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The last 50 bibliographies

A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities

Author(s) : Cluet D, Amri I, Vergier B, Léault J, Audibert A, Grosjean C, Calabrési D, Spichty M,
Journal : Molecular & Cellular Proteomics
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pM to 17 µM. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition.

Author(s) : Torres-Campana D, Kimura S, Orsi G, Horard B, Benoit G, Loppin B,
Journal : PLoS Genet
Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. Here, we haveperformed an shRNA-based genetic screen in Drosophila to identify genes requiredto form a diploid zygote. We found that the Lid/KDM5 histone demethylase and itspartner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favorthe accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. We propose that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

Author(s) : Shah S, Hartono S, Piazza A, Som V, Wright W, Chedin F, Heyer W,
Journal : Biorxiv
Displacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54’s translocation activity, limiting D-loop formation and D-loop length.

Saccharomyces Cerevisiae Mus81-Mms4 Prevents Accelerated Senescence in Telomerase-Deficient Cells

Author(s) : Scwartz E, Hung S, Meyer D, Piazza A, Yan K, Fu B, Heyer W,
Journal : PLoS Genetics
Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

Cell-to-cell expression dispersion of B-cell surface proteins is linked to genetic variants in humans.

Author(s) : Triqueneaux G, Burny C, Symmons O, Janczarski S, Gruffat H, Yvert G,
Journal : Commun Biol
Variability in gene expression across a population of homogeneous cells is known toinfluence various biological processes. In model organisms, natural genetic variantswere found that modify expression dispersion (variability at a fixed mean) but veryfew studies have detected such effects in humans. Here, we analyzed single-cellexpression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derivedfrom individuals of the Yoruba population. Using data from over 30 million cells, wefound substantial inter-individual variation of dispersion. We demonstrate, via denovo cell line generation and subcloning experiments, that this variation exceedsthe variation associated with cellular immortalization. We detected a geneticassociation between the expression dispersion of CD63 and the rs971 SNP. Our resultsshow that human DNA variants can have inherently-probabilistic effects on geneexpression. Such subtle genetic effects may participate to phenotypic variation anddisease outcome.

Guidelines for cell-type heterogeneity quantification based on a comparative analysis of reference-free DNA methylation deconvolution software.

Author(s) : Decamps C, Prive F, Bacher R, Jost D, Waguet A, Houseman E, Lurie E, Lutsik P, Milosavljevic A, Scherer M, Blum M, Richard M,
Journal : BMC Bioinformatics
BACKGROUND: Cell-type heterogeneity of tumors is a key factor in tumor progression and response to chemotherapy. Tumor cell-type heterogeneity, definedas the proportion of the various cell-types in a tumor, can be inferred from DNAmethylation of surgical specimens. However, confounding factors known to associate with methylation values, such as age and sex, complicate accurate inference of cell-type proportions. While reference-free algorithms have been developed to infer cell-type proportions from DNA methylation, a comparative evaluation of the performance of these methods is still lacking. RESULTS: Here we use simulations to evaluate several computational pipelines based on the software packages MeDeCom, EDec, and RefFreeEWAS. We identify that accounting for confounders, feature selection, and the choice of the number of estimated cell types are critical steps for inferring cell-type proportions. We find that removal of methylation probes which are correlated with confounder variables reduces the error of inference by 30-35%, and that selection of cell-type informative probes has similar effect. We show that Cattell's rule based on the scree plot is a powerful tool to determine the number of cell-types. Once the pre-processing steps are achieved, the three deconvolution methods provide comparable results. We observe that all the algorithms' performance improves when inter-sample variation of cell-type proportions is large or when the number of available samples is large. We find that under specific circumstances the methods are sensitive to the initialization method, suggesting that averaging different solutions or optimizing initialization is an avenue for future research. CONCLUSION: Based on the lessons learned, to facilitate pipeline validation and catalyze further pipeline improvement by the community, we develop a benchmark pipeline for inference of cell-type proportions and implement it in the R package medepir.

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

Author(s) : Sati S, Bonev B, Szabo Q, Jost D, Bensadoun P, Serra F, Loubiere V, Papadopoulos G, Rivera-Mulia J, Fritsch L, Bouret P, Castillo D, Gelpi J, Orozco M, Vaillant C, Pellestor F, Bantignies F, Marti-Renom M, Gilbert D, Lemaitre J, Cavalli G,
Journal : Mol Cell
To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. ThisOIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging canidentify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Chromosome dynamics during interphase: a biophysical perspective.

Author(s) : Tortora M, Salari H, Jost D,
Journal : Curr Opin Genet Dev
The dynamic nature of chromosome organization plays a central role in the regulation of many crucial processes, such as DNA transcription and replication.However, the molecular bases of the link between genomic function, structure anddynamics remain elusive. In this review, we focus on how biophysical modelling can be instrumentally used to rationalize experimental studies of chromosome dynamics, and to probe the impact of putative mechanisms on genome folding kinetics during interphase. We introduce the general connection between chromatin internal organization and dynamics, and outline the potential effects of passiveinteractions mediated by architectural proteins and of active, energy-dependent processes on chromatin motion. Finally, we discuss current ambiguities emerging from in vivo observations, in particular related to ATP depletion and transcriptional activation, and highlight future perspectives.

PenDA, a rank-based method for personalized differential analysis: Application to lung cancer.

Author(s) : Richard M, Decamps C, Chuffart F, Brambilla E, Rousseaux S, Khochbin S, Jost D,
Journal : PLoS Comput Biol
The hopes of precision medicine rely on our capacity to measure various high-throughput genomic information of a patient and to integrate them for personalized diagnosis and adapted treatment. Reaching these ambitious objectives will require the development of efficient tools for the detection of molecular defects at the individual level. Here, we propose a novel method, PenDA, to perform Personalized Differential Analysis at the scale of a single sample. PenDA is based on the local ordering of gene expressions within individual cases and infers the deregulation status of genes in a sample of interest compared to a reference dataset. Based on realistic simulations of RNA-seq data of tumors, we showed that PenDA outcompetes existing approaches with very high specificity andsensitivity and is robust to normalization effects. Applying the method to lung cancer cohorts, we observed that deregulated genes in tumors exhibit a cancer-type-specific commitment towards up- or down-regulation. Based on the individual information of deregulation given by PenDA, we were able to define two new molecular histologies for lung adenocarcinoma cancers strongly correlated tosurvival. In particular, we identified 37 biomarkers whose up-regulation lead tobad prognosis and that we validated on two independent cohorts. PenDA provides arobust, generic tool to extract personalized deregulation patterns that can thenbe used for the discovery of therapeutic targets and for personalized diagnosis.An open-access, user-friendly R package is available at

Developmental variability channels mouse molar evolution.

Author(s) : Hayden L, Lochovska K, Semon M, Renaud S, Delignette-Muller M, Vilcot M, Peterkova R, Hovorakova M, Pantalacci S,
Journal : Elife
Do developmental systems preferentially produce certain types of variation that orient phenotypic evolution along preferred directions? At different scales, from the intra-population to the interspecific, the murine first upper molar shows repeated anterior elongation. Using a novel quantitative approach to compare thedevelopment of two mouse strains with short or long molars, we identified temporal, spatial and functional differences in tooth signaling center activity,that arise from differential tuning of the activation-inhibition mechanisms underlying tooth patterning. By tracing their fate, we could explain why only the upper first molar reacts via elongation of its anterior part. Despite a lack of genetic variation, individuals of the elongated strain varied in tooth length and the temporal dynamics of their signaling centers, highlighting the intrinsic instability of the upper molar developmental system. Collectively, these resultsreveal the variational properties of murine molar development that drive morphological evolution along a line of least resistance.