AbstractIn plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNAs and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components have been uncovered that are involved in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated.

摘要在植物中，植物特异性RNA聚合酶V（Pol V）转录非编码RNA，并为RNA定向DNA甲基化（RdDM）途径中辅助蛋白的缔合提供了对接平台。已经发现了参与DNA甲基化过程的各种成分，但仍不清楚Pol V的转录是如何调控的。

Here, we report that the conserved RNA polymerase II (Pol II) elongator, SPT6L, binds to thousands of intergenic regions in a Pol II-independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupies with the largest subunit of Pol V (NRPE1) and mutation of SPT6L leads to the reduction of DNA methylation but not Pol V enrichment.

在这里，我们报告说，保守的RNA聚合酶II（Pol II）延伸子SPT6L以不依赖Pol II的方式与数千个基因间区域结合。有趣的是，SPT6L的基因间富集与Pol V的最大亚基（NRPE1）共同占据，SPT6L的突变导致DNA甲基化减少，但不导致Pol V富集。

Furthermore, the association of SPT6L at Pol V loci is dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 is compromised in spt5l. Finally, Pol V RIP-seq reveals that SPT6L is required to maintain the amount and length of Pol V transcripts.

此外，SPT6L在Pol V基因座上的关联取决于Pol V相关因子SPT5L，而不是Pol V的存在，并且SPT6L和NRPE1之间的相互作用在SPT5L中受到损害。最后，Pol V RIP-seq揭示了SPT6L是维持Pol V转录本的数量和长度所必需的。

Our findings thus uncover the critical role of a Pol II conserved elongator in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and II in plants..

因此，我们的发现揭示了Pol II保守延伸子在Pol V介导的DNA甲基化和转录中的关键作用，并阐明了植物中Pol V和II之间的相互调节。。

IntroductionIn eukaryotic cells, transcription elongation is a dynamic and highly regulated process, in which a variety of functionally distinct transcript elongation factors are involved in Pol II progression1,2. Among them, the conserved elongator, SPT6, is recruited by the phosphorylated Pol II3 and involved in the enhancement of elongation rate4,5,6, repression of intragenic initiation7,8, and transcription termination5 in yeast and animal cells.

引言在真核细胞中，转录延伸是一个动态且高度调控的过程，其中多种功能不同的转录延伸因子参与Pol II进展1,2。其中，保守的延伸因子SPT6被磷酸化的Pol II3募集，并参与酵母和动物细胞中延伸率的提高4,5,6，基因内启动的抑制7,8和转录终止5。

In plants, the functional homolog of SPT6, SPT6-like (SPT6L), interacts with phosphorylated Pol II and plays conserved roles in Pol II progression9. The mutation of SPT6L causes pleiotropic defects in embryogensis10 and post-germination stages9. Recently, it was found that SPT6L was able to recruit chromatin remodelers SWI2/SNF2 at transcription start sites (TSS) in a Pol II-independent manner11, indicating a potential role of SPT6L in transcription initiation/early elongation in plants.Different from animal and yeast cells, in plants, two plant-specific RNA polymerases (Pol IV and V) have evolved and they play essential roles in the establishment and maintenance of DNA methylation through the RdDM pathway12,13.

在植物中，SPT6的功能同源物SPT6样（SPT6L）与磷酸化的Pol II相互作用，并在Pol II进展中起保守作用9。SPT6L的突变导致胚胎发生和萌发后阶段的多效性缺陷9。最近，发现SPT6L能够以不依赖Pol II的方式在转录起始位点（TSS）募集染色质重塑剂SWI2/SNF2 11，表明SPT6L在植物转录起始/早期延伸中的潜在作用。与动物和酵母细胞不同，在植物中，两种植物特异性RNA聚合酶（Pol IV和V）已经进化，它们在通过RdDM途径建立和维持DNA甲基化中起着至关重要的作用12,13。

In general, the canonical RdDM pathway is composed of two parts: the production of 24-nt siRNA and the establishment of DNA methylation13. The production of 24-nt siRNA is accomplished by Pol IV’s transcription, RNA-DEPENDENT RNA POLYMERASE2 (RDR2)’s generation of double-stranded RNA, and DICER-LIKE PROTEINs (DCLs) dependent cleavage13.

通常，经典的RdDM途径由两部分组成：24-nt siRNA的产生和DNA甲基化的建立13。24-nt siRNA的产生是通过Pol IV的转录，RNA依赖性RNA聚合酶2（RDR2）产生双链RNA和DICER样蛋白（DCLs）依赖性切割13完成的。

In the second part, Pol V transcripts serve as a docking platform to recruit AGOs-siRNA complex and other accessory proteins to establish DNA methylation. The RdDM pathway is a self-reinforcing loop14 and the reduced siRNA and DNA methylation negatively affect the transcription of Po.

在第二部分中，Pol V转录本充当对接平台，以募集AGOs siRNA复合物和其他辅助蛋白以建立DNA甲基化。RdDM途径是一个自我增强的环14，减少的siRNA和DNA甲基化对Po的转录产生负面影响。

Data availability

数据可用性

The data that support the findings of this study are available from the corresponding author upon request. The high-throughput sequencing data generated in this study have been deposited in Gene Expression Omnibus with the accession code GSE233781. Source data are provided with this paper.

支持本研究结果的数据可应要求从通讯作者处获得。本研究中产生的高通量测序数据已保存在Gene Expression Omnibus中，登录号为GSE233781。本文提供了源数据。

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Download referencesAcknowledgementsWe thank the Arabidopsis Biological Resource Centre for providing the mutant seeds used in this study. The mutant seeds of nrpd1-3, nrpe1-11, and drm1/2 were kindly provided by Dr. Shulin Deng. This work was supported by the National Natural Science Foundation of China to C.C.

下载参考文献致谢我们感谢拟南芥生物资源中心提供本研究中使用的突变种子。nrpd1-3，nrpe1-11和drm1/2的突变种子由邓树林博士提供。这项工作得到了国家自然科学基金委员会的支持。

(32070648) and J.S. (32100474), Guangdong Pearl River Talent Program to C.C. (2021QN020018), Science and Technology Projects in Guangzhou to C.C. (E3330900-01), and the Natural Science and Engineering Council of Canada to Y.C. (RGPIN/04625-2017).Author informationAuthors and AffiliationsState Key Laboratory of Plant Diversity and Specialty Crops, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, ChinaYujuan Liu, Zhi Zhang, Jinyuan Liu, Changhu Wang & Chen ChenKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, ChinaYujuan Liu, Jie Shu, Zhi Zhang, Ning Ding, Jinyuan Liu, Changhu Wang & Chen ChenUniversity of the Chinese Academy of Sciences, Beijing, 100049, ChinaYujuan Liu, Zhi Zhang, Jinyuan Liu, Changhu Wang & Chen ChenGuangdong Academy of Agricultural Sciences, Guangzhou, Guangdong, 510640, ChinaJie Shu & Jun LiuMOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, 730000, ChinaNing DingAgriculture and Agri-Food Canada, London Research and Development Centre, London, ON, N5V 4T3, CanadaYuhai CuiDepartment of Biology, Western University, London, ON, N6A 5B7, CanadaYuhai CuiAuthorsYujuan LiuView author publicationsYou can also search for this author in.

（32070648）和J.S.（32100474），广东珠江人才计划（2021QN020018），广州科技项目（E3330900-01），加拿大自然科学与工程委员会（RGPIN/04625-2017）。作者信息作者及所属单位中国科学院华南植物园植物多样性与特色作物国家重点实验室，广东广州，510650，刘玉娟，张志，刘金元，王昌虎，陈晨华南农业植物分子分析与遗传改良重点实验室，中国科学院华南植物园应用植物学广东省重点实验室，广东广州，510650，刘玉娟，舒洁，张志，丁宁，刘金元，王昌虎，陈晨中国科学院大学，北京，100049，刘玉娟，张志，刘金元，王昌虎，陈光山东农业科学院，广东广州，510640，中国兰州大学生命科学学院细胞活动与应激适应重点实验室，兰州，730000，中国宁丁农业和农业食品加拿大，伦敦研究与发展中心，伦敦，安大略省，N5V 4T3，CanadaYuhai Cui，伦敦，安大略省，N6A 5B7，CanadaYuhai Cui生物学系作者刘玉娟（Yujuan LiuView）作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsC.C. conceived the project; C.C., C.H.W., and Y.J.L. designed the experiments; N.D. performed SPT6LdeltaWG/GW-related ChIP-seq and immunoblots; J.S. performed the all the ChIP-qPCR assays; Z.Z. examined qPCR and Chop-PCR experiments; Y.J.L. performed all the rest of the experiments; Y.J.L., J.Y.L., and J.L.

PubMed谷歌学术贡献中心。C、 构思了这个项目；C、 C.，C.H.W。和Y.J.L.设计了实验；N、 D.进行SPT6LdeltaWG/GW相关的ChIP-seq和免疫印迹；J、 S.进行了所有ChIP-qPCR分析；Z、 Z.检查了qPCR和Chop PCR实验；Y、 J.L.完成了所有其余的实验；Y、 J.L.，J.Y.L。和J.L。

analyzed ChIP-seq data; C.C. and Y.H.C. analyzed rest of high-throughput data; C.C. and Y.J.L. wrote the paper.Corresponding authorsCorrespondence to.

分析ChIP-seq数据；C、 C.和Y.H.C.分析了其余的高通量数据；C、 C.和Y.J.L.写了这篇论文。通讯作者通讯。

Changhu Wang or Chen Chen.Ethics declarations

王昌虎或陈晨。道德宣言

Competing interests

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Reprints and permissionsAbout this articleCite this articleLiu, Y., Shu, J., Zhang, Z. et al. A conserved Pol II elongator SPT6L mediates Pol V transcription to regulate RNA-directed DNA methylation in Arabidopsis.

转载和许可本文引用本文Liu，Y.，Shu，J.，Zhang，Z。等人。保守的Pol II延伸子SPT6L介导Pol V转录以调节拟南芥中RNA定向的DNA甲基化。

Nat Commun 15, 4460 (2024). https://doi.org/10.1038/s41467-024-48940-8Download citationReceived: 04 July 2023Accepted: 14 May 2024Published: 25 May 2024DOI: https://doi.org/10.1038/s41467-024-48940-8Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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Chromatin immunoprecipitationDNA methylationNext-generation sequencingPlant molecular biology

染色质免疫沉淀DNA甲基化下一代测序植物分子生物学

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