Abstract
Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.
Highlights
R NA-mediated chromatin regulation has emerged as a key mechanism in gene regulation in eukaryotes [1]
The interaction between AGO1 and SDG26 was further confirmed by coimmunoprecipitation using nuclear extracts from a stable transgenic line expressing SDG26 fused to a FLAG-HA TAP tag (SDG26-TAP) (Fig. 1A)
Genetic screens uncovered FLOWERING LOCUS C (FLC) activators and repressors that identified cotranscriptional regulators involved in RNA processing and chromatin modification
Summary
R NA-mediated chromatin regulation has emerged as a key mechanism in gene regulation in eukaryotes [1]. Functioning antagonistically to these are repressors of FLC expression: conserved RNA binding proteins (FCA, FPA, and FLK), RNA 30-end processing and splicing factors, and chromatin modifiers These repressors were grouped into the autonomous pathway that regulates FLC through an RNAmediated chromatin-silencing mechanism Our current understanding of this mechanism is that the RNAbinding protein FCA concentrates 30-end processing factors in nuclear condensates and promotes proximal polyadenylation of a set of antisense long noncoding transcripts transcribed from FLC, called COOLAIR [9, 10]. These 30-end processing factors dynamically interact with a histone-modifying complex FLD/ SDG26/LD, thereby removing H3K4me over the FLC gene body. The detailed dissection of the FLC/COOLAIR transcriptional unit has enabled an understanding that differential assembly of cotranscriptional machineries with RNA Pol II results in changes in RNA processing that feeds back to alter local chromatin environment and influence subsequent transcriptional output
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