In living organisms, gene expression is finely regulated by the joint action of regulatory proteins. Among these proteins, transcription factors (TFs hereafter) play a key role since they bind specific sequences in the promoters of genes to initiate their regulation. TFs can combine to form complexes and regulate the expression of new genes or to alter the direction or level of regulation of genes already targeted by one of the two TFs alone. The complexes thus diversify the repertoire and regulatory levels of genes targeted by the transcription factors. Little is known about the extent of this phenomenon, the number of complexes, the identity of the partners and the way they bind to DNA. This project proposes to develop a bioinformatics model to predict the existence of protein complexes formed by transcription factors and likely to regulate gene expression in the plant Arabidopsis thaliana. In a second phase, the project will explore the predictions of the model to verify the existence of the predicted complexes, and to characterize their DNA binding mode and their target genes. The discovery of new complexes will be done by developing a model that integrates clues scattered in different types of genomic data. These clues are (i) the common binding of the TFs on promoter regions, the motifs and combinations of DNA motifs bound by the TFs on these bound regions, (i) the co-expression of the TFs, (iii) the target genes common to both TFs, and (iv) the co-evolution of amino acid residues between the two TFs forming a complex. The model will be obtained by machine learning on these data: the model will be built and its parameters adjusted to optimize the predictions against a set of TFs known to form complexes. Newly predicted interactions, in particular those between transcription factors studied in our lab and new partners, will be explored in detail to understand how these complexes form (interaction surface), how they bind DNA and to know which genes and functions they regulate. The results of the model will be represented in the form of an interaction network for all Arabidopsis thaliana TFs.. This network will be made available to the community so that biologists can in turn explore the potential partners of their favorite TFs. In the medium term, the model could be applied to other plant species such as rice and maize, two species characterized by extensive genomic data. This approach represents a considerable time saving compared to the genetic method and works even in the case where several TFs play a redundant role.

How do higher plants deploy flexible and adjustable developmental programs? Considerable advances have been made in identifying the responsible genetic and epigenetic regulators. However thus far, mutant-based approaches have mainly generated correlative conclusions between transcriptional activities and chromatin contexts, thereby revealing limited information on the direct function of epigenomes during development. To convert correlation-based findings into mechanistic principles, a current challenge in functional epigenomics is to develop tools for the precise manipulation of epigenetic marks. With REWIRE, we propose to construct and use such tools for understanding the impact of epigenetic marks on plant architecture. To this aim, we will enforce novel molecular editing technologies to rewrite epigenetic marks on histone protein residues, both genome-wide and at targeted key genes. With in cyto imaging and high-throughput molecular analyses we will probe structure-function links between chromatin and transcription, at an unprecedented spatio-temporal resolution. REWIRE is the stepping-stone to discoveries of epigenetic determinants for plant plasticity, and should also bring answers to prevailing questions in epigenetics of higher eukaryotes. Moreover, because the epigenetic marks we study correlate with plant fitness, flowering time and progeny setting, our discoveries should bring levers for improving agronomic traits. In conclusion, REWIRE will have multiple impacts, generating knowledge for fundamental science, providing teaching material for a wide public audience, as well as delivering tools for patent development in the agricultural sector.

Chloroplasts (cp) are a defining feature of plant cells, carrying out the fundamental reaction of photosynthesis and metabolic functions essential for plant productivity, fertility and stress adaptation. Cp genomes in land plants retain ~100 genes whose expression is primarily controlled by imported nuclear-encoded nucleic acid binding proteins. Recently, members of the mTERF protein-family in plants have emerged as key regulators of organellar genes and plant abiotic stress responses. Our results have demonstrated an unexpected dual protein- and RNA-scaffolding function for mTERF9, a previously uncharacterized member of the mTERF family in Arabidopsis. mTERF9 plays an important role in stimulating ribosomal assembly and translation in cps where it forms in vivo puncta and, unlike most mTERF family proteins, mTERF9 contains a phosphorylated N-terminal intrinsically disordered region (IDR). Based on preliminary unpublished data, the N-terminal IDR and its phosphorylation are required for mTERF9 in vivo function, however the molecular mechanisms for this are unknown. The proposed project will directly address the underlying molecular determinants of mTERF9 function, identify its core protein and RNA binding partners and determine the role of the IDR and phosphorylation state in mTERF9 activity and puncta formation during cp ribosomal assembly and translation. To address these ambitious goals, we will use an interdisciplinary approach combining plant molecular genetics, proteomics, ribonomics and cell biology (P1) coupled with cutting edge biophysics and structural biology including small angle X-ray scattering and electron microscopy (P2). These experiments will demonstrate how mTERF9 is able to fulfill multiple roles in cp ribosomal assembly, stability and translation, major outstanding questions in the plant biology field.

The ChromAuxi project aims at understanding how the interplay between Auxin Response Factors (ARFs) and chromatin dynamics orchestrates the multiplicity of transcriptional responses to this phytohormone in Arabidopsis. First, we propose to determine using in vitro approaches the binding rules of the different classes of ARFs on genomic DNA and to characterize their mode of interaction with proteins affecting chromatin organization. Then, we will determine how the chromatin context influences the binding of ARFs along the genome and, conversely, how this binding affects chromatin accessibility in response to auxin. The functional significance of these molecular rules will be tested in planta using (epi)genome engineering methods. Decrypting auxin response at the ARF-chromatin interface should provide unprecedented insights into the principles governing the numerous yet specific auxin-triggered regulations that underpin most aspects of plant development.

Light has two roles for photosynthetic organisms: sensed by photoreceptors it pro-vides spatiotemporal information; absorbed by chloroplast pigments it supplies energy for photosynthesis, crucial for life on Earth. To thrive, plants and algae evolved the ability to integrate these two functions, through complex mechanisms currently not well understood. A main goal is to prevent lethal photodamage caused by excess light and they do so mainly via the photoprotection mechanism qE (energy quenching). Our data show that in the green alga Chlamydomonas reinhardtii there is a complex regulatory interconnection between qE, the blue light photoreceptor PHOTOTROPIN, photosynthesis and metabolic CO2 which we aim to elucidate within MetaboLIGHT at the molecular level, using biochemical, genetic, chemical genetic, and genome-wide transcriptomic approaches. MetaboLIGHT will advance our fundamental knowledge on the regulation of photosynthesis under fluctuating environmental conditions.