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

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: Comparison of DNA methylation profile among different lines

调控性元件（CREs）调控邻近基因的空间时序表达和组织特异性。然而，在转基因状态下，许多调控性元件对RNA指导的DNA甲基化（RdDM）极为敏感，而RdDM通常具有转录性损害和生物学上不利的影响。这种针对转基因的RdDM易感性提示了存在抗RdDM元件（AREs），以防御CREs在体内的新发甲基化。在本研究中，我们在拟南芥AGAMOUS（AG）基因座上鉴定了一个这样的ARE，该基因座包括一个物理上分离的增强子和启动子，它们都对转基因沉默极为敏感。我们证明该ARE通过抑制具有强大非编码RNA（ncRNAs）转录和加工的方式，有效地抑制了其同源和异源CREs上的RdDM活性，这些ncRNAs作为引导RdDM的24-nt小干扰RNA（siRNAs）的生物发生底物。此外，我们确立了ARE利用108-bp内部区域（被称为M1）的超甲基化作为调控信号，以招募甲基阅读器SU(VAR)3-9同源物1（SUVH1），以及与HDP1相关的Harbinger转座子衍生的蛋白2（HDP2），以执行由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、甲基化易感区域（称为M2，长度为737-bp）的甲基化是必不可少的，以保障ARE的功能完整性。综上所述，本研究揭示了一种新型抗RdDM元件，它通过防御依赖于ncRNA、由siRNA驱动和甲基化引起的表观遗传干扰，来保护CREs的调控完整性。总体设计：比较不同品系间的DNA甲基化谱。