EVOLUTION OF TRANSCRIPTIONAL EFFECTORS AND REGULATORS

Group Leader: Jorge Hernández García - Young Investigator Researcher (YIR)
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Personnel:

 

https://sites.google.com/view/eterlab 

 

Background

Plant growth is dependent on the precise coordination between developmental programs and the integration of environmental information. A big number of signalling systems involved in mediating this coordination have evolved independently from those in animals. Recent studies indicate that, at least in plants, transcriptional regulation is the eventual target of most signalling systems that integrate endogenous and environmental information. Therefore, defining the intimate molecular mechanisms that modulate transcriptional activity is key to understanding adaptation.


Transcription factors (TFs), as key transcriptional effectors able to bind DNA, provide both target gene selectivity and regulatory function (i.e., effector function). Many studies have focused on understanding their DNA-binding properties, or, separately, studying the molecular mechanisms linking effector function to gene expression (i.e., through recruitment of co-activators or co-repressors). However, these functions occur concurrently and likely influence one another through chromatin rearrangements and/or interaction with other regulatory proteins (Fig. 1). Studying how both properties interact to define a final transcriptional output is essential to understanding which and how genes are regulated by certain signals and TFs.




Figure 1. Interplay between transcription factor properties. Transcription factors (TFs) exhibit two key functions: DNA binding specificity, encoded in the DNA binding domains (DBD) and transcriptional regulatory function, encoded in effector domains (ED). DNA binding allows TFs to target specific genomic loci, while the effector domain modulates transcriptional activity through recruitment of co-effectors (CoE). Additional interacting partners, such as other TFs or transcriptional regulators, in turn, can modulate the former TF effector functionality, and/or the chromatin local environment, thus further contributing to both ED and DBD function. These functions are interconnected and influence each other through establishing specific interactions and rearranging chromatin.

 


Research interests

Our goal is to characterize the evolution and the molecular mechanisms behind plant transcription factor (TF) properties (i.e. transcriptional effector, interacting, DNA-binding) using an interdisciplinary perspective, including genetics, biochemistry, single molecule studies, and synthetic biology combined with high-resolution imaging, as well as evolutionary approaches as phylogenetics and ancestral sequence prediction. We currently 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 (Fig. 2).


Figure 2. Phylogenetic tree of ARF proteins. The tree highlights the five ARF classes we have uncovered, with each ancestral branching node colour-shaded. The predicted ARF ancestor is marked (predicted to have occurred around 800 mya – 1 bya). Tree is built with protein sequences from DD-to-AD regions, using PHIP sequences as outgroup. Bootstrap values are indicated as color-coded bubbles in branch nodes, with the main ARF class ancestral nodes highlighted. Scale bar represents distance in substitutions per residue. Adapted from Hernández-García et al. 2024.

 

We will 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:

  1. 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 ( 3), 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.

    Figure 3. Marchantia polymorpha nucleus showing native ARF proteins localization. Knock-in line (endogenous loci integration) with MpARF1 tagged with mScarlet-I, shown in violet, MpARF2 tagged with mNeonGreen, shown in green. Zoom in shows independent clusters or condensates. Image represents a reconstructed Z-stack.

  2. 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 hence aim to analyze 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
  3. 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.

 

Genetic models

In our group, we study a wide range of plants, but our main workhorse is Marchantia polymorpha, a thalloid liverwort (Fig. 4). We are developing a toolkit of synthetic reporters to study transcriptional dynamics and chromatin status using life imaging for this plant. 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. We have recently created an electroporation-mediated platform to analyze protein functions in streptophyte algae, you can read more about that here.




Figure 4. Marchantia polymorpha gametophyte in the wild. Styria, Austria. Credits to Walter Obermayer, Karl-Franzens-Universität Graz.

 

 

Funding

Call: Ayudas Atracción Talento Investigador ‘César Nombela’

Title: Deciphering the Antagonistic mechanisms of Transcriptional Effectors (2024-T1/BIO-31319)

Funding agency: Comunidad de Madrid

Period: 2025-2030

 

 


Representative Publications

Carrillo-Carrasco, V.P., Hernandez-Garcia, J., Girou, C., Grubor, I., Keller, J., Lim, E., Schmidt, V., Sørensen, I., Vosolsobe, S., Buschmann, H., Delaux, P.-M., Domozych, D., Holzinger, A., Nakagami, H., Nishiyama, T., Petrasek, J., Renault, H., Rensing, S.A., Rose, J.K.C., Sekimoto, H., Delwiche, C.D., Weijers, D., Vries, J. de 2025. A roadmap to developing unified streptophyte algal model systems. Current Biology 35, R725–R738. DOI: 10.1016/j.cub.2025.05.023

Carrillo-Carrasco, V.P., van Galen, M., Bronkhorst, J., Mutte, S., Kohlen, W., Sprakel, J., Hernández-García, J., Weijers, D. 2025. Auxin and tryptophan trigger common responses in the streptophyte alga Penium margaritaceum. Current Biology 35, 2078-2087.e4. DOI: 10.1016/j.cub.2025.03.037

de Roij, M., Hernández García, J., Das, S., Borst, J.W., Weijers, D. 2025. ARF degradation defines a deeply conserved step in auxin response. Nature Plants 11, 717–724. DOI: 10.1038/s41477-025-01975-1

Rienstra, J., Carrillo-Carrasco, V.P., de Roij, M., Hernandez-Garcia, J., Weijers, D. 2025. A conserved ARF–DNA interface underlies auxin-triggered transcriptional response. Proceedings of the National Academy of Sciences 122, e2501915122. DOI: 10.1073/pnas.2501915122

Hernández-García, J., Carrillo-Carrasco, V.P., Rienstra, J., Tanaka, K., de Roij, M., Dipp-Álvarez, M., Freire-Ríos, A., Crespo, I., Boer, R., van den Berg, W.A.M., Lindhoud, S., Weijers, D. 2024. Evolutionary origins and functional diversification of Auxin Response Factors. Nature Communications 15, 10909. DOI: 10.1038/s41467-024-55278-8

Rienstra, J., Hernández-García, J., Weijers, D. 2023. To bind or not to bind: how AUXIN RESPONSE FACTORs select their target genes. Journal of Experimental Botany 74, 6922–6932. DOI: 10.1093/jxb/erad259

Carrillo-Carrasco, V.P., Hernández-García, J., Weijers, D. 2023. Electroporation-based delivery of proteins in Penium margaritaceum and other zygnematophycean algae. Physiologia Plantarum 175, e14121. DOI: 10.1111/ppl.14121

Carrillo‐Carrasco, V.P., Hernandez‐Garcia, J., Mutte, S.K., Weijers, D. 2023. The birth of a giant: evolutionary insights into the origin of auxin responses in plants. The EMBO Journal 42, e113018. DOI: 10.15252/embj.2022113018

Briones-Moreno, A., Hernández-García, J., Vargas-Chávez, C., Blanco-Touriñán, N., Phokas, A., Úrbez, C., Cerdán, P.D., Coates, J.C., Alabadí, D., Blázquez, M.A. 2023. DELLA functions evolved by rewiring of associated transcriptional networks. Nature Plants 9, 535–543. DOI: 10.1038/s41477-023-01372-6

Hernández-García, J., Diego-Martin, B., Kuo, P.H., Jami-Alahmadi, Y., Vashisht, A.A., Wohlschlegel, J., Jacobsen, S.E., Blázquez, M.A., Gallego-Bartolomé, J. 2022. Comprehensive identification of SWI/SNF complex subunits underpins deep eukaryotic ancestry and reveals new plant components. Communications Biology 5, 1–11. DOI: 10.1038/s42003-022-03490-x

Hernández-García, J., Sun, R., Serrano-Mislata, A., Inoue, K., Vargas-Chávez, C., Esteve-Bruna, D., Arbona, V., Yamaoka, S., Nishihama, R., Kohchi, T., Blázquez, M.A. 2021. Coordination between growth and stress responses by DELLA in the liverwort Marchantia polymorpha. Current Biology 31, 3678-3686.e11. DOI: 10.1016/j.cub.2021.06.010

Li, F.-W., Nishiyama, T., Waller, M., Frangedakis, E., Keller, J., Li, Z., Fernandez-Pozo, N., Barker, M.S., Bennett, T., Blázquez, M.A., Cheng, S., Cuming, A.C., de Vries, J., de Vries, S., Delaux, P.-M., Diop, I.S., Harrison, C.J., Hauser, D., Hernández-García, J., Kirbis, A., Meeks, J.C., Monte, I., Mutte, S.K., Neubauer, A., Quandt, D., Robison, T., Shimamura, M., Rensing, S.A., Villarreal, J.C., Weijers, D., Wicke, S., Wong, G.K.-S., Sakakibara, K., Szövényi, P. 2020. Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts. Nature Plants 6, 259–272. DOI: 10.1038/s41477-020-0618-2

Hernández-García, J., Briones-Moreno, A., Dumas, R., Blázquez, M.A. 2019. Origin of Gibberellin-Dependent Transcriptional Regulation by Molecular Exploitation of a Transactivation Domain in DELLA Proteins. Molecular Biology and Evolution 36, 908–918. DOI: 10.1093/molbev/msz009