A Family of Transcriptional Co-repressors Controlling Multiple Processes in Plants

Living organisms need to turn expression of their genes on and off in response to their environment. Transcription factors bind to specific DNA sequences and control whether genes are activated or repressed. Turning off gene expression is not simply the absence of activation: actively shutting down expression of target genes is of vital importance. For example, in this proposal we will look at two cases where repressing target genes is required for essential plant processes. In one example, we will investigate the role of repression in hormone signalling and in the other we will look at the role of repression in maintaining the pool of undifferentiated cells at the growing tip of the shoot (the meristem) that is essential for plants to modulate their development in response to age and environment. Transcription factors repress gene expression using a class of proteins called transcriptional co-repressors. These proteins cannot bind DNA and must interact with transcription factors to be recruited to the regulatory regions of genes. Target gene specificity is contributed by the transcription factor and the repressive capacity is provided by the co-repressor. We identified the first members of a small family of plant co-repressors that here we call the TTW family. There are five TTW proteins in the model plant Arabidopsis. Others have shown that this family is required as a central part of the signalling mechanism for the plant hormone auxin. The ability of each cell to perceive its auxin environment and to regulate expression of its genes in response to that environment plays a huge part in shaping the plant and allowing it to function. Understanding how auxin levels are translated into instructions to turn genes on and off has been one of the major scientific achievements of the last 5 years. We now know that one of the key players in this signalling process is able to shut down gene expression by recruiting members of the TTW family. The TTW family is therefore already linked directly to two distinct processes that are central to plant life: meristem maintenance and auxin signalling. We have new data that indicate that the influence of the TTW family is much wider than this. Our evidence shows that just one member of the TTW family helps a minimum of 110 different transcription factors to repress target genes. It also implicates the TTW family in another phytohormone signalling pathway; suggesting that the remarkable organisational similarities between the jasmonic acid and auxin signalling pathways extend to a common use of this class of co-repressors. Given that many aspects of gene regulation appear to converge on this small family of proteins it is important that we understand how they work. To this end we propose three objectives that address the following questions: 1. Is the TTW family of co-repressors required to enact the repressive function in the jasmonic acid signalling pathway, as it is in the auxin signalling pathway? 2. Can we use the connection between the TTW corepressors and meristem maintenance to understand the mode of action of the TTW co-repressors? 3. Can we define the full extent of the interactions between the TTW proteins and plant transcription factors so that we can define exactly what allows any transcription factor to recruit a TTW co-repressor? The first question allows us to understand more about a vital hormone signalling pathway and reveals a remarkable evolutionary similarity between the way in which different proteins become organised in a similar way. The second question, explains the link between meristem maintenance and the TTW proteins, and shows how the TTW proteins actively shut down gene expression. The final question will produce a community resource to catalogue TTW - transcription factor interactions and should also help us to define the rules that determine which transcription factors interact with which co-repressors.

Recent discoveries, from our group and others, indicate that a small family of transcriptional co-repressors (called TTW here) are recruited by many transcription factors, belonging to different families, to actively shut off expression of specific target genes. We identified the first two members of this family by their interactions with the meristem maintenance factor WUSCHEL. Subsequently, a total of five family members have been identified in Arabidopsis. They have been shown to be required for the establishment of shoot fate and to be a key part of the auxin signalling pathway, since they mediate the transcriptional repression inherent to the AUX/IAA proteins, whose stability in the cell is dependent on the auxin levels. It has also been established that the TTW co-repressors are recruited by protein motifs including the well-known EAR-domain, which is a sequence as short as 6 amino acids that, when attached to an activating transcription factor, converts it into a dominant repressor. Further unpublished work from our group shows that the TTW co-repressors have both overlap and specificity in their protein-protein interaction potential and that the interactions are defined by amino acids within the EAR-domain. Some of the many new transcription factors we have identified as TTW interactors are known to act as repressors, including the JAZ factors which occupy an analogous position in the jasmonate signalling pathway as the AUX/IAA factors occupy in the auxin pathway. In summary, the TTW co-repressors are recruited by many different transcription factors to influence known processes such as meristem fate, meristem maintenance and phytohormone signalling and potentially many more yet to be discovered. In this proposal we use two biological processes, meristem maintenance and jasmonate signalling to ask three questions: what sort of roles does TTW co-repression play, how does TTW co-repression work and what transcription factors use the TTW co-repression system?

This proposal aims to build on basic research, to understand how transcription factors use a family of co-repressors to actively turn off gene expression. Transcription factors are prime targets to manipulate in order to allow the rational design of novel plant biomass. The ability of the co-repressors under study in this proposal to be recruited by small peptide motifs present in a wide range of transcription factors, provides a link between small motifs that are amenable to engineering into existing proteins and controllable regulation of gene expression. We envisage outputs of this project coming in 5 main areas. 1. Defining the role of TTW corepressors in JA signalling., allowing the manipulation of this phytohormone signalling pathway. 2. Revealing the role of TTW corepressors in meristem maintenance. 3. Identification of the mechanism of transcriptional repression by TTW co-repressors, opening up new opportunities in artificially regulating gene expression in plants. 4. Defining the minimum requirements for TTW repression in a heterologous system; opening up the possibility of its use in other kingdoms. 5. A catalogue of TTW co-repressor - transcription factors, revealing new processes controlled by TTW co-repressors and helping to model the plant transcriptional network. Impact will be delivered according to the attached impact plan, largely in the following ways: Engagement with the wider scientific establishment through publications, online resources and presentations at meeting and seminars. Interaction, translation and dissemination to those concerned with crop security in the developing world by our formal, established links in China, India and Africa. Interaction with industry and commercialisation of our research through the action of our dedicated office of Enterprise and Innovation. Engagement with the wider public through a range of individual activities in schools, higher education and popular science initiatives and also via the specific activities of a dedicated publicity company, CampusPR. Training of a PhD student for future employment in academic and/or commercial environments and research-led teaching to undergraduate students. Continuing and broadening the training of the appointed fellow, Dr. Causier.