Control over gene expression is essential for life. This is especially evident during development when the switching of genes between active and repressed states drives fate determination. Mutations that interfere with repression lead to or exacerbate numerous cancers (Wong et al., 2014) and cause developmental defects in diverse organisms (Grbavec et al., 1998; Long et al., 2006), yet many questions remain about how cells induce, maintain, and relieve transcriptional repression. Transcriptional repression is controlled in part by a class of proteins known as corepressors that interact with DNA-binding transcription factors and actively recruit repressive machinery. Transcriptional corepressors are found in all eukaryotes and include the animal SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) complexes (Mottis et al., 2013; Oberoi et al., 2011), the yeast Tup1 (Keleher et al., 1992; Matsumura et al., 2012; Tzamarias and Struhl, 1994), and its homologs Drosophila Groucho (Gro) and mammalian transducing-like enhancer (TLE) (Agarwal et al., 2015).

In plants, the role of Gro/TLE-type corepressors is played by TOPLESS (TPL), TOPLESS-RELATED (TPR1-4), LEUNIG (LUG) and its homolog (LUH), and High Expression of Osmotically responsive genes 15 (HOS15) (Causier et al., 2012; Lee and Golz, 2012; Liu and Karmarkar, 2008; Long et al., 2006; Zhu et al., 2008). Plant corepressors share a general structure, where at the N-terminus a LIS1 homology (LisH) domain contributes to protein dimerization (Delto et al., 2015; Kim et al., 2004). At the C-terminus, WD40 repeats form beta-propeller structures that are involved in protein-protein interactions (Collins et al., 2019; Liu et al., 2019). In TPL family corepressors, the LisH is followed by a C-terminal to LisH (CTLH) domain that binds transcriptional repressors through an Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif found in partner proteins (Causier et al., 2012; Kagale et al., 2010). The N-terminal domain also contains a CT11-RanBPM (CRA) domain, which provides a second TPL dimerization interface and stabilizes the LisH domain (Ke et al., 2015; Martin-Arevalillo et al., 2017). The beta-propellers bind to the non-EAR TPL recruitment motifs found in a subset of transcriptional regulators (RLFGV- and DLN-type motifs; Collins et al., 2019; Liu et al., 2019) and may control protein interaction with other repressive machinery. Defects in the TPL family have been linked to aberrant stem cell homeostasis (Busch et al., 2010), organ development (Gonzalez et al., 2015), and hormone signaling (Causier et al., 2012; Kagale et al., 2010), especially the plant hormone auxin (Long et al., 2006).

We have previously demonstrated the recapitulation of the auxin response pathway in Saccharomyces cerevisiae (yeast) by porting individual components of the Arabidopsis auxin nuclear response (Pierre-Jerome et al., 2014). In this Arabidopsis thaliana Auxin Response Circuit in Saccharomyces cerevisiae (AtARC^Sc; Figure 1A), an auxin-responsive transcription factor (ARF) binds to a promoter driving a fluorescent reporter. In the absence of auxin, the ARF protein activity is repressed by interaction with a full-length Aux/IAA protein fused to the N-terminal domain of TPL. Upon the addition of auxin, the TPL-IAA fusion protein is targeted for degradation through interaction with a member of the Auxin Signaling F-box protein family and releases the transcriptional repression of the fluorescent reporter. Reporter activation can be quantified after auxin addition by microscopy or flow cytometry (Pierre-Jerome et al., 2014). In the original build and characterization of AtARC^Sc, it was noted that the two N-terminal truncations of TPL (N100 or N300) behave differently (Pierre-Jerome et al., 2014). While both truncations are able to repress the function of a transcriptional activator fused to an Aux/IAA, only the TPLN100 fusion shows alleviation of repression after auxin addition. TPLN300 fusions to Aux/IAAs maintain strong durable repression even under high concentrations of auxin. This disparity is not due to differential rates of protein degradation as both proteins appear to be turned over with equal efficiency after auxin addition (Pierre-Jerome et al., 2014).

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The conservation of TPL’s repressive function in yeast suggests that the protein partners that enact repression are conserved across eukaryotes. Consistent with this speculation, the series of alpha-helices that form the N-terminal portion of TPL (Figure 1B; Martin-Arevalillo et al., 2017) is highly reminiscent of naturally occurring truncated forms of mammalian TLE (Gasperowicz and Otto, 2005), such as Amino-terminal Enhancer of Split (AES) (Zhang et al., 2010), the Groucho ortholog LSY-22 (Flowers et al., 2010), and the unrelated mouse repressor protein MXI1 (Schreiber-Agus et al., 1995). Gro/TLE family members are generally considered to repress by recruiting histone deacetylases (HDACs) to control chromatin compaction and availability for transcription (Chen and Courey, 2000; Long et al., 2006). An alternative hypothesis has been described for Tup1 in yeast, where Tup1 blocks the recruitment of RNA polymerase II (Pol II) (Wong and Struhl, 2011), possibly through contacts with Mediator complex subunits Med21 or Med3 (Gromöller and Lehming, 2000; Papamichos-Chronakis et al., 2000). However, like many of these family members, multiple repression mechanisms have been described for TPL at different genetic loci. For example, TPL has been found to recruit the repressive CDK8 Mediator complex (Ito et al., 2016), chromatin remodeling enzymes such as Histone Deacetylase 19 (HD19) (Long et al., 2006), and directly bind to histone proteins (Ma et al., 2017).

Here, we leveraged the power of yeast genetics to interrogate the mechanism of TPL repression. Using AtARC^Sc, we discovered that the N-terminal domain of TPL contains two distinct repression domains that can act independently. We mapped the first, weaker repression domain to the first 18 amino acids of the LisH domain (Helix 1), and the second, more potent domain to Helix 8, which falls within the CRA domain. Full repression by Helix 8 required the Mediator complex, specifically direct interaction with Med21 and Med10. The Med21 residues that interact with TPL are the same ones required for transcriptional repression by the yeast corepressor Tup1. In addition, we found that multimerization of TPL was not required for repression in yeast or in plants. Our yeast results were validated with plant assays and extended to include evidence that interaction with the middle domain of Mediator was required for TPL repression of the auxin-regulated development program giving rise to lateral roots. Our findings point to a conserved functional connection between Tup1/TPL corepressors and the Mediator complex that together create a repressed state poised for rapid activation.

To understand how TPL represses transcription, we first localized repressive activity within the protein using the AtARC^Sc (Figure 1A). The extent of auxin-induced turnover of TPLN100 and TPLN300 fusion proteins appears similar, although neither are completely degraded (Pierre-Jerome et al., 2014). In this way, auxin sensitizes the AtARC^Sc to even subtle differences in the strength of repressive activity by reducing the relative concentration of the TPL fusion proteins. To pinpoint the region conferring the strong repression of TPLN300, we generated a deletion series of the N-terminus guided by the available structural information (Figure 1B, C; Ke et al., 2015; Martin-Arevalillo et al., 2017).

We started by identifying a shorter truncation, TPLN188, which behaved identically to TPLN300 (Figure 1D, Pierre-Jerome et al., 2014). Subsequently, we deleted each alpha helical domain starting with Helix 9 (constructs are named in the format Helix x – Helix y or Hx-Hy). We found that Helix 8 was required for the maximum level of repression activity and for the maintenance of repression after auxin addition (Figure 1D). All constructs lacking Helix 8 retained the ability to repress transcription, but this repression was lifted in the presence of auxin (Figure 1D) as had been observed for the original TPLN100 construct (Pierre-Jerome et al., 2014). In addition to the repressive activity of Helix 8, further deletions revealed that the 18 amino acids of Helix 1 were sufficient to confer repression on their own (H1, Figure 1E). To test whether Helix 8 activity depended on Helix 1, we analyzed a construct consisting of Helix 3 through Helix 9 (H3-H9, Figure 1E), which was able to repress transcription. Thus, Helix 1 (LisH) and Helix 8 (CRA) could contribute to TPL-mediated repression on their own (Figure 1D).

To identify the minimal domain required for Helix 8-based repression, we generated additional deletions (Figure 1C, F, Figure 1—figure supplement 1). Helix 8 and the following linker were not sufficient for repression (Figure 1F), and removal of Helix 9 or of the linker between Helix 8 and Helix 9 slightly increased sensitivity to auxin compared to TPLN188 (H1-H8Δ8L, Figure 1—figure supplement 1). A deletion that removed both the LisH and Helix 8 repression domains (H3-H7) was only able to weakly repress reporter expression (Figure 1—figure supplement 1). These results demonstrate that Helix 8, in combination with the linker between Helix 8 and Helix 9 (which folds over Helix 1), was required for maintaining repression following addition of auxin. Moreover, the repressive activity of Helix 8 and the linker were only functional in the context of the larger Helix 3-Helix 8 truncation that carries the CTLH domain and a portion of the CRA domain.

To determine which of the many known or predicted TPL-binding partners could mediate the repression activity of Helix8, we identified known interactors with either TPL or other Gro/TLE co-repressors, and then introduced the Arabidopsis homologs of these genes into the cytoplasmic split-ubiquitin system (cytoSUS) (Asseck and Grefen, 2018). We chose cytoSUS over yeast two-hybrid because in cytoSUS the interaction between target proteins takes place in the cytoplasm, and we had observed that the TPL N-terminus could repress activation of yeast two hybrid prototrophy reporters (Figure 2—figure supplement 1A). Putative direct interactors include HDACs (AtHDAC9, AtHDAC6; Long et al., 2006), Histone proteins (Histone H3, Histone H4; Ma et al., 2017), and the Mediator components MED13 (AtMED13; Ito et al., 2016) and MED21, which has been demonstrated to interact with Tup1, the yeast homolog of TPL (Gromöller and Lehming, 2000). We did not observe any interactions between TPLN188 and the HDACs HDA6 and HDA9; the histone protein AtHIS4; or the Mediator subunit AtMED13 (Figure 2A, Figure 2—figure supplement 1B). HDAC interaction with TPL has been previously hypothesized to occur through indirect interactions with partner proteins (Krogan et al., 2012); however, direct interactions with histones and MED13 have been detected (Ito et al., 2016; Ma et al., 2017). The absence of interaction between TPLN188 and these proteins may be due to differences between methods or interaction interfaces in the C-terminal WD40 repeats.

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Strong interaction was detected between TPLN188 and AtMED21, a component of the Mediator middle domain (Figure 2A). MED21 is one of the most highly conserved Mediator subunits (Bourbon, 2008) and has a particularly highly conserved N-terminus (Figure 2—figure supplement 2A, C–E). In yeast, Tup1 interacts with the first 31 amino acids of ScMed21, with the first 7 amino acids being absolutely required for interaction and transcriptional repression (Gromöller and Lehming, 2000). We observed that the equivalent truncation of AtMED21 (AtMED21-N31) was sufficient for interaction with TPLN188 (Figure 2A). We next created truncations of the N-terminal domain of AtMED21 to closely match those that had been made in yeast (Figure 2B) where deletion of the first five amino acids of ScMed21 (ScΔ5Med21) severely reduce the ability of the Mediator complex to co-purify with Pol II and CDK8 kinase complex (Sato et al., 2016). Interaction between TPLN188 and AtMED21 similarly required the first five amino acids of AtMED21 (Figure 2B), and, as in yeast, this was not a result of destabilization of the AtMED21 protein (Figure 2B). In fact, N-terminal Med21 deletions increased protein levels with no interaction with TPLN188; this results in high confidence that the Med21 N-terminus is required for interaction. AtMED21 interaction was specific to the Helix8-based repression domain as it interacted with TPLH3-H9 (Figure 2E), and not TPLH1-H7 (Figure 2—figure supplement 1C). Further screening of middle domain Mediator components identified an additional interaction with AtMED10B, the predominantly expressed MED10 isoform in Arabidopsis (Klepikova et al., 2016; Figure 2C). Interactions between TPLN188 and both AtMED21 and AtMED10B were confirmed by immunoprecipitation (Figure 2D).

The MED21 N-terminus lies in the hinge region of the middle module and has residues that are exterior facing and could be docking points for protein-protein interactions (Figure 2—figure supplement 2C–E). A manual juxtaposition of the yeast Mediator structure with the Arabidopsis TPL N-terminal structure shows that Helix 8 and the linker following face away from the tetramer and are therefore optimally placed to interact with Mediator components (Figure 2F). To pinpoint which residues of Helix 8 coordinate repression through interaction with MED21, we identified solution-facing amino acids (Martin-Arevalillo et al., 2017). We reasoned that such residues were most likely not involved in stabilizing the hydrophobic interactions between intra-TPL helical domains and could therefore be available to interact with partner proteins. Eight amino acids in Helix 8 were mutated to alanine in the context of the H3-H8-IAA3 fusion protein (Figure 3A, light green residues) to enable assessment of repression activity in the absence (Figure 3B) or presence (Figure 3C) of auxin. No single amino acid was essential for repression. Two mutations (R140A and K148A) slightly increased baseline expression of the reporter (Figure 3B, C). With the exception of E152A, which behaved similarly to controls, all of the mutations altered the stability of repression after auxin addition, either by increasing (S138A, V145A, E146A, K149A) or decreasing (I142A) the final fluorescence level (Figure 3C). Mutating E146 and K149 also increased the speed with which the reporter responded to auxin (Figure 3C), suggesting that these two neighboring residues could be a critical point of contact with co-repressive machinery. S138A had a small increase in auxin sensitivity, while I142 reduced auxin sensitivity (Figure 3E).

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We next tested whether the residues in Helix 8 that were required for repression (V145, E146, K148, K149; Figure 3A–C) were also required for interaction with AtMED21. Single-alanine mutations of these four amino acids in the context of TPLN188 significantly reduced interaction with AtMED21, while the quadruple mutation (here called Quad^AAAA) completely abrogated AtMED21 binding (Figure 3D). These mutations had little effect on interaction with AtMED10B (Figure 2—figure supplement 1D). Introduction of Quad^AAAA mutations into the Helix 3 through Helix 8 context (H3-H8-Quad^AAAA) in the AtARC^Sc largely phenocopied a deletion of Helix 8 (yellow and pink, Figure 3E, compare to H3-H7, Figure 1—figure supplement 1). In contrast, TPLN188-Quad^AAAA largely retained the repressive activity of wild-type N188 (red and black, Figure 3E), consistent with the observation that Helix 1 is sufficient for full repression (Figure 1E). These results indicate that the CRA domain (H3-H8) requires contact with MED21 to repress, and that this is independent of repression via Helix 1.

The large Mediator complex stabilizes the pre-initiation complex (PIC) enabling transcriptional activation (Kornberg, 2005; Nozawa et al., 2017; Roeder, 1996; Schilbach et al., 2017). The connection we found between TPL, two Mediator components, and transcriptional repression raised the possibility that other parts of the Mediator complex might also contribute to corepressor function. To test this, we needed a way to measure whether loss of function of individual Mediator components led to de-repression at an individual locus. In the case of the yeast corepressor Tup1, a standard approach has been to test the level of transcription at target genes in the presence of deletion mutations of Tup1-interacting proteins (Gromöller and Lehming, 2000; Lee et al., 2000; Zhang and Reese, 2004). One challenge to such an approach for Mediator components is that loss-of-function mutants can be lethal or exhibit drastic physiological phenotypes (Biddick and Young, 2005).

To avoid these complications, we turned to the well-established Anchor Away system for inducible protein depletion (Haruki et al., 2008) and combined it with quantification of transcriptional activity at the synthetic locus in the AtARC^S (Figure 4A, B). However, AtARC^Sc integrates components at four genomic locations using prototrophic markers that are not compatible with those needed for Anchor Away. To overcome this limitation, we re-created the ARC on a single plasmid (we refer to this plasmid as SPARC) using the Versatile Genetic Assembly System (VEGAS, Mitchell et al., 2015). SPARC behaved with similar dynamics to the original AtARC^Sc on both solid and liquid growth conditions (Figure 4—figure supplement 1A–C). As a first test of the Anchor Away system with SPARC, we fused Tup1 and its partner protein Cyc8 to two copies of the FKBP12-rapamycin-binding (FRB) domain of human mTOR (Haruki et al., 2008). Rapamycin treatment of strains targeting either of these proteins caused no release of repression on the SPARC reporter, providing confirmation of orthogonality of the synthetic system in yeast (Figure 4—figure supplement 1D).

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Before testing repressive function, we first performed chromatin immunoprecipitation using the Anchor Away FRB tag to ask whether Mediator proteins or specific Mediator modules were detectable at the promoters of TPL-repressed genes. We began by assaying the integrated AtARC^Sc locus and comparing it to a Tup1-repressed locus, SUC2 (Carlson and Botstein, 1982; Fleming and Pennings, 2007; Trumbly, 1992), and to an active locus enriched for Mediator, PMA1 (Petrenko et al., 2017; Schmitt et al., 2006; Serrano et al., 1986). We generated a yeast strain with an integrated MED21-FRB fusion protein and an integrated AtARC^Sc locus with a 2xHA epitope-tagged TPLN188-IAA3 repressor protein. We observed enrichment of both MED21-FRB and the 2xHA-TPLN188-IAA3 fusion protein at the AtARC^Sc locus (Figure 4C, Figure 4—figure supplement 2A–C). We also observed a modest enrichment of MED21-FRB at the SUC2 promoter (approximately twofold) and higher enrichment of MED21-FRB at the active PMA1 promoter (approximately eightfold). Next, we introduced the fully repressed SPARC plasmid containing TPLN188 (SPARC^N188) into a library of Anchor Away yeast strains that allow specific depletion of Mediator components (see Figure 4A, B; Haruki et al., 2008; Petrenko et al., 2017). We tested representatives of the mediator complex (tail – Med15, head – Med18, middle – Med21 and Med14, kinase – CDK8, general transcription factors – TBP1, TFIIA, and RNA Pol II – Rpb1) by ChIP-qPCR. We observed enrichment of all tested core mediator complex members, as well as general transcription factors, at both the SPARC and the SUC2 loci, with very little enrichment of RNA Pol II (Figure 4D). In general, MED21 was detected in lower levels at repressed loci than at the active PMA1 promoter (Figure 4D). Consistent with this observation, Mediator is highly enriched at the SPARC promoter when TPL is absent (Figure 4—figure supplement 2D). Higher enrichment of members of the middle module at repressed promoters (i.e., Med21, Med14, Figure 4D) may point to these subunits nucleating assembly of the entire complex.

We next tested whether the association of Mediator complex components was required for TPL-mediated repression (Figure 4A, B). Nuclear depletion of Mediator components from the tail, head, and middle domain triggered clear activation of the SPARC^N188 reporter (Figure 4E–I). Depletion of the Mediator kinase module component CDK8 had a more modest effect (Figure 4H). One caveat to this approach is that nuclear depletion of components that are absolutely required for transcriptional activation, such as RNA Pol II Anchor Away (Rpb1; Figure 4I), cannot be assayed for impacts on repression using transcription of the reporter as the output.

To further interrogate the impact of Mediator on repression, we next focused on the other side of the interaction, namely which region of MED21 was required for interaction with TPL. Deletion of the first seven amino acids of ScMed21 (Δ7Med21) partially activates genes that are normally repressed by Tup1 (Gromöller and Lehming, 2000), so we first tested if the same held true for TPL-mediated repression. We introduced SPARCs with different TPL constructs into strains where wild-type ScMed21 or N-terminal deletions were targets of Anchor Away. Importantly, the addition of the FRB tag did not alter ScMED21 function (Figure 4J, Figure 4—figure supplement 1E, F). We observed that deletion of either five or seven of the N-terminal residues of ScMed21 increased the expression of the reporter in SPARC^N188 to a level similar as what is observed with TPL H1-H5 (Figure 4J, Figure 4—figure supplement 1E, F). No mutation increased the SPARC’s sensitivity to auxin. As Δ7ScMed21 had a noticeable impact on growth, as has been reported previously (Gromöller and Lehming, 2000; Hallberg et al., 2006), we removed it from further studies. Δ5ScMed21 had no observable growth defects, although this deletion is known to be sufficient to alter Mediator assembly and disrupt binding of Pol II and the CDK8 kinase module (Hallberg et al., 2006; Sato et al., 2016).

The fully repressed SPARC^N188 in Δ3ScMed21 or Δ5ScMed21 mutants showed elevated reporter transcription when compared to strains carrying wild-type ScMED21 (Figure 4K). The addition of rapamycin further increased reporter expression, particularly in the Δ3ScMed21 strains, suggesting that this deletion could only partially disrupt the TPLN188-Med21 interaction. We used chromatin immunoprecipitation to directly test whether Δ5Med21 showed a change in association with the SPARC^N188 promoter. While this deletion would be expected to reduce Med21 association with TPL, the resulting de-repression of the locus should lead to an increase in Mediator association with the activated promoter. Indeed, we observed an ~1.6×-fold increase in Δ5Med21-FRB promoter binding compared to wild type (Figure 4L). While this enrichment was modest compared to a repressor-free SPARC^IAA14 (Figure 4L, no TPL; dark gray bar), it was similar to the magnitude of transcriptional activation of the reporter in the Δ5Med21 genotype (Figure 4J, K). The well-documented PMA1 promoter had a substantial enrichment of wild-type MED21, as expected, and was unaffected by the presence of TPL (Figure 4L, dark and light gray bars). To confirm that the interaction with TPL was not unique to ScMed21, we replaced the first five amino acids of ScMed21-FRB with the corresponding sequence from AtMED21. The strain carrying this chimeric protein had an identical repression profile as the one with native ScMed21 (Figure 4—figure supplement 3B) and showed no difference in growth or viability (Figure 4—figure supplement 3C).

To minimize any possible off-target impact of ScMed21 deletions, we introduced estradiol-inducible versions of ScMed21 (iScMed21) into the Anchor Away SPARC^N188 strains (Figure 4—figure supplement 4; McIsaac et al., 2013). The combination of all three synthetic systems – ARC^Sc, Anchor Away, and estradiol inducibility – made it possible to rapidly deplete the wild-type ScMed21-FRB from the nucleus while simultaneously inducing ScMed21 variants and visualizing the impact on a single auxin-regulated locus. Depletion of nuclear ScMed21 by rapamycin increased levels of the reporter in all genotypes examined (Figure 4G, K) while increasing cell size even in short time courses, consistent with its essential role in many core pathways (Figure 4—figure supplement 4A; Gromöller and Lehming, 2000). When wild-type iScMed21 was induced, there was a rescue of both reporter repression and cell size (Figure 4—figure supplement 4C, D), whereas induction of either Δ3 and Δ5 variants resulted in significantly less reporter repression (Figure 4—figure supplement 4D). iΔ3Med21 was induced and accumulated at a comparable level to wild-type Med21, while iΔ5Med21 is less stable (Figure 4—figure supplement 4E, F). In the time courses with both rapamycin and estradiol, we did not observe the cell size increases observed in the rapamycin treatments alone (populations were evenly distributed around a single mean), suggesting that we were observing the immediate effects of the Med21 deletions (Figure 4—figure supplement 4G, H).

Several lines of evidence suggest that, in addition to interactions with other partners, homomultimerization modulates TPL repression potential. First, structures of the N-terminal domains of TPL (Martin-Arevalillo et al., 2017) and a rice homolog OsTPR2 (Ke et al., 2015) reveal high conservation of residues that coordinate formation of homotetramers and connect tetramer formation to Aux/IAA binding. Second, the dominant TPL mutant tpl-1 altered a single amino acid in the ninth helix of the TPL-N terminus (N176H) that induces aggregation of TPL and its homologs (TPR1-4), reducing total activity (Long et al., 2006; Ma et al., 2017). Third, TPL recruitment motifs found in the rice strigolactone signaling repressor D53 induce higher-order oligomerization of the TPL N-terminus, which increases histone binding and transcriptional repression (Ma et al., 2017). Our studies in yeast suggest that there may be a more complex relationship between tetramer formation and repression as we have measured strong repressive activity in several constructs that are unlikely (TPLN100; Pierre-Jerome et al., 2014) or unable (H1, H3-8; Figure 1C) to form tetramers (compare Figure 1B with Figure 5A). To quantify the potential for interaction among our constructs, we used the cytoSUS assay (Asseck and Grefen, 2018). Helix 8 was required for strongest interaction between TPL constructs (Figure 5—figure supplement 1A), although this assessment was complicated by the fact that some of the shorter constructs accumulated to significantly lower levels (Figure 5—figure supplement 1B). The weak interaction we could observe between full-length TPL-N and the Helix 1 through Helix 3 construct (H1-3) indicated that the TPL LisH domain is sufficient for dimerization. Therefore, while auxin-insensitive repression may require multimeric TPL, this higher-order complex was not required for auxin-sensitive repression mediated by Helix 1 (Figure 1E).

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To avoid any potential artifacts from analysis of truncated forms of the N-terminus, we next generated site-specific mutations that disrupted multimerization in the context of TPLN188. Martin-Arevalillo et al. had previously identified a quadruple mutation (K102S-T116A-Q117S-E122S) that abrogated the ability of the CRA domain (Helix 6 and Helix 7) to form inter-TPL interactions (Martin-Arevalillo et al., 2017). As this mutant form of TPL is only capable of dimerizing through its LisH domain, we refer to it here as LDimer (Figure 5A). The LDimer mutations in TPLN188 retained the same auxin-insensitive repression behavior as wild-type TPLN188 (Figure 5D), supporting the finding from the deletion series.

To make a fully monomeric form of TPL, we introduced mutations into the dimerization interface of the LisH domain in the context of LDimer. We first mutated one of a pair of interacting residues (F15) to a series of amino acids (tyrosine – Y, alanine – A, arginine – R, or aspartic acid – D) in the context of either LDimer (Figure 5D), or H1-2 (Figure 5B, E). Conversion of F15 to the polar and charged aspartic acid (D) completely abolished repression activity, while the positively charged arginine was better tolerated (Figure 5D, E). The conversion of F15 to tyrosine had no effect on LDimer (Figure 5D), and only a minimal increase in auxin sensitivity in the context of H1-2 (Figure 5E). We then combined LDimer-F15Y with a mutation of the coordinating residue L8 to serine with the intention of stabilizing the now solvent-facing residues. The repressive behavior of this mutant was indistinguishable from that of LDimer (Figure 5F).

To further push the LDimer towards a monomeric form, we introduced two additional mutations (S5A, E19S, Figure 5C, G). Size-exclusion chromatography confirmed that this combination of mutations (S5A-L8S-F15Y-E19S-K102S-T116A-Q117S-E122S, hereafter called Monomer) successfully shifted the majority of the protein into a monomeric state (Figure 5H); however, this shift had no observable impact on repression strength before or after auxin addition (Figure 5G). To test whether these mutations had a similar impact on in vivo TPL complexes, we introduced the LDimer and Monomer mutations into the cytoSUS assay. In contrast to the in vitro chromatography results with purified proteins, Monomer expressed in yeast retained measurable interaction with wild-type TPL, LDimer or Monomer, albeit at a reduced level than what was observed between wild-type TPLN188 constructs (Figure 5I). A caveat to this apparent difference between assays is that the Monomer mutations led to a striking increase in protein concentration in yeast (Figure 5—figure supplement 1C), likely partially compensating for the decrease in affinity.

To ascertain which of our findings about TPL required the sensitivity and simplicity of the synthetic context and which could be observed in the full complexity of intact plant systems, we performed a set of experiments in Nicotiana benthamiana (tobacco) and Arabidopsis. Bimolecular fluorescence complementation (BiFC) confirmed the interaction between TPL and MED21 (Figure 6A), which was further validated by co-immunoprecipitation using tobacco extracts (Figure 6B). We were also able to pull down MED21 and TPL using MED10B (Figure 6—figure supplement 1A). BiFC also confirmed the importance of the same TPL Helix 8 residues for the TPL-AtMed21 interaction (Figure 6A, TPL^H8QuadA). Similarly, the Δ5AtMED21 N-terminal truncation eliminated interaction with full-length TPL (Figure 6A). We next developed a quantitative repression assay based on UAS/GAL4-VP16 (Brand and Perrimon, 1993; Figure 6C). To block potentially confounding interactions with endogenous TPL/TPRs or TIR1/AFBs, we engineered a variant of IAA14 with mutations in the two EAR domains (EAR^AAA) and in the degron (P306S) (IAA14^mED; Figure 6C). After prototyping the system in yeast (Figure 6—figure supplement 1B, C), we quantified repression strength of constructs carrying TPLN-IAA14^mED variants using the well-characterized synthetic auxin-responsive promoter DR5 (Ulmasov et al., 1997). As expected, DR5 was strongly induced by co-transformation with AtARF19, and this induction was sharply reduced by the inclusion of UAS-TPLN188-IAA14^mED and GAL4-VP16 (Figure 6—figure supplement 1D). Overall, we observed strong correlation in repression activity between what was observed in yeast and in tobacco.

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To connect the observed differences in repression strength to a developmental context, we generated transgenic Arabidopsis lines where the UAS-TPL-IAA14^mED constructs were activated in the cells where IAA14 normally acts to regulate the initiation of lateral root primordia (Figure 6D; Gala et al., 2021; Laplaze et al., 2005). Expression of functional TPL-IAA14^mED fusion proteins in these xylem pole pericycle cells should strongly suppress production of lateral roots, phenocopying the solitary root (slr) mutant, which carries an auxin-resistant form of IAA14 (Fukaki et al., 2002). Indeed, TPLN188 fusion constructs sharply decreased lateral root density (Figure 6E), while transformants expressing either IAA14^mED (with no TPL fusion) or TPLN188 (with no IAA14 fusion) had no effect on lateral root production (Figure 6E). TPLH3-H9 decreased lateral root density albeit not as effectively as TPLN188, suggesting that Helix 1 is required for full repression in a native context (Figure 6E). Both LDimer and Monomer constructs (Figure 6E) were able to repress lateral root development to the same extent as TPLN188, meaning that multimer formation is not required for TPL-mediated repression in this context. The fusion containing the Helix 8 quadruple mutant demonstrated a clear loss of repression, indicating that the TPL-MED21 interaction is critical for repression when expressed in lateral root-forming cells (Figure 6E).

Given this result, we wanted to directly test the role of AtMed21 in auxin-regulated development in the presence of native isoforms of TPL and Aux/IAAs. This was complicated by the fact that, as in yeast, AtMED21 is essential in plants. While homozygous loss-of-function mutations are embryo lethal (Dhawan et al., 2009), plants heterozygous for Atmed21 mutations appear wild type (Figure 6—figure supplement 2A, B). To overcome this obstacle, we took two approaches that relied on the same xylem pole pericycle driver as described for the TPL functional assays. First, we expressed N-terminal deletion variants of MED21 that should weaken or sever interaction with TPL (Figure 6F; Δ3MED21, Δ5MED21, Δ7MED21). If MED21-TPL interaction is critical to maintain normal expression of the lateral root program, reduced interaction should trigger an increase in lateral root density. This was exactly what we observed for transgenic lines expressing any of the three deletions. Second, we repressed transcription of AtMED21 by introducing a dCAS9-TPLN300 synthetic repressor under the control of a UAS promoter along with three sgRNAs complementary to the AtMED21 promoter. Similar to the predictions above, reduced expression of AtMED21 in xylem pole pericycle cells should stimulate lateral root development. This is indeed what we observed (Figure 6—figure supplement 2C–E).

Previous reports connected the Mediator kinase module, and specifically the function of AtMED13/GRAND CENTRAL (GCT), to repression by TPL (Ito et al., 2016). We did not find evidence for this relationship in our yeast assays and wanted to investigate further. Although embryo lethality is seen in the majority of homozygous med13/gct individuals, a small percentage survive. This made it possible to examine the effect of expressing wild-type and monomeric forms of TPLN188 in xylem pole pericycle cells in the absence of MED13/GCT activity (Figure 6—figure supplement 1E, F). Loss of med13 function was unable to rescue the production of lateral roots in these lines, leading to the conclusion that TPLN188 requires AtMED21 but not AtMED13 for repression, at least in this context.

A review of the current literature on corepressors gives the conflicting impressions that (a) corepressor function is broadly conserved and (b) that every organism (and perhaps even every corepressor) has a distinct mode for transcriptional repression (Adams et al., 2018; Mottis et al., 2013; Perissi et al., 2010; Wong and Struhl, 2011). We hoped that the AtARC^Sc could facilitate a resolution to this apparent contradiction by targeting repression to a single synthetic locus. We focused our initial efforts on the analysis of the N-terminal portion of TPL, which has multiple known protein-protein interaction surfaces (Ke et al., 2015; Martin-Arevalillo et al., 2017). Experiments with the AtARC^Sc identified two independent repression domains (Figure 1), and we focused additional study on the stronger of the two that was localized to the CRA domain. Within this domain, we were able to identify two interacting partners that are part of the middle module of the Mediator complex: MED21 and MED10 (Figure 2). Four amino acids within Helix 8 with R-groups oriented away from the hydrophobic core of the TPL structure were found to be required for both Med21-binding and repressive function (Figure 3). Indeed, the entire core Mediator complex (head, middle, tail) appears to be recruited to TPL-repressed loci and required to maintain repression (Figure 4). Contrary to our initial hypothesis, the monomeric form of TPL was sufficient for strong repression in yeast and in plants (Figure 5), leaving open the question of the role of higher-order TPL complex formation. Finally, we were able to confirm that our insights from synthetic assays in yeast were relevant to regulation of the auxin-mediated lateral root development pathway in intact plants (Figure 6).

Corepressors coordinate multiple mechanisms of repression through discrete protein interactions, leading to robust control over eukaryotic transcription by combining repression modalities. Corepressor function has variously been linked to (a) altering chromatin confirmation, often through interaction with histone-modifying proteins or histone proteins themselves, (b) direct interference with transcription factor binding or function, and (c) physical spreading of long-range oligomeric corepressor complexes across regions of regulatory DNA (Perissi et al., 2010). Dissection of the importance of each modality in Tup1 repression has been challenging (Lee et al., 2000; Zhang and Reese, 2004). The tour-de-force of corepressor mechanism studies in yeast concluded that the primary function of Tup1 was to physically block activators (Wong and Struhl, 2011). In their work, the authors utilized the Anchor Away approach to correlate the importance of HDACs, transcriptional machinery, and chromatin remodeling enzymes to the repression state of endogenously repressed Cyc8-Tup1 target genes. They observed that Tup1 did not block the binding of transcription factors but inhibited the recruitment of one Mediator component in the tail domain, GAL11/MED15, as well as Pol II and the chromatin remodelers Snf2 and Sth1. They additionally observed that HDACs had only a supportive role in reinforcing Tup1 repression. These results led to their hypothesis that Tup1 blocks the activation domains of transcription factors and suggested this was through direct binding to activation domains (Wong and Struhl, 2011).

The synthetic system used here allowed us to build on this model and further refine our understanding of TPL’s repressive activity. In our experiments, we see a similar set of conditions, with TPL recruited to the DNA-bound transcriptional activator (ARF), and several possible mechanisms of repression. Unlike Tup1, we have subdivided the TPL protein to identify interactions between TPL and individual protein interactors with no effect on yeast function. In these experiments, we can eliminate the possibility that TPL blocks ARF activation by directly blocking the transcription factor activation domains because we see a loss of repression only when TPL-MED21 binding is eliminated through specific point mutations (Figure 4J–L). Our estradiol-inducible replacement assays where different isoforms of Med21 are expressed also corroborate these findings (Figure 4—figure supplement 4) as the SPARC remains genetically identical in these strains, indicating that TPL-MED21 interaction is regulating Mediator activity not a TPL-ARF interaction. Furthermore, our results correlate well with findings that repressed targets are reactivated when this portion of MED21 is deleted in yeast (TPL, Figure 4; Tup1; Gromöller and Lehming, 2000). Therefore, we suggest that instead of directly binding activation domains that TPL (and likely Tup1) binds to components of Mediator (MED21, MED10B, and possibly others) recruited by the transcription factor. Indeed, it is easier to rationalize that the repressor binds the same domains of the Mediator complex recruited by the transcription factor’s activation domain (with the same affinity) as opposed to binding each diverse activation domain (with varying affinity). In this model, corepressor binding blocks formation of a fully active Mediator complex, thereby limiting Pol II recruitment and promoter escape (Petrenko et al., 2017).

The Mediator complex is a multi‐subunit complex that connects DNA‐bound transcription factors and the RNA Pol II complex to coordinate gene expression (Flanagan et al., 1991; Kim et al., 1994; Kornberg, 2005). The yeast Mediator subunits are organized into four separate modules, head, middle, tail, and kinase, with a strong conservation of module components in plants (Dolan and Chapple, 2017; Maji et al., 2019; Malik et al., 2017; Samanta and Thakur, 2015). Med21 forms a heterodimer with Med7 and interacts with Med10, among others, to create the central region of the middle region of the Mediator complex. The Med21 N-terminus is centered on a flexible hinge region (Baumli et al., 2005), which is required for recruitment of Pol II and the CDK8 kinase module (Sato et al., 2016). The protein interaction between TPL and MED21 occurs at the N-terminus of MED21, highlighting the importance of this region as a signaling hub (Sato et al., 2016). Other lines of evidence support this role as this region binds the yeast homolog of TPL, Tup1 (Gromöller and Lehming, 2000), through a completely different protein domain as no homology can be found between TPL Helix 8 and Tup1 in any region by primary amino acid homology (i.e., BLAST).

As suggested by Ito and colleagues (Ito et al., 2016) and supported by our synthetic system, auxin-induced removal of TPL is sufficient to induce changes in the activity of the Mediator complex; however, multiple points of contact likely exist between the Mediator complex and other parts of the transcriptional machinery in both transcriptionally repressed and active states. For auxin response, specifically, there are several lines of evidence to support this model, including documented association between the structural backbone of Mediator, MED14, and activated and repressed auxin loci in Arabidopsis (Ito et al., 2016). In addition, MED12 and MED13 are required for auxin-responsive gene expression in the root, and MED12 acts upstream of AUX1 in the root growth response to sugar (Raya-González et al., 2018). MED18 in the head module represses auxin signaling and positively regulates the viability of the root meristem (Raya-González et al., 2018). PFT1/MED25 regulates auxin transport and response in the root (Raya-González et al., 2014). MED7, MED21’s partner protein in the hinge domain, is required for normal root development, and loss of MED7 function impacts expression of auxin signaling components (Kumar et al., 2018). Previous research identified the Mediator CDK8 module, specifically MED13 (MAB2), as an interactor with the full-length TPL protein (Ito et al., 2016). We could not observe interaction between the N-terminal domain of TPL and AtMED13, AtCYC8, or AtCYCC (Figure 2—figure supplement 1B), suggesting that any direct interactions occur outside the N-terminal region.

The conserved interaction of both TPL and Tup1 with Mediator has implications for modeling eukaryotic transcription (e.g., Estrada et al., 2016). By stabilizing the Mediator complex, TPL (and by extension Tup1) may create a ‘pre-paused’ state that allows rapid recruitment of Pol II and activation once TPL is removed. This would be compatible with the multiple repression mechanisms described for TPL at different genetic loci. TPL recruitment of the repressive CDK8 Mediator complex (Ito et al., 2016), chromatin remodeling enzymes such as HD19 (Long et al., 2006), and contact with histone proteins (Ma et al., 2017) would be removed with TPL upon relief of repression. It will be critical in the future to understand how these various forms of repression interact, and especially to map the dynamics of assembly and disassembly of complexes as loci transition from repressed to active states and back to repressed once again.

All flow cytometry data is deposited at https://flowrepository.org/. Repository IDS: FR-FCM-Z2GM, FR-FCM-Z2GR, FR-FCM-Z2GQ, FR-FCM-Z2GX, FR-FCM-Z2GT, FR-FCM-Z2W2. All protein interactions have been deposited to IntAct, under the accession code, IM-28972. All code is available through Github: https://github.com/achillobator/TPL_Structure_Function/ (copy archived at https://archive.softwareheritage.org/swh:1:rev:141d7d05fe0c23be55af5050563d160f019d6d65).

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Author details

1. Alexander R Leydon

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3034-1482

2. Wei Wang

   Department of Pharmacology, Seattle, United States

   Present address

   Key Laboratory of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Science, Jinming Campus, Henan University, Kaifeng, China

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

3. Hardik P Gala

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Formal analysis, Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

4. Sabrina Gilmour

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

5. Samuel Juarez-Solis

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

6. Mollye L Zahler

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

7. Joseph E Zemke

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

8. Ning Zheng

   1. Department of Pharmacology, Seattle, United States
   2. Howard Hughes Medical Institute, University of Washington, Seattle, United States

   Contribution

   Resources, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

9. Jennifer L Nemhauser

   Department of Biology, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   jn7@uw.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8909-735X

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