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