A novel anti-RdDM element that defends cis-regulatory element against ncRNA-dependent, siRNA-driven, and methylation-imposed transcriptional silencing in plants [ARE ssRNAseq]

Cis-regulatory elements (CREs) dictate spatiotemporal expression and tissue specificity of proximal genes. However, when in a transgenic state, many of them become highly vulnerable to RNA-Directed DNA Methylation (RdDM) that is often transcriptionally deleterious and biologically detrimental. This transgene-specific RdDM vulnerability suggests the existence of anti-RdDM elements (AREs) to defend CREs against de novo methylation in vivo. In this work, we identify such an ARE at the Arabidopsis AGAMOUS (AG) locus, which includes a physically separated enhancer and promoter, both of which are highly vulnerable to transgene silencing. We demonstrate that this ARE effectively represses RdDM activity at its cognate and heterologous CREs via the inhibition of transcription and processing of potent non-coding RNAs (ncRNAs), which act as substrates for the biogenesis of 24-nt small interfering RNAs (siRNAs) that guide RdDM. Furthermore, we establish that the ARE exploits hypermethylation in a 108-bp internal region (referred to as M1) as a regulatory signal to recruit methyl reader SU(VAR)3-9 homolog 1 (SUVH1), as well as Harbinger transposon-derived protein 2 (HDP2) associated with HDP1, to carry out ARE-imposed transcriptional and post-transcriptional repression, with the former mediating the repression of ncRNA transcription and the latter repressing both ncRNA transcription and processing. We also show that M1 methylation is indispensable for the repression of methylation in an adjacent, methylation-vulnerable 737-bp region (dubbed M2) to safeguard the ARE’s functional integrity. Taken together, the present study uncovers a novel anti-RdDM element that defends CREs against ncRNA-dependent, siRNA-driven, and methylation-imposed epigenetic interference to safeguard their regulatory integrity. Overall design: Analyses of ncRNA expression and profile in various Arabidopis lines

顺式调控元件（CREs）规定邻近基因的空间-时间表达和组织特异性。然而，当处于转基因状态时，其中许多元件对RNA导向的DNA甲基化（RdDM）高度敏感，这种甲基化通常对转录有害，并从生物学角度带来损害。这种转基因特异性RdDM的易感性暗示了存在抗RdDM元件（AREs），以防御CREs在体内的去甲基化。在本研究中，我们鉴定了位于拟南芥AGAMOUS（AG）位点的一个ARE，该位点包括一个物理上分离的增强子和启动子，两者都极易受到转基因沉默的影响。我们证明了此ARE通过抑制强效非编码RNA（ncRNAs）的转录和加工，有效地抑制了其同源和异源CREs中的RdDM活性，这些ncRNAs作为24-nt小干扰RNA（siRNAs）的生物发生底物，引导RdDM。此外，我们确立该ARE利用一个108-bp内部区域（称为M1）的过度甲基化作为一个调控信号，以招募甲基阅读器SU(VAR)3-9同源体1（SUVH1），以及与HDP1相关的Harbinger转座子衍生的蛋白2（HDP2），以执行ARE施加的转录和转录后抑制，前者介导ncRNA转录的抑制，后者抑制ncRNA的转录和加工。我们还表明M1甲基化对于抑制相邻的、易受甲基化影响的737-bp区域（称为M2）的甲基化至关重要，以确保ARE的功能完整性。综上所述，本研究揭示了抗RdDM元素的新发现，该元素通过防御非编码RNA依赖性、siRNA驱动和甲基化诱导的表观遗传干扰，以保护CREs的调控完整性。总体设计：分析不同拟南芥品系中的ncRNA表达和谱系。