2017年5月31日，国际著名学术期刊《Genome biology》在线发表了中国科学院上海植物逆境生物学研究中心张蘅研究组题为“The developmental regulator PKL is required to maintain correct DNA methylation patterns at RNA-directed DNA methylation loci”的研究论文。该研究揭示了染色质重塑因子PKL在RNA介导的DNA甲基化过程中的重要调控作用。杨荣博士是该文章的第一作者，张蘅研究员为通讯作者。
在植物中，RNA介导的DNA甲基化(RdDM)是一种重要的建立全新DNA甲基化式样和转录基因沉默的机制，通过小干扰RNA(siRNA)与支架RNA(scaffold RNA)的碱基配对引导DNA甲基转移酶到特定的位点进行全新DNA甲基化。RdDM在外源基因沉默、维持基因组稳定性、生殖细胞DNA甲基化模式建立等生物学过程中起重要作用，解析其分子机理对于实现特定基因的转录沉默或激活具有重要意义。
在这项研究中，张蘅研究组通过正向遗传学筛选到了两个新的参与转基因沉默的突变体。图位克隆发现这两个突变体是由于一个重要的发育调控基因PICKLE(PKL)功能缺失造成的。全基因组甲基化分析发现PKL影响大约一半RdDM位点的正常DNA甲基化，其中上升和下降的位点大致相当。在RNA介导的DNA甲基化过程中，DNA甲基化的位点特异性主要由两类非编码rna界定：小干扰RNA(siRNA)和支架RNA(scaffold RNA)，分别由植物特有的RNA聚合酶Pol IV和Pol V参与生成。因此研究人员也分析了全基因组的小干扰RNA和特定位点的支架RNA水平。他们发现在pkl突变体中，部分位点的DNA甲基化水平的变化伴随着相同趋势的小干扰RNA水平和支架RNA水平的变化。同时PKL还促进多个受Pol V调节的核小体的定位，说明与Pol V相关的功能可能受到了影响。
通过分析mRNA转录组与全基因组DNA甲基化的相关性，研究人员发现在pkl突变体中，虽然一定数量的转座子和基因的转录产物上升伴随着DNA甲基化的下降，大部分位点的DNA甲基化变化不足以释放转录沉默。因此研究人员设想，在RdDM的靶位点区域，PKL能够通过其核小体重塑活性改变染色质环境，从而影响非编码RNA的产生和转录沉默。此论文揭示了CHD家族蛋白PKL在RNA介导的DNA甲基化过程中的作用并暗示RNA聚合酶Pol IV/V可以使用与Pol II相同的染色体重塑因子进行转录调控。
原文链接：
The developmental regulator PKL is required to maintain correct DNA methylation patterns at RNA-directed DNA methylation loci
原文摘要：
Background
The chromodomain helicase DNA-binding family of ATP-dependent chromatin remodeling factors play essential roles during eukaryote growth and development. They are recruited by specific transcription factors and regulate the expression of developmentally important genes. Here, we describe an unexpected role in non-coding RNA-directed DNA methylation in Arabidopsis thaliana.
Results
Through forward genetic screens we identified PKL, a gene required for developmental regulation in plants, as a factor promoting transcriptional silencing at the transgenic RD29A promoter. Mutation of PKL results in DNA methylation changes at more than half of the loci that are targeted by RNA-directed DNA methylation (RdDM). A small number of transposable elements and genes had reduced DNA methylation correlated with derepression in the pkl mutant, though for the majority, decreases in DNA methylation are not sufficient to cause release of silencing. The changes in DNA methylation in the pkl mutant are positively correlated with changes in 24-nt siRNA levels. In addition, PKL is required for the accumulation of Pol V-dependent transcripts and for the positioning of Pol V-stabilized nucleosomes at several tested loci, indicating that RNA polymerase V-related functions are impaired in the pkl mutant.
Conclusions
PKL is required for transcriptional silencing and has significant effects on RdDM in plants. The changes in DNA methylation in the pkl mutant are correlated with changes in the non-coding RNAs produced by Pol IV and Pol V. We propose that at RdDM target regions, PKL may be required to create a chromatin environment that influences non-coding RNA production, DNA methylation, and transcriptional silencing.
作者：张蘅 点击：次
