The intersection of DNA replication with antisense 3' RNA processing in <i>Arabidopsis FLC</i> chromatin silencing.

Abstract

How noncoding transcription influences chromatin states is still unclear. The <i>Arabidopsis</i> floral repressor gene <i>FLC</i> is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The <i>FLC</i> antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the <i>Arabidopsis</i> genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that <i>FLC</i> R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at <i>FLC</i>.