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Summary The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties., Graphical Abstract, Highlights • Stable transcriptional reprogramming toward endoderm within one cell cycle • Replication timing can change in the first S phase of a cell fate change • Early discordance between Replication timing and Hi-C compartments • Alignment of replication timing to compartments increases in later cell cycles, The temporal order of DNA replication is regulated during development, and is highly correlated with gene expression, chromatin structure, and 3D-genome architecture. Gilbert and colleagues find that stable transcriptional changes of human embryonic stem cells to endoderm can occur within a single cell cycle, accompanied by discordance in replication timing and chromatin compartment that align in later differentiation stages.

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. HiC is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any HiC experimental design. Here, we present the mathematical concepts to estimate the resolution of a library and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to human and mouse HiC libraries.

Prostate cancer is one of the most commonly diagnosed cancers in men. A number of genomic and clinical studies have led to a better understanding of prostate cancer biology. Still, the care of patients as well as the prediction of disease aggressiveness, recurrence and outcome remain challenging. Here, we showed that expression of the gene ZBTB38 is associated with poor prognosis in localised prostate cancer and could help discriminate aggressive localised prostate tumours from those who can benefit only from observation. Analysis of different prostate cancer cohorts indicates that low expression levels of ZBTB38 associate with increased levels of chromosomal abnormalities and more aggressive pathological features, including higher rate of biochemical recurrence of the disease. Importantly, gene expression profiling of these tumours, complemented with cellular assays on prostate cancer cell lines, unveiled that tumours with low levels of ZBTB38 expression might be targeted by doxorubicin, a compound generating reactive oxygen species. Our study shows that ZBTB38 is involved in prostate cancer pathogenesis and may represent a useful marker to identify high risk and highly rearranged localised prostate cancer susceptible to doxorubicin.

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. Hi-C is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any Hi-C experimental design. Here, we present the mathematical concepts to estimate the resolution of a dataset and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to several Hi-C libraries.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms. Different genomic regions are replicated at different times during the S phase of the cell cycle, forming early- and late-replicating domains that occupy different locations in the nucleus. The recent identification of specific DNA sequences and long non-coding RNAs that regulate DNA replication timing is providing key insights into the roles of replication timing and into timing and 3D organization.

Literature searches are essential for scientists. Thus, courses on how to do a good literature search have been integrated in studies at CPE Lyon for many years. Recently, we modified our pedagogical approach in order to initiate students progressively in the search for chemical information. In addition, this new teaching organization is now based on a “learn and prompt practice” concept.

Comment in :"DNA Methylation and Chromatin: Role(s) of Methyl-CpG-Binding Protein ZBTB38. [Epigenet Insights. 2018]"; International audience; Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.

DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles.

Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia , OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro . We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.

Four picolinate building blocks were implemented into the multidentate linker N,N',N'-tetrakis[(6-carboxypyridin-2-yl)methyl]butylenediamine (H(4)tpabn) with a linear flexible spacer to promote the assembly of lanthanide-based 1D coordination polymers. The role of the linker in directing the geometry of the final assembly is evidenced by the different results obtained in the presence of Htpabn(3-) and tpabn(4-) ions. The tpabn(4-) ion leads to the desired 1D polymer {[Nd(tpabn)]H(3)O x 6 H(2)O}(infinity) (12). The Htpabn(3-) ion leads to the assembly of Tb(III) and Er(III) ions into 1D zigzag chains of the general formula {[M(Htpabn)] x xH(2)O}(infinity) (M = Tb, x = 14 (1); M = Tb, x = 8 (11); M = Er, x = 14 (2); M = Er, x = 5.5 (4)), a 2D network is formed by the Eu(III) ion (i.e., {[Eu(Htpabn)] x 10 H(2)O}(infinity) (7)), and both supramolecular isomers (1D and 2D) are obtained by the Tb(III) ion. The high flexibility of the polymeric chains results in a dynamic behavior with a solvent-induced reversible structural transition. The Tb(III)- and Eu(III)-containing polymers display high-luminescence quantum yields (38 and 18%, respectively). A sizeable near-IR luminescence emission is observed for the Er(III)- and Nd(III)-containing polymers when lattice water molecules are removed.

International audience; Keywords: human T-cell leukemia virus type-1, adult T-cell leukemia, HBZ, histone acetyl transferase activity, p53 ABSTRACT Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53 +/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples. INTRODUCTION The tumor suppressor, p53, is a central regulator of genome stability in mammalian cells. Following DNA damage, p53 becomes acetylated and phosphorylated at multiple sites, thereby shifting the protein from an unstable, latent form to one that is stable, active and concentrated in the nucleus [1]. This transition allows p53 to bind to promoters of genes involved in cell cycle arrest, such as p21/CDKN1A and GADD45A, and activate their transcription [2–4]. p53 is also able to regulate expression of genes involved in the DNA damage response, apoptosis and in its own regulation [5, 6]. Although certain posttranslational modifications may serve redundant functions, there appears to be separate patterns of acetylation and phosphorylation of p53 that favor either cell cycle arrest and DNA repair or, alternatively, apoptosis [6]. Regardless of these complexities, it is clear that acetylation of p53 is essential in promoting either of the two cellular fates [7]. In general, acetylation has been shown to augment the transcriptional activity and stability of p53. For example, acetylation of lysine residues within the C-terminal domain of the protein have been found

International audience; DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles.

(Figure Presented) A homochiral triangle: An enantiopure europium triangle selectively selfassembles from a new bipyoxazolinecarboxylate ligand in a concentrationdependent process (see scheme). © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Six bidentate building blocks have been used to form a tripodal ligand with a flexible C3 symmetric spacer to direct the assembly of large octametallic clusters covalently linked to form a 1-D highly luminescent terbium tube-like polymer.

The implementation of four bidentate building blocks into a high-denticity linker with a flexible spacer leads to a predisposed ligand that allows one to direct the self-assembly of 1D functional coordination polymers. This is illustrated by the assembly under mild conditions of the luminescent metal-organic framework [Tb(Htpabn)] . 14H2O infinity (1; H4tpabn = N,N,N',N'-tetrakis[(6-carboxypyridin-2-yl)methyl]butylenediamine). The X-ray crystal structure shows that the monoprotonated Htpabn binds two equivalent lanthanide ions to form a one-directional staircase chain. The high ligand denticity prevents solvent coordination and leads to a high luminescence quantum yield (Q = 39%), which is maintained after solvent removal.

Marchal Claire, Ribery Claudine. Rapports de sexage et opérations énonciatives. In: Langage et société, n°8, 1979. Juin 1979. pp. 31-54.

Marchal Claire. "Tout homme doit gagner sa vie, j'ai une femme et des enfants". In: Langage et société, supplément au n°17, 1981. Pratiques langagières et stratégies de communication. Terrains, méthodes d'enquête et d'ananlyse. pp. 87-90.

Ribery Claudine, Marchal Claire. Thèse de 3° cycle DRL Paris VII. In: Langage et société, supplément au n°9, 1979. Sociétés dominées, pratiques langagières dominées et stratégies alternatives. pp. 140-144.

Two building blocks, 2-(1H-tetrazol-5-yl)quinoline-8-ol (8-hydroxyquinoline-2-tetrazole) (H(2)hqt) and 8-hydroxyquinoline-2-carboxylic acid (H(2)hqa) provide convenient dianionic tridentate O,N,N- and O,N,O-chelating units containing the 8-hydroxyquinoline chromophore. These ligands form tris-chelate tris-anionic complexes [Ln(hqa)(3)]K(3) and [Ln(hqt)(3)]K(3) in water at pH = 12. At lower pH partially protonated species are formed. The Nd(III), Yb(III) and Er(III) complexes of hqa, and hqt and the partially protonated complex [Nd(H(1/2)hqa)(3)](2)(Et(3)NH)(3).Et(3)NHOTf have been crystallised from methanol solution. The X-ray crystal structure ([Nd(hqa)(3)]K(3).7MeOH(∞)·MeOH, [Er(hqa)(3)]K(3).4MeOH(∞)·3MeOH and [Nd(hqt)(3)]K(3).5MeOH(∞)·MeOH) show a helical arrangement of both ligands with an "anti" geometry for hqa and a "syn" geometry for hqt. Only tris-chelate complexes are formed in water and 0.04 M methanol solutions for both ligands. A "syn" geometry is found for the partially protonated dimeric complexes which is preserved in methanol solution. A statistical distribution of "anti" and "syn" species is found for [Ln(hqa)(3)]K(3) complexes in solution while the major solution geometry of [Ln(hqt)(3)]K(3) complexes is "syn". Sizable near-IR emission quantum yields were measured for the [Ln(hqa)(3)]K(3) complexes in solid state (0.06%, 0.18%, 0.0051% for Nd(III), Yb(III) and Er(III) respectively) and in methanol (0.063%, 0.28%, 0.0019% for Nd(III), Yb(III) and Er(III) respectively). All the values of quantum yields obtained for the hqt complexes are 5-17% higher than those measured for the hqa complexes. The trianionic complexes present a high solubility in organic and aqueous solvents and a good resistance to ligand dissociation compared to neutral tris 8-hydroxyquinoline complexes.

DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.