Macrophages are immune cells playing a central role in maintaining tissue homeostasis and orchestrating repair after tissue injury. Macrophages can originate from two different pathways, deriving from embryonic precursors or from adult blood monocytes. While the biology of embryo-derived macrophages is well studied, the properties of monocyte-derived macrophages remain incompletely understood. In this project, we will focus on monocyte-derived macrophages that develop during sterile inflammation. Using in vitro models with human cells and in vivo models in mouse, we will address the differentiation mechanisms and functional roles of monocyte-derived macrophages during synovial joint inflammation and cutaneous wound healing. Our results should create new opportunities for ameliorating the treatment of autoimmune inflammation including rheumatoid arthritis.

Protein complexes are at the heart of most biological functions. The CHIPSeT project aims at a deeper understanding of interaction surfaces and will integrate large-scale experimental and computational strategies to disentangle the complexity underlying protein-protein interaction (PPI) networks. It will first focus on newly discovered physical interactions ensuring the cross-talk between chromatin remodelers (INO80) and proteostasis pathways (Cdc48/VCP/p97). Combining large scale proteomic screens and genome-wide CHIP-seq technologies, partner 2 found that both machineries act in a concerted manner for the disassembly and subsequent proteolysis of large complexes involved in DNA-metabolic processes. A key limitation of the current approach is that the PPI-network of both INO80 and Cdc48 is highly intricate. To tackle these challenging systems and reveal the molecular logic associated with PPI networks, we propose to develop two connected strategies that exploit co-evolution information and rely on computational and experimental settings. First, partner 1 showed that the use of multiple sequence alignments to reveal co-evolutionary constraints acting at PPI interfaces could critically increase the reliability of structural models in docking simulations. The main future challenge will be to integrate co-evolutionary constraints not only at the coarse-grained step of the docking process but also at the fine-grained, during optimization of models. The second strategy has a highly innovative potential and consists in producing experimentally and artificially co-evolution events at high rates at protein complex interfaces. Next generation sequencing methods are providing us with the possibility to reach a very rich landscape of compensatory mutants which can be used with great potency in the modeling process. Generating this artificial coevolution at high rate is a significant challenge. In our project, we devised a strategy that takes the best from the expertise of partner 3 and 4 to propose a robust pipeline likely to fulfil our goals. On the one hand, we set up a two-hybrid system running with a variety of fluorescent probes to report for gain or loss of interactions in a relatively quantitative manner (partner 3). Next, compensatory mutations can be screened and prepared for sequencing very efficiently thanks to the cutting-edge droplet-based microfluidic technology (partner 4). Both co-evolution-based strategies should provide unprecedented power to explore and tune the properties of complex interfaces, in vivo. This work will provide invaluable tools for modeling protein interaction networks at large scale and analyzing the effect of mutations in pathologies connected to Cdc48/P97/VCP system or to specific subunits of the INO80 machinery.

Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. Fundamental to centromere function is a histone H3 variant, CenH3, that initiates kinetochore assembly on centromeric DNA. In all organisms that have been studied, CenH3 deletions are lethal and lead to catastrophic defects in chromosome segregation. Moreover, the incorporation of CenH3 defines both canonical centromeres and neocentromeres in diverse organisms. The presence of CenH3 homologs in all animals, fungi and plants, together with their identification in distantly branching protist lineages have established the paradigm that CenH3-containing chromatin is an absolute requirement for centromere function. Our recent findings undermined this paradigm of CenH3 essentiality. We showed that CenH3 was lost independently in four lineages of insects. These losses are concomitant with dramatic changes in their centromeric architecture, in which each lineage independently transitioned from monocentric chromosomes (where microtubules attach to a single chromosomal region) to holocentric chromosomes (where microtubules attach along the entire length of the chromosome). This implies that CenH3 function must have been acquired by another protein that localizes chromosome-wide, resulting in the spread of the kinetochore over large parts of the chromosome, and ultimately giving rise to holocentromeres. I aim to characterize this unique CenH3-deficient chromosomes segregation pathway. My goal is to determine how kinetochore assembly can occur in a CenH3-independent manner. Specifically, I will test three models of CenH3 replacement in initiating kinetochore assembly by (1) a pre-existing kinetochore component that has DNA or chromatin binding activity, (2) another DNA or chromatin binding factor that acquired kinetochore function, or (3) a novel histone variant or modification. To evaluate these three models, I have established lepidopteran cell lines as a model for the study of their CenH3-deficient centromeres. Using these cell lines, I will use proteomic approaches to determine the composition and assembly hierarchy of their kinetochores. Using genomic approaches, I will also determine the underlying DNA sequence preferences of these lepidopteran holocentromeres. This will complement my proteomic analyses and determine whether the factor initiating kinetochore assembly recognizes discrete DNA sequence motifs (genetically defined holocentromere) or acts independently of the underlying DNA sequence (suggesting an epigenetically defined holocentromere). Finally, I will expand my analyses to additional holocentric insects derived from independent transitions to determine whether this kinetochore assembly pathway has recurrently evolved. Our discovery of CenH3 loss in holocentric insects establishes a new class of centromeres. Despite this paradigm-overturning discovery, our findings suggest that CenH3-deficient centromeres are not rare; instead holocentric insects comprise more that 16% of the known eukaryotic biodiversity. My research will elucidate this novel CenH3-independent chromosome segregation pathway. This will reveal how CenH3 that is essential in most other eukaryotes, could have become dispensable in holocentric insects. Since the evolution of this CenH3-independent chromosome segregation pathway occurred concomitant with the rise of holocentric architectures, my research will also provide the first insights into the transition from a monocentromere to a holocentromere.