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We focus on understanding the molecular basis of specific eukaryotic transcription factors function and evolution. Transcription factors are the elements of signalling pathways that directly affect and regulate gene expression. Transcription factors recognize specific DNA sequences, and exert an effect on transcription in many different ways (usually binary categorized as positive or negative).
Our research focuses on characterizing the evolution and molecular mechanisms behind plant transcription factor (TF) effector (i.e. transcriptional) properties using an interdisciplinary perspective, including genetics, biochemistry, single molecule studies, and high-resolution imaging, as well as evolutionary approaches as phylogenetics and ancestral sequence prediction and resurrection. We now focus on different Auxin Response Factor (ARF) classes as model proteins, predicted to have different effector functions, using a variety of model organisms covering key lineages in the phylogeny of streptophyte plants. We plan on eventually adventure into studying the function of additional plant TF families and transcriptional co-effectors to finally unravel the mechanism behind the coordination of multiple cis-regulatory inputs into a single transcriptional output, and how this evolves to integrate novel inputs, as a core component of signal complexity in plants. Our immediate lines of work can be split into three differentiated topics:
A) Understanding the effector properties of ARFs, using A- and B-classes as antagonist TFs in auxin signalling, and the algal A/B-class as a proxy to their ancestral state prior to divergence. For this, we study the molecular mechanisms behind transcriptional function following different approaches: (i) Search for co-effector candidates using available techniques. For example, we have set up Marchantia TurboID biotin-based nuclear labelling and obtained candidates for A-class co-activation partners. We also use targeted approaches using known co-effectors as baits for ARFs, such as TPL for the repressors. (ii) Characterize the interactions, validating the physiological and molecular relevance using established genetic systems whenever possible. (iii) Analyse the aggregate behaviour and the physicochemical properties of the TF/co-effector complexes. TFs as ARFs form nuclear biocondensates that behave as liquid-like particles, as our preliminary work has shown for TPL and the putative co-activators. For this, multiple techniques can be implemented, from simple confocal imaging to more robust high-resolution imaging, and in vitro analysis techniques.
B) Transcriptional condensates commonly form to initiate transcription, as has been shown in animal systems, suggesting this mechanism can also contribute to transcriptional activation in plants. We aim to analyse the relevance of ARF transcriptional condensates, which facilitate either transcriptional activation or repression, and study their relevance at a chromatin-wide level. For this, we combine (i) imaging of higher-order complexes and analysis of the physicochemical properties of the aggregates, as mentioned above, including RNA polymerase and nascent RNA imaging techniques, with (ii) chromatin accessibility, histone-mark, and TF DNA-binding profiling. Altogether, the integration of this data will help us understand the map of gene activation and repression in correlation with their epigenetic footprint. These approaches will allow for uncovering distinct modes of chromatin context-dependent transcriptional activation.
C) Uncovering common effector mechanisms and new transcriptional co-effectors. Widening the focus from only TFs to transcriptional co-effectors, such as the plant repressor TPL, the Mediator complex subunits, or another kind of known co-effector proteins as baits in screening approaches would allow to find transcriptional effectors functioning by common mechanisms, and open the door for novel lines. Likewise, extending this kind of study to more TFs and transcriptional effectors to understand the basic principles of transcriptional integration. Ultimately, we want to learn how multiple co-occurring effectors coordinate the transcriptional machinery to reach a unique decision in terms of transcription.
Underlying A) and B), we study the evolutionary divergence between the activator and repressive ARF functions by assessing the importance of key ARF gene split and divergence points for the neofunctionalization of new transcriptional activities. We have predicted this specific mechanism is relevant for the evolution of the NAP, as activator/repressor divergence was a key event in the evolution of the pathway as pinpointed above. This will also serve to study the relevance of the establishment of new interactions for the assembly of new hormonal pathways.
We study a wide range of plants, but our main workhorse is Marchantia polymorpha, a thalloid liverwort.
While we focus on bryophytes, we also include phylogenetically-informed models for comparative analyses, and we are working on introducing streptophyte algae as genetic systems.
Marchantia polymorpha gametophyte in the wild
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