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

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE228515 | GEO | 2023/09/21

REPOSITORIES: GEO

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Project description: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 Cis-regulatory elements (CREs) control gene expression amplitude, tissue specificity and timing, whereby they directly impact phenotypic variation. However, in plants, CREs frequently become prone to RNA-directed DNA methylation (RdDM) when in a transgenic state (particularly in a tandem-repeat configuration), and the mechanism driving this phenomenon is currently unknown. In this study, we demonstrate that three of six flower-specific CREs, including the AGAMOUS enhancer (AGe), undergo transcriptional silencing when in a transgenic state, leading to the production of mutant flowers in 28-44% of transgenic lines. This silencing is dependent on the transgene self-transcribed non-coding RNAs (ncRNAs) that feed into biogenesis of 24-nt small interfering RNAs (siRNAs) and the canonical RdDM pathway, but not RNAs transcribed by the RNA polymerase II promoter. Strikingly, this ncRNA-dependent, siRNA-driven RdDM is prone to the enhancing action of an adjacent CaMV35S enhancer (35Se), which was shown to promiscuously interact with, and ectopically activate, numerous CREs including the AGe. This enhancement corresponded with an increase in ncRNAs specifically transcribed from the antisense strand of the transgene. We further demonstrate that the same 35Se inserted approximately 3.5 kb upstream of the native AGe within a genome promiscuously interacts with and activating de novo RdDM activity in the AGe, resulting in a mutant flower. These findings provide the first evidence that promiscuous CRE-CRE interactions serve as a causative force driving RdDM/silencing, and novel insight into the mechanism underlying the methylation-vulnerablity of the CRE transgenes, duplicated genes and repetitive genomic regions rich in transposon elements, where promiscuous CRE-CRE interactions are common.

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

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