Michel Dron’s research while affiliated with University of Paris-Saclay and other places

Vanilla planifolia, the species cultivated to produce one of the world’s most popular flavors, is highly prone to partial genome endoreplication (PE) which leads to highly unbalanced DNA content in cells. We report here first molecular evidence of PE at chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59,128 predicted protein-coding genes. The atypical k-mers frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. On the contrary, low coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns suggesting a complex molecular regulation of endoreplication along the chromosomes. This high-quality anchored assembly represented 83% of the estimated V. planifolia genome. It provides a significant step towards the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that allows centralized access to high-throughput genomic and other omics data, and interoperable use of bioinformatics tools.

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of beta-sheet-rich, insoluble and protease-resistant self-replicating assemblies (PrP Sc ). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously-forming prions are currently lacking. Here, extending studies on the role of H2 alpha-helix C-terminus of PrP, we found that deletion of the highly conserved 190 HTVTTTT 196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase-K resistant, insoluble and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of PK-resistant core of the spontaneous prion (∆ Spont ) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely ∆190-196 C1 PrP construct, in absence of the full-length protein, were susceptible to ∆ Spont prions. ∆ Spont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ∆Spont prion. In conclusion this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30 years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.

DNA remodelling during endoreplication appears to be a strong developmental characteristic in orchids. In this study, we analysed DNA content and nuclei in 41 species of orchids to further map the genome evolution in this plant family. We demonstrate that the DNA remodelling observed in 36 out of 41 orchids studied corresponds to strict partial endoreplication. Such process is developmentally regulated in each wild species studied. Cytometry data analyses allowed us to propose a model where nuclear states 2C, 4E, 8E, etc. form a series comprising a fixed proportion, the euploid genome 2C, plus 2 to 32 additional copies of a complementary part of the genome. The fixed proportion ranged from 89% of the genome in Vanilla mexicana down to 19% in V. pompona, the lowest value for all 148 orchids reported. Insterspecific hybridisation did not suppress this phenomenon. Interestingly, this process was not observed in mass-produced epiphytes. Nucleolar volumes grow with the number of endocopies present, coherent with high transcription activity in endoreplicated nuclei. Our analyses suggest species-specific chromatin rearrangement. Towards understanding endoreplication, V. planifolia constitutes a tractable system for isolating the genomic sequences that confer an advantage via endoreplication from those that apparently suffice at diploid level.

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrPC structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrPC nor for the formation of a stable prion structure.

Importance: Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date resolution of prion structure remains elusive. Thus to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C-terminus of alpha-helix H2, did not hamper prion formation, but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

Prions are formed of misfolded assemblies (PrP(Sc)) of the variably N-glycosylated cellular prion protein (PrP(C)). In infected species, prions replicate by seeding the conversion and polymerization of host PrP(C). Distinct prion strains can be recognized, exhibiting defined PrP(Sc) biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrP(Sc) assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrP(C) glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrP(C) species of interest as substrate. Applying the technique to PrP(C) glycosylation mutants expressing cells revealed that neither PrP(C) nor PrP(Sc) glycoform stoichiometry was instrumental to PrP(Sc) formation and strainness perpetuation. Our study supports the view that strain properties, including PrP(Sc) glycotype are enciphered within PrP(Sc) structural backbone, not in the attached glycans.