A conserved function of corepressors is to nucleate assembly of the preinitiation complex

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Methods
Two independent crosses of YNL3669 (MATa SPARCH1-H5[pTDH3-AtTPLH1-5-IAA14-ttACS, pRPS2-AtAFB2-ttCIT1, LEU2, pADH1-AtARF19-ttADH1, pP3(2x)-UbiVenus-ttCYC1] HO::ACT1pr-tdTomato::hphMX, can1∆::STE2pr-Sphis5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0) and YNL3670 (MATa SPARCTPLN188[pTDH3-AtTPLN188-IAA14-ttACS, pRPS2-AtAFB2-ttCIT1, LEU2, pADH1-AtARF19-ttADH1, pP3(2x)-UbiVenus-ttCYC1 ] HO::ACT1pr-tdTomato::hphMX, can1∆::STE2pr-Sphis5 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0) with the yeast nonessential deletion collection and a set of conditional temperature-sensitive alleles of essential genes were performed following standard SGA procedures104. Final arrays were pinned in duplicate on either SD/MSG–his–leu+ 200mg/mL G418 (untreated) or YPD supplemented with 50mM NAA and grown for 24hr before fluorescence scanning. The Typhoon TrioVariable Mode Imager (GEHealthcare) was used to acquire Venus (488-nmlaser, 520/40BP emission filter) and tdTomato (532-nmlaser, 610/30BPemission filter) fluorescence values. For the essential temperature-sensitive mutants, all growth was conducted at 23°C until the final growth before imaging, where they were grown at 30°C. After fluorescence imaging, colony size data were acquired by individually photographing plates with a Canon PowerShotG 24.0 megapixel digital camera using Remote Capture software. Data analysis followed essentially what is described in Kainth et al. (2009)43, with small variations. To summarize, background-subtracted yellow fluorescent protein (Venus) and tdTomato intensities were computed for each colony from .GEL images using GenePixPro version 7.0 software. Colony size was imaged on SPimager from S&P Robotics, Inc, and size information was calculated from individual photographs SGAtools. Border colonies, small colonies (colony area < 500pixels), and colony size information was calculated from individual photographs. Border colonies, small colonies (colony area < 500pixels), and colonies with aberrantly low tdTomato values (bottom 0.05%) were removed before further analysis. log2(Venus/tdTomato) values were calculated and LOESS normalized for each plate. Using the log2(Venus /tdTomato) ratio as a metric for Venus abundance has the advantage that dividing by tdTomato corrects for any colony size dependent intensity effects. Finally, normalized log2(Venus/tdTomato) values were averaged across all replicate experiments and a Z-score calculated (See Supporting Information). All analyses were performed in R.

For Arabidopsis thaliana experiments using the GAL4-UAS system (Laplaze et al., 2005), J0121 was introgressed eight times into Col-0 accession from the C24 accession and rigorously checked to ensure root growth was comparable to Col-0 before use. UAS-TPL-IAA14mED constructs were introduced to J0121 introgression lines by floral dip method112. T1 seedlings were selected on 0.5× LS (Caisson Laboratories, Smithfield, UT)+ 25 μg/ml Hygromycin B (company) + 0.8% phytoagar (Plantmedia; Dublin, OH). Plates were stratified for 2 days, exposed to light for 6 hr, and then grown in the dark for 3 days following a modification of the method of Harrison et al., 2006. Hygromycin-resistant seedlings were identified by their long hypocotyl, enlarged green leaves, and long root. Transformants were transferred by hand to fresh 0.5× LS plates + 0.8% Bacto agar (Thermo Fisher Scientific) and grown vertically for 14 days at 22°C. Plates were scanned on a flatbed scanner (Epson America, Long Beach, CA) at day 14. slr seeds were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). For integrase switch experiments T2 plant lines harboring T-DNAs for either MED21 (med21–1, WiscDsLox461–464K13), SPT6L (spt6l-7, SAIL_59_G06) and TAF5 (taf5–4, SAIL_274_A04) were transformed with the floral dip method to generate integrase target lines, and then used to introduce each integrase construct into these established target lines. For T1 selection: 120 mg of T1 seeds (~2000 seeds) were sterilized using 70% ethanol and 0.05% Triton-X-100 and then washed using 95% ethanol. Seeds were resuspended in 0.1% agarose and spread onto 0.5X LS Bacto selection plates, using 25 μg/mL of kanamycin for target lines and 25 μg/mL kanamycin and 25 μg/mL hygromycin for lines with both the integrase and the target. The plates were stratified at 4 °C for 48 h then light pulsed for 6 h and covered for 48 h. They were then grown for 4–5 days. To select transformants, tall seedlings with long roots and a vibrant green color were picked from the selection plate with sterilized tweezers and transferred to a new 0.5X LS Phyto agar plate for characterization.