R-Loop Stabilization Represses Antisense Transcription at the Arabidopsis FLC Locus

Abstract

Roles for long noncoding RNAs (lncRNAs) in gene expression are emerging, but regulation of the lncRNA itself is poorly understood. We have identified a homeodomain protein, AtNDX, that regulates COOLAIR, a set of antisense transcripts originating from the 3' end of Arabidopsis FLOWERING LOCUS C (FLC). AtNDX associates with single-stranded DNA rather than doublestranded DNA non-sequence-specifically in vitro, and localizes to a heterochromatic region in the COOLAIR promoter in vivo. Single-stranded DNA was detected in vivo as part of an RNA-DNA hybrid, or R-loop, that covers the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR transcription, which in turn modifies FLC expression. Differential stabilization of R-loops could be a general mechanism influencing gene expression in many organisms.

Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization

“…In addition to this processing of the COOLAIR transcript, the COOLAIR promoter is regulated by the presence of an R loop (13). Stabilization of the R loop by the homeodomain protein NDX1 reduces COOLAIR transcription (13). COOLAIR transcription is also regulated by cold, with a transient expression peak after 2-3 wk of cold exposure (11).…”

“…Distal polyadenylation is associated with a high expression state of the locus, but it is still unclear whether use of the distal site causes, or is a consequence of, increased FLC transcription (9,12). In addition to this processing of the COOLAIR transcript, the COOLAIR promoter is regulated by the presence of an R loop (13). Stabilization of the R loop by the homeodomain protein NDX1 reduces COOLAIR transcription (13).…”

LncRNAs: Bridging environmental sensing and gene expression

“…61,62 In contrast, R-loops play beneficial roles by mediating CRISPR interference, 63,64 promoting IgG class switching, 65,66 and by regulating transcription. 67,68 These structures can form in cis 60 or trans 69 in vivo, either by "threading back" of nascent RNAs during transcription by RNA Pol II, through the action of the homologous recombination machinery, or associated RNA-binding proteins as in the case of CRISPR. 60,70,71 Given that RNP assembly defects cause R-loops, 60,70 we speculated that loss of DBP2 might promote formation of these structures between the GAL lncRNAs and GAL gene promoters.…”

Epigenetic setting and reprogramming for neural cell fate determination and differentiation

“…The R-loop structure is favoured by H3K4 methylation, but not by DNMT activity, which may explain the unmethylated characteristics of CpG island type promoters. However, it should be noted that Rloop structure constitutes a repressed state in a different context in plants [53]. The mechanisms of switching between opening and closing of chromatin structure via lncRNA might be coupled with association with different components in steps 2 and 3.…”