A single helix repression domain is functional across diverse eukaryotes

Abstract

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL—and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1)—can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae . Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across many eukaryotes.

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

“…In contrast, mLisH failed to rescue repression, indicating that the conserved residues in the H1 domain that direct LisH dimerization are required for the activity of ScTAF5 in its role in repression. This finding is consistent with cross-kingdom analyses of other LisH domains 22 . We engineered an Auxin inducible degron (AID) version of ScTaf5 (AID-Taf5, Figure 5I) where the degron was fused to the N-terminus of ScTaf5 and observed that this approach did not reduce function of the Taf5 protein (Figure 5I).…”

“…We observed a significant reduction in CD4 abundance when TPLN188 was recruited to the CD4 promoter region, compared to a control guide (Figure 5K). Interestingly, the HsTBL1 N-terminal domain (N98), which we previously found to be able to replace TPL in our synthetic yeast assay 22 , was equally efficient at repressing CD4 as ZIM3 (Figure 5K).…”

“…We observed that ScSpt4 interacted with bait constructs containing only the TPL LisH domain (H1- H2) (Figure 3F). The human protein Transducin Beta-like 1 (HsTBL1) contains a LisH domain with a similar structural fold as TPL 49 , and we have previously demonstrated that the HsTBL1 N-terminal domain can repress transcription in yeast 22 . Like TPLN188, the HsTBL1 N-terminal domain strongly interacted with ScSpt4 (Figure 3G).…”

“…H8 makes direct contact with Mediator 21 (Med21) and Mediator 10 (Med10), likely leading to assembly of the entire Mediator complex at a promoter while simultaneously inhibiting the transition to active transcription 21 . Despite its broad functional conservation 22 and importance to TPL activity, the mechanism of H1/LisH-based repression on transcription is unknown.…”

“…Structure-function studies indicate that the modes of TPL action are distinct from other well-characterized modes of repression in plants and fungi, such as those that rely exclusively on epigenetic marks on histones or DNA methylation 23 . TPL appears to act as a priming agent for transcriptional activation, facilitating the assembly of Mediator and other components of the transcriptional preinitiation complex (PIC) at promoters bound by transcriptional activators 22,24 . This form of repression is reminiscent of RNA Polymerase II proximal promoter pausing in metazoans 25 .…”