Éditeur(s) : HAL CCSD Landes Bioscience
Résumé : International audience
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Autophagy

hal-00735751
https://hal.archives-ouvertes.fr/hal-00735751
PUBMED : 22966490

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in refs 1-5). To allow a comprehensive analysis of hotspot-associated DNA and chromatin characteristics, we developed a direct molecular approach for mapping meiotic DNA double-strand breaks that initiate recombination. Here we present the genome-wide distribution of recombination initiation sites in the mouse genome. Hotspot centres are mapped with approximately 200-nucleotide precision, which allows analysis of the fine structural details of the preferred recombination sites. We determine that hotspots share a centrally distributed consensus motif, possess a nucleotide skew that changes polarity at the centres of hotspots and have an intrinsic preference to be occupied by a nucleosome. Furthermore, we find that the vast majority of recombination initiation sites in mouse males are associated with testis-specific trimethylation of lysine 4 on histone H3 that is distinct from histone H3 lysine 4 trimethylation marks associated with transcription. The recombination map presented here has been derived from a homogeneous mouse population with a defined genetic background and therefore lends itself to extensive future experimental exploration. We note that the mapping technique developed here does not depend on the availability of genetic markers and hence can be easily adapted to other species with complex genomes. Our findings uncover several fundamental features of mammalian recombination hotspots and underline the power of the new recombination map for future studies of genetic recombination, genome stability and evolution.
ISSN: 0028-0836

hal-00877681
https://hal.archives-ouvertes.fr/hal-00877681
DOI : 10.1038/nature09869
PUBMED : 21460839

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : 4 pages
International audience
This article documents the addition of 268 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alburnoides bipunctatus, Chamaerops humilis, Chlidonias hybrida, Cyperus papyrus, Fusarium graminearum, Loxigilla barbadensis, Macrobrachium rosenbergii, Odontesthes bonariensis, Pelteobagrus vachelli, Posidonia oceanica, Potamotrygon motoro, Rhamdia quelen, Sarotherodon melanotheron heudelotii, Sibiraea angustata, Takifugu rubripes, Tarentola mauritanica, Trimmatostroma sp. and Wallago attu. These loci were cross-tested on the following species: Alburnoides fasciatus, Alburnoides kubanicus, Alburnoides maculatus, Alburnoides ohridanus, Alburnoides prespensis, Alburnoides rossicus, Alburnoides strymonicus, Alburnoides thessalicus, Alburnoides tzanevi, Carassius carassius, Fusarium asiaticum, Leucaspius delineatus, Loxigilla noctis dominica, Pelecus cultratus, Phoenix canariensis, Potamotrygon falkneri, Trachycarpus fortune and Vimba vimba.
ISSN: 1755-098X

hal-00823716
https://hal.archives-ouvertes.fr/hal-00823716
DOI : 10.1111/1755-0998.12095
PUBMED : 23521844

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. RESULTS: We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes -- normally the hottest region in the genome. CONCLUSIONS: Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.
ISSN: 1471-2164

inserm-00849779
http://www.hal.inserm.fr/inserm-00849779
http://www.hal.inserm.fr/inserm-00849779/document
http://www.hal.inserm.fr/inserm-00849779/file/1471-2164-14-493.pdf
DOI : 10.1186/1471-2164-14-493

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
ISSN: 1088-9051

hal-00877675
https://hal.archives-ouvertes.fr/hal-00877675
DOI : 10.1101/gr.130583.111
PUBMED : 22367190

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
ISSN: 1354-1013

hal-01080954
https://hal-agroparistech.archives-ouvertes.fr/hal-01080954
DOI : 10.1111/j.1365-2486.2007.01439.x

Éditeur(s) : HAL CCSD
Résumé : International audience
o abstract
The International Liver Congress™ 2015 – 50th Annual meeting of the European Association for the Study of the Liver
Vienne, Austria

hal-01162775
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01162775
DOI : 10.1016/S0168-8278(15)30674-7

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
o abstract
ISSN: 0168-8278

hal-01519257
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01519257
DOI : 10.1016/S0168-8278(16)00178-1

Éditeur(s) : HAL CCSD American Association for the Advancement of Science
Résumé : International audience
DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.
ISSN: 0036-8075

hal-01123717
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01123717
PUBMED : 25395542
DOI : 10.1126/science.1256442

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation
ISSN: 0890-9369

hal-01281480
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01281480
DOI : 10.1101/gad.270009.115
PUBMED : 26833728

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases. © The Author(s) 2016.
ISSN: 2041-4889

hal-01414916
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01414916
DOI : 10.1038/cddis.2016.362
PUBMED : 27831558

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.
ISSN: 0028-0836

hal-00877666
https://hal.archives-ouvertes.fr/hal-00877666
DOI : 10.1038/nature11089
PUBMED : 22660327

Éditeur(s) : HAL CCSD
Résumé : In this paper we report a study of the mating system and gene flow of Sextonia rubra, a hermaphroditic, insect pollinated tropical tree species with a geographic distribution in the Guyana Plateau and the Amazon. Using five microsatellites we analysed 428 seeds of 27 open pollinated families at the experimental site "Paracou" in French Guiana. We observed, compared to other tropical tree species, a high level of genetic diversity. We estimated parameters of the mating system and gene flow by using the mixed mating model and the TwoGenerapproach. The estimated multilocus outcrossing rate, tm, was 0.992 indicating nearly complete outcrossing. A significant level of biparental inbreeding and a small proportion of full-sibs were estimated for the 27 seed arrays. The differentiation of allelic frequencies among the pollen pools was ΦFT = 0.061. We estimated mean pollen dispersal distances between 65 m and 89 m according to the dispersal models used. The joint estimation of pollen dispersal and density of reproductive trees gave an effective density estimate of 2.1-2.2 trees/ha
Silvae genetica

hal-01031483
https://hal.archives-ouvertes.fr/hal-01031483

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Hepatocyte death is a central event during liver disease progression, in which immune cells play key roles by activating members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), including TNFR1 (TNFRSF1A), Fas (TNFRSF6) and TRAIL-R2 (TNFRSF10B). Receptor Interacting Protein Kinase 1 (RIPK1) emerged as a signaling node downstream of these receptors. In the case of TNFR1, RIPK1 has been demonstrated to paradoxically serve as a scaffold to promote the survival of hepatocytes and as a kinase to kill them. To evaluate whether RIPK1 also protects hepatocytes from death in response to FasL or TRAIL, we took advantage of liver parenchymal cell-specific Ripk1 knockout mice (Ripk1 (LPC-KO)). We found that Ripk1 (LPC-KO) mice, as well as primary hepatocytes derived from them, were more susceptible to Fas-mediated apoptosis than their respective WT counterparts. Fas-induced hepatocyte death was independent of TNF-α signaling. Interestingly, while TRAIL administration did not induce hepatitis in Ripk1 (LPC-KO) mice or in their WT counterparts, its combination with IFN-γ only induced TNF-α dependent apoptosis in the Ripk1 (LPC-KO) mice. Together, our data demonstrate the protective role of RIPK1 downstream of Fas and highlight the general protective function of RIPK1 in hepatocytes exposed to inflammatory conditions, where TNF-α, FasL and/or TRAIL are present.
ISSN: 2045-2322

hal-01579612
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01579612
DOI : 10.1038/s41598-017-09789-8
PUBMED : 28835677