轉(zhuǎn)座子元件和其他類型的DNA重復(fù)序列上。散在分布轉(zhuǎn)座子相關(guān)的DNA甲基化同樣在真核生物染色體臂上存在。

Establishment of DNA methylation by the RNA-directed DNA methylation pathway

In plants, de novo DNA methylation is mediated through the RNA-directed DNA methylation (RdDM) pathway, which involves small interfering RNAs (siRNAs) and scaffold RNAs in addition to an array of proteins (Fig. 1). According to the current understanding of canonical RdDM in A. thaliana7,11,15,16, the production of 24-nucleotide siRNAs is initiated through transcription by RNA POLYMERASE IV (POL IV), which is followed by RNA-DEPENDENT RNA POLYMERASE 2 (RDRP2; also known as RDR2)-dependent copying of the transcript to generate a double-stranded RNA (dsRNA) and by DICER-LIKE PROTEIN 3 (DCL3)-dependent cleavage of the dsRNA into siRNAs. The siRNAs are loaded onto ARGONAUTE (AGO) proteins, mainly AGO4 and AGO6, and pair with complementary scaffold RNAs, which are nascent (初生的) transcripts produced by POL V. AGO4 interacts with the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2)17, which catalyses de novo DNA methylation in a sequence-independent manner. This reaction may be assisted by RNA-DIRECTED DNA METHYLATION 1 (RDM1), which associates with both AGO4 and DRM2 and may bind single-stranded methylated DNA18 (Fig. 1).

在植物中，從頭DNA甲基化是由RNA指導(dǎo)的DNA甲基化通路（RdDM）所介導(dǎo)的，除了一系列的蛋白還涉及到了小干擾RNA（siRNA）和支架RNA（圖1）。根據(jù)目前對于擬南芥中經(jīng)典的RdDM通路理解，24核苷酸siRNA的產(chǎn)生是由RNA聚合酶POL IV通過轉(zhuǎn)錄起始的，起始后由RNA依賴性的RNA聚合酶RDRP2（也叫RDR2）進(jìn)行轉(zhuǎn)錄本拷貝以形成雙鏈的RNA（dsRNA），再由類DICER蛋白DCL3將dsRNA剪切成siRNA。這些siRNA會(huì)裝載到AGO蛋白上，主要是AGO4和AGO6，然后通過與支架RNA互補(bǔ)配對，這些支架RNA是由POL V. AGO4與DNA甲基轉(zhuǎn)移酶DRM2互作產(chǎn)生的，進(jìn)而通過一種不依賴于序列的方式催化從頭甲基化。該反應(yīng)可能由RDM1所協(xié)助，該蛋白可同時(shí)關(guān)聯(lián)AGO4和DRM2，還可能能夠結(jié)合單鏈的被甲基化的DNA（圖1）。

In addition to the sequence-specific pairing between siRNAs and scaffold RNAs, protein interactions between AGO4 and the AGO hook-containing proteins DNA-DIRECTED POL V SUBUNIT 1 (also known as NRPE1) and RDM3 are also important for RdDM. NRPE1 is the largest subunit of POL V, and RDM3 is a POL V-associated putative transcription elongation factor19,20. POL V-transcribed ncRNAs must remain on the chromatin to function as scaffold RNAs; this association seems to be facilitated by RRP6-LIKE 1 (RRP6L1), which is a homologue of the yeast and mammalian ribosomal RNA-processing 6 (RRP6) proteins that can function in RNA retention21. In addition, the siRNA-scaffold RNA pairing may be stabilized by the INVOLVED IN DE NOVO 2 (IDN2)-IDN2 PARALOGUE (IDP) complex, which binds RNA and interacts with the SWI/SNF chromatin-remodelling complex that contains SWI/SNF COMPLEX SUBUNIT SWI3B and participates in POL V-mediated transcriptional silencing by altering nucleosome positioning22-28.

除了siRNA和支架RNA之間序列特異性的配對外，AGO4和NRPE1及RDM之間的蛋白互作對于RdDM也是非常重要的。NRPE1是POL V最大的亞基，而RDM3是POL V相關(guān)的轉(zhuǎn)錄延伸因子。POL V轉(zhuǎn)錄的ncRNA肯定保留在染色質(zhì)上，作為支架RNA發(fā)揮功能，這個(gè)過程可能受到了RRP6L1的促進(jìn)，RRP6L1蛋白是酵母和哺乳動(dòng)物核糖體RNA加工RRP6蛋白的同源物，主要作用于RNA保留。另外，IDP復(fù)合物能夠穩(wěn)定siRNA與支架RNA之間的配對，IDP結(jié)合RNA，并與SWI/SNF染色質(zhì)重塑復(fù)合物互作，該復(fù)合物主要包含SWI/SNF復(fù)合物亞基SWI3B，并參與POL V介導(dǎo)的核小體位置改變引起的轉(zhuǎn)錄沉默。

The recruitment of POL IV and POL V to RdDM target loci can be facilitated by pre-existing chromatin modifications. POL IV is recruited by SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1), which binds dimethylated histone H3 lysine 9 (H3K9me2) through its Tudor domain29,30. SHH1 also interacts with SNF2 DOMAIN-CONTAINING PROTEIN CLASSY 1 (CLSY1), which is a chromatin-remodelling protein associated with POL IV and is required for POL IV-dependent siRNA production30,31 (Fig. 1). The association of POL V with chromatin for scaffold-RNA production requires the chromatin-remodelling DDR, which comprises the chromatin-remodelling protein DEFECTIVE IN RNADIRECTED DNA METHYLATION 1 (DRD1), the putative structural maintenance of chromosomes protein DEFECTIVE IN MERISTEM SILENCING 3 and RDM118,32-35 (Fig. 1). The DDR complex physically interacts with SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG PROTEIN 2 (SUVH2) and SUVH9, which are SUPPRESSOR OF VARIEGATION (SU(VAR)) 3-9 histone methyltransferase family proteins but lack histone methyltransferase activity36,37 (Fig. 1). SUVH2 and SUVH9 recognize methylated cytosine through theirSET and RING finger-associated (SRA) domains and are required for the proper genome-wide chromatin occupancy of POL V, and were therefore proposed to recruit POL V through pre-existing DNA methylation37. However, the tethering (用繩子拴住) of SUVH9 by a zinc-finger to unmethylated DNA is sufficient to recruit POL V and to establish DNA methylation and gene silencing37.

預(yù)先存在的染色質(zhì)修飾可以促進(jìn)招募POL IV和POL V到RdDM的靶向位點(diǎn)。POL IV由SHH1蛋白招募，該蛋白通過本身的Tudor結(jié)構(gòu)域結(jié)合二甲基化的組蛋白H3賴氨酸9（H3K9me2）。SHH1還能夠與CLSY1互作，而CLSY1蛋白是與POL IV相關(guān)的染色質(zhì)重塑蛋白，并且對于POL IV-依賴性的siRNA合成是必要的（圖1）。POL V與染色質(zhì)之間的關(guān)聯(lián)生成支架RNA這個(gè)過程需要染色質(zhì)重塑DDR復(fù)合物，主要由染色質(zhì)重塑蛋白DRD1、染色體結(jié)構(gòu)維持蛋白DMS3和RDM1構(gòu)成（圖1）。DDR復(fù)合物物理上能夠與SUVH2和SUVH9互作，這兩個(gè)蛋白是屬于SU(VAR)3-9組蛋白甲基轉(zhuǎn)移酶家族蛋白的成員，但缺失足蛋白甲基轉(zhuǎn)移酶活性（圖1）。SUVH2和SUVH9通SRA結(jié)構(gòu)域識別甲基化的胞嘧啶，對于POL V在全基因組范圍染色質(zhì)上的正確分布是必需的，因此被認(rèn)為通過預(yù)先存在的DNA甲基化來招募POL V。然而，通過鋅指將SUVH9栓到未甲基化的DNA上已經(jīng)能夠招募POL V，并建立起DNA甲基化和基因沉默。

Given that POL V can generate ncRNAs with different 5? ends from the same locus, it seems to initiate transcription independently of promoters38. Genomewide POL V or POL IV chromatin occupancy mapping did not reveal consensus promoter motifs29,39. Some POL V-transcribed ncRNAs have 7-methylguanosine caps at the 5? ends38, indicating that POL V-generated transcripts can be subjected to certain RNA-processing activities that are known to modify POL II-transcribed mRNAs. Nevertheless, POL V-generated transcripts are devoid (缺乏) of polyadenylation at their 3? ends and thus are different from mRNAs38. Unlike POL V transcripts, which are long enough to be detected by regular PCR38, POL IV-transcribed ncRNAs (P4 RNAs) are mostly 26–50 nucleotides in length and were thus identified only recently by small-RNA deep sequencing in A. thaliana with mutant dcl2, dcl3and dcl4(dcltriple mutant) and in A. thalianawith mutant dcl1, dcl2, dcl3and dcl4 (dclquadruple mutant)40-44, in which the cleavage of POL IV-dependent and RDR2-dependent dsRNAs into 24-nucleotide siRNAs is presumably blocked. P4 RNAs accumulate in dcltriple mutants and can be processed into 24-nucleotide siRNAs by exogenous DCL3. Because P4 RNAs are small, each 24-nucleotide siRNA could be produced from one slicing of a precursor P4 RNA42.

既然POL V能夠在同一位點(diǎn)產(chǎn)生帶有不同5?端的ncRNA，貌似起始轉(zhuǎn)錄的過程并不依賴于啟動(dòng)子。全基因范圍的POL V或POL IV染色質(zhì)分布圖譜并未發(fā)現(xiàn)一致的啟動(dòng)子基序。一些POL V轉(zhuǎn)錄的ncRNA在5?端具有7-甲基鳥苷的帽子，顯示POL V產(chǎn)生的轉(zhuǎn)錄本可以適用于一些已知的用于修飾POL II轉(zhuǎn)錄的mRNA加工過程。然而，POL V產(chǎn)生的轉(zhuǎn)錄本在3?端缺少多聚腺苷酸，因此與mRNA不同。與POL V轉(zhuǎn)錄本足夠長能夠被常規(guī)的PCR檢測到不一樣，POL IV轉(zhuǎn)錄的ncRNA（P4 RNA）大多只有46-50個(gè)核苷酸，因此只有近期對擬南芥的dcl三突和四突植株采用小RNA深度測序檢測到了這一類ncRNA，這兩個(gè)突變體植株中POL IV依賴性和RDR2依賴性的dsRNA剪切成24核苷酸siRNA可能失效了。P4 RNA在dcl三突植株中積累，而且可以被外源的DCL3處理成24核苷酸siRNA。因?yàn)镻4 RNA非常小，每一個(gè)24核苷酸siRNA都可能來自于一個(gè)P4 RNA前體的切割。

In addition to the canonical POL IV–RDR2–DCL3 pathway that generates 24-nucleotide siRNAs, paralogues of these proteins can also produce siRNAs that trigger non-canonical RdDM (Fig. 1). POL II-mediated transcription can not only generate 24-nucleotide siRNAs and scaffold RNAs but also recruits POL IV and POL V to promote siRNA production at some RdDM target loci45. POL II also has spatially distinct associations with different AGO proteins when compared with POL V46. At Trans- acting siRNA genes and at some regions of transcriptionally active transposons, RdDM depends on POL II and RDR6 rather than on POL IV and RDR247-49. RDR6-dependent RdDM can be mediated either through 21-nucleotide or 22-nucleotide siRNAs, which are produced by DCL2 and DCL4, or through 24-nucleotide siRNAs produced by DCL349,50.

除了經(jīng)典的POL IV–RDR2–DCL3通路產(chǎn)生24核苷酸siRNA，這些蛋白的旁系同源物同樣可以通過非典型RdDM通路產(chǎn)生siRNA（圖1）。POL II介導(dǎo)的轉(zhuǎn)錄不僅可以產(chǎn)生24核苷酸siRN和支架RNA，同時(shí)還能在某些RdDM靶向位點(diǎn)招募POL IV和POL V促進(jìn)siRNA的產(chǎn)生。POL II與POL V相比，能夠與不同的AGO蛋白有空間上不同的聯(lián)系。在反式激活siRNA基因和一些轉(zhuǎn)錄激活轉(zhuǎn)座子區(qū)域中，相比于POL IV和RDR2，RdDM更加依賴于POL II和RDR6。由DCL2和DCL4產(chǎn)生的21核苷酸或22核苷酸或者由DCL2產(chǎn)生的24核苷酸可以影響RDR6依賴性RdDM。

Genome wide, most siRNAs in A. thalianaare 24-nucleotide siRNAs, which disappear almost completely in the dclquadruple mutant; however, DNA methylation at approximately two-thirds of RdDM target regions still remains43,44, indicating the existence of DCL-independent RdDM that may be mediated by some DCL-independent siRNAs or directly by P4 RNAs (Fig. 1). RNase III enzymes other than DCL proteins could dice dsRNAs51, and in wild-type plants, they may work with DCLs in processing POL II, POL IV or POL V transcripts into siRNAs.

擬南芥中的siRNA大多數(shù)是24核苷酸的，在dcl四突變體植株中幾乎完全消失；然而，RdDM靶區(qū)域大約三分之二的DNA甲基化仍然保留，表明存在不依賴于DCL的RdDM存在，可能由一些不依賴于DCL的siRNA或直接由P4 RNA所介導(dǎo)（圖1）。除了DCL蛋白外的RNase III酶能夠切割dsRNA，并且在野生型植株中，RNase III酶可能與DCL蛋白一同發(fā)揮作用，將POL II、POL IV或POL V轉(zhuǎn)錄本加工成siRNA。

Genetic screens have identified some pre-mRNA splicing factors whose mutations reduce the levels of POL IV-dependent siRNAs to varying degrees52-55,although it remains largely unclear how these splicing factors affect siRNA levels. Similarly, mutations in two splicing factors, STABILIZED 1 and RDM16, reduce the accumulation of POL V-dependent scaffold RNAs53,54. Presumably, some of the splicing factors that normally bind pre-mRNAs may interact with the non-coding transcripts generated by POL IV and POL V and affect their processing or stability, thus influencing siRNA or scaffold RNA abundance.

遺傳篩選已經(jīng)鑒定了一些mRNA前體剪切因子，這些因子突變掉會(huì)不同程度地降低POL IV依賴性siRNA的水平，而這些剪切因子是如何影響siRNA的水平暫時(shí)還不清楚。比如說，STABILIZED 1和RDM16這兩個(gè)剪切因子的突變會(huì)降低POL V依賴性支架RNA的積累。據(jù)推測，一些能夠結(jié)合mRNA前體的剪切因子可能會(huì)跟由POL IV和POL V產(chǎn)生的非編碼轉(zhuǎn)錄本結(jié)合，并影響它們的加工或是穩(wěn)定性，進(jìn)而影響siRNA或支架RNA的豐度。

doi: https://doi.org/10.1038/s41580-018-0016-z

Journal: Nature Reviews Molecular Cell Biology

Published date: 21 May, 2018