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A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin chromatin is found in subtelomeric regions leading to telomeric position effect (TPE) in yeast, fly and man. Silent chromatin generally initiates at defined loci and tends to propagate from those sites by self-recruitment mechanisms implying the requirement for processes preventing ectopic spreading of silencing. Barrier elements that can block the spread of silent chromatin have been documented, but their relative efficiency is not known. Here we explore the dose-dependency of silencing factors for the extent of TPE in budding yeast. We characterized genome wide the impact of overexpressing the silencing factors Sir2 and Sir3 on the spreading of Sir3 and its impact on coding and non-coding transcription. We thus reveal that extension of silent domains can reach saturation. Analysis of published data sets enabled to uncover that the extension of Sir3 bound domains stops at zones corresponding to transitions of specific histone marks including H3K79 methylation that is deposited by the conserved enzyme Dot1. Importantly, DOT1 is essential for viability when Sir3 is in excess indicating that this transition actively block Sir3 spreading. Our work uncovers previously uncharacterized discrete chromosomal domains associated with specific chromatin features and demonstrates that TPE is efficiently restricted to subtelomeres by the preexisting chromatin landscape.

International audience

Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF-1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we investigated the role of CAF-1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF-1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I-element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF-1 play an important role in both the regulation of stem cell identity and genome integrity.

International audience; In mammals, CENP-A, a histone H3 variant found in the centromeric chromatin, is critical for faithful chromosome segregation and genome integrity maintenance through cell divisions. Specifically, it has dual functions, enabling to define epigenetically the centromere position and providing the foundation for building up the kinetochore. Regulation of its dynamics of synthesis and deposition ensures to propagate proper centromeres on each chromosome across mitosis and meiosis. However, CENP-A overexpression is a feature identified in many cancers. Importantly, high levels of CENP-A lead to its mislocalization outside the centromere. Recent studies in mammals have begun to uncover how CENP-A overexpression can affect genome integrity, reprogram cell fate and impact 3D nuclear organization in cancer. Here, we summarize the mechanisms that orchestrate CENP-A regulation. Then we review how, beyond its centromeric function, CENP-A overexpression is linked to cancer state in mammalian cells, with a focus on the perturbations that ensue at the level of chromatin organization. Finally, we review the clinical interest for CENP-A in cancer treatment.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain., Author summary Multicellular development depends on a tight control of gene expression in each cell type. This relies on the coordinated activities of nuclear proteins that interact with DNA or its histone scaffold to promote or restrict gene transcription. For example, we previously showed that the histone modifying enzymes Lid and Sin3A/Rpd3 are required in Drosophila ovaries for the massive expression of deadhead (dhd), a gene encoding for a thioredoxin that is essential for fertility. In this paper, we have further identified two additional dhd regulators, Snr1 and Mod(mdg4) and dissected the mechanism behind hyperactivation of this gene. Using the epigenomic profiling method Cut&Run with a dedicated data analysis approach, we unexpectedly found that dhd is embedded in an unusual chromatin mini-domain featuring repressive histone modifications H3K27me3 and H3K9me3 and flanked by two regulatory elements. However, we further showed that Lid, Sin3A, Snr1 and Mod(mdg4) behave like obligatory activators of dhd independently of this mini-domain. Our study unveils how multiple broad-acting epigenomic effectors operate in non-canonical manners to ensure a critical and specialized gene activation event. These findings challenge our knowledge on these regulatory mechanisms and their roles in development and pathology.

How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process.

International audience; In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

International audience; One striking feature of eukaryotic nuclei is the existence of discrete regions, in which specific factors concentrate while others are excluded, thus forming microenvironments with different molecular compositions and biological functions. These domains are often referred to as subcompartments even though they are not membrane enclosed. Despite their functional importance the physical nature of these structures remains largely unknown. Here, we describe how the Saccharomyces cerevisiae nucleus is compartmentalized and discuss possible physical models underlying the formation and maintenance of chromatin associated subcompartments. Focusing on three particular examples, the nucleolus, silencing foci, and repair foci, we discuss the biological implications of these different models as well as possible approaches to challenge them in living cells.

International audience; The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones.; L'absence d'une séquence d'ADN consensuelle définissant les origines de réplication chez les mammifères a conduit les chercheurs à considérer la chromatine comme un moyen de spécifier ces régions. Cependant, à ce jour, il n'y a pas de compréhension mécaniste de la façon dont cela pourrait être réalisé et maintenu étant donné que la rupture des nucléosomes se produit à chaque passage de fourche et avec la transcription. Ici, en cartographiant à l'échelle du génome le dépôt de novo des variants d'histone H3.1 et H3.3 dans les cellules humaines pendant la phase S, nous avons identifié comment leur mode de dépôt double assure un marquage stable avec H3.3 flanqué des deux côtés par H3.1. Ces frontières H3.1/H3.3 correspondent aux zones d'initiation des origines précoces. La perte du chaperon H3.3 HIRA conduit à la perturbation concomitante des frontières H3.1/H3.3 et des zones d'initiation. Nous proposons que le dépôt de H3.3 dépendant de HIRA préserve les frontières H3.1/H3.3 en les protégeant de l'invasion de H3.1 liée à la progression des fourches, contribuant ainsi à une définition chromatinienne des zones de réplication précoce.

Despite their essentiality for chromosome segregation, centromeres are diverse among eukaryotes and embody two main configurations: mono- and holocentromeres, referring respectively to a localized or unrestricted distribution of centromeric activity. Previous studies revealed that holocentricity in many insects coincides with the loss of the otherwise essential centromere component CenH3 (CENP-A), suggesting a molecular link between the two events. In this study, we leveraged recently-identified centromere components to map and characterize the centromeres of Bombyx mori. This uncovered a robust correlation between centromere profiles and regions of low chromatin dynamics. Transcriptional perturbation experiments showed that low chromatin activity is crucial for centromere formation in B. mori. Our study points to a novel mechanism of centromere formation that occurs in a manner recessive to the chromosome-wide chromatin landscape. Based on similar profiles in additional Lepidoptera, we propose an evolutionarily conserved mechanism that underlies the establishment of holocentromeres through loss of centromere specificity.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns., The boundaries of topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. Here the authors find that CTCF positions cohesin through its N-terminus but does not control its overall binding dynamics on chromatin, and show how the orientation of CTCF binding sites translates into genome folding patterns.

International audience; Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1–HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.; Les nucléosomes représentent un défi en ce qui concerne la transcription. L'éviction des histones permet la progression de l'ARN polymérase II (RNAPII) dans l'ADN, mais compromet l'intégrité de la chromatine. Ici, nous avons utilisé le système SNAP-tag pour distinguer les nouvelles et les anciennes histones et surveiller le réassemblage de la chromatine couplé à la transcription dans les cellules humaines. Nous avons découvert une perte dépendante de la transcription des anciens variants d'histones H3.1 et H3.3. Au niveau des domaines transcriptionnellement actifs, l'enrichissement en H3.3 reflétait à la fois la rétention de l'ancien H3.3 et le nouveau dépôt. Sur le plan mécanique, nous avons constaté que le chaperon du régulateur d'histone A (HIRA) est essentiel pour traiter à la fois le nouveau et l'ancien H3.3 par des voies différentes. Le dépôt de novo de H3.3 dépend totalement de la trimérisation de l'HIRA ainsi que de son partenaire l'ubinucléine 1 (UBN1), tandis que l'interaction de la fonction antisilencing 1 (ASF1) avec l'HIRA peut être contournée. En revanche, le recyclage du H3.3 nécessite l'AERH mais se fait indépendamment de la trimérisation de l'UBN1 ou de l'AERH et montre une dépendance absolue de l'interaction ASF1-AERH. Nous proposons un modèle dans lequel l'ARHI coordonne ces voies distinctes pendant la transcription pour affiner les états de la chromatine.

Background Maintaining a proper supply of soluble histones throughout the cell cycle is important to ensure chromatin and genome stability. Following their synthesis, histones undergo a series of maturation steps to prepare them for deposition onto chromatin. Results Here, we identify the lysine demethylase JMJD1B as a novel player in the maturation cascade that contributes to regulate histone provision. We find that depletion of JMJD1B increases the protein levels of the histone chaperone tNASP leading to an accumulation of newly synthesized histones H3 and H4 at early steps of the histone maturation cascade, which perturbs chromatin assembly. Furthermore, we find a high rate of JMJD1B mutations in cancer patients, and a correlation with genomic instability. Conclusions Our data support a role for JMJD1B in fine-tuning histone supply to maintain genome integrity, opening novel avenues for cancer therapeutics.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis., Epigenetic modifications are a key contributor to cell identity, and their propagation is crucial for proper development. Here the authors use a super-resolution microscopy approach to reveal how histone variants are faithfully transmitted during genome duplication, and reveal an important role for the histone chaperone ASF1 in the redistribution of parental histones.

International audience; During cell division, maintenance of chromatin features from the parental genome requires their proper establishment on its newly synthetized copy. The loss of epigenetic marks within heterochromatin, typically enriched in repetitive elements, endangers genome stability and permits chromosomal rearrangements via recombination. However, how histone modifications associated with heterochromatin are maintained across mitosis remains poorly understood. KAP1 is known to act as a scaffold for a repressor complex that mediates local heterochromatin formation, and was previously demonstrated to play an important role during DNA repair. Accordingly, we investigated a putative role for this protein in the replication of heterochromatic regions. We first found that KAP1 associates with several DNA replication factors including PCNA, MCM3 and MCM6. We then observed that these interactions are promoted by KAP1 phosphorylation on serine 473 during S phase. Finally, we could demonstrate that KAP1 forms a complex with PCNA and the histone-lysine methyltransferase Suv39h1 to reinstate heterochromatin after DNA replication.

International audience; The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.

International audience; Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

International audience; Within the cell nucleus, the organization of the eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. This chromatin organization contributes to the regulation of all DNA template-based reactions impacting genome function, stability and plasticity. Histones and their variants endow chromatin with unique properties and show a distinct distribution into the genome that is regulated by dedicated deposition machineries. The histone variants have important roles during early development, cell differentiation and chromosome segregation. Recent progress has also shed light on how mutations and transcriptional deregulation of these variants participate in tumorigenesis. In this chapter we introduce the organization of the genome in chromatin with a focus on the basic unit, the nucleosome, which contains histones as the major protein component. Then we review our current knowledge on the histone H3 family and its variants-in particular H3.3 and CenH3 CENP-Afocusing on their deposition pathways and their dedicated histone chaperones that are key players in histone dynamics.; Dans le noyau de la cellule, l'organisation de l'ADN eucaryote en chromatine utilise les histones comme composants de son élément constitutif, le nucléosome. Cette organisation de la chromatine contribue à la régulation de toutes les réactions basées sur les modèles d'ADN qui ont un impact sur la fonction, la stabilité et la plasticité du génome. Les histones et leurs variantes confèrent à la chromatine des propriétés uniques et présentent une distribution distincte dans le génome qui est régulée par des mécanismes de dépôt dédiés. Les variants d'histones jouent un rôle important dans le développement précoce, la différenciation des cellules et la ségrégation des chromosomes. Les progrès récents ont également mis en lumière la manière dont les mutations et la dérégulation transcriptionnelle de ces variants participent à la tumorigenèse. Dans ce chapitre, nous présentons l'organisation du génome dans la chromatine en nous concentrant sur l'unité de base, le nucléosome, qui contient des histones comme principal composant protéique. Ensuite, nous passons en revue nos connaissances actuelles sur la famille des histones H3 et ses variants - en particulier H3.3 et CenH3CENP-A - en nous concentrant sur leurs voies de dépôt et leurs chaperons d'histones dédiés qui sont des acteurs clés dans la dynamique des histones.

International audience; A hallmark of Wnt/β-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in β-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/β-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/β-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.; L'une des caractéristiques de la signalisation de Wnt/β-Catenin est l'extrême diversité de sa réponse transcriptionnelle, qui varie en fonction de la cellule et du contexte de développement. Ce qui contrôle cette diversité est mal compris. Dans tous les cas, le passage de la répression transcriptionnelle à l'activation dépend d'une augmentation nucléaire de la β-Caténine, qui détache le facteur de transcription T (Tcf7l1) lié aux co-répresseurs transcriptionnels de Groucho (Gro) de ses sites de liaison ADN et transforme temporairement Tcf7 en un activateur de la transcription. L'une des premières fonctions de signalisation de Wnt/β-Catenin, conservée au cours de l'évolution, est l'induction de l'organiseur de lSpemann. Nous démontrons ici que la protéine BarH-like Homeobox-2 (Barhl2), conservée au cours de l'évolution, stabilise le complexe Tcf7l1-Gro et maintient l'expression réprimée des gènes cibles du Tcf par un mécanisme qui dépend de l'activité des histone désacétylases 1 (Hdac-1). De cette façon, Barhl2 désactive la réponse transcriptionnelle précoce dépendante de Wnt/β-Catenin, limitant ainsi la formation de l'organisateur dans le temps et/ou l'espace. Cette étude révèle un nouveau mécanisme d'inhibition nucléaire de la signalisation Wnt/Tcf qui désactive la détermination du sort de l'organisateur.

The closely related replicative H3 and non-replicative H3.3 variants show specific requirement during development in vertebrates. Whether it involves distinct mode of deposition or unique roles once incorporated into chromatin remains unclear. To disentangle the two aspects, we took advantage of the Xenopus early development combined with chromatin assays. Our previous work showed that in Xenopus, depletion of the non-replicative variant H3.3 impairs development at gastrulation, without compensation through provision of the replicative variant H3.2. We systematically mutated H3.3 at each four residues that differ from H3.2 and tested their ability to rescue developmental defects. Surprisingly, all H3.3 mutated variants functionally complemented endogenous H3.3, regardless of their incorporation pathways, except for one residue. This particular residue, the serine at position 31 in H3.3, gets phosphorylated onto chromatin in a cell cycle dependent manner. While the alanine substitution failed to rescue H3.3 depletion, a phosphomimic residue sufficed. We conclude that the time of gastrulation reveals a critical importance of the H3.3S31 residue independently of the variant incorporation pathway. We discuss how this single evolutionary conserved residue conveys a unique property for this variant in vertebrates during cell cycle and cell fate commitment.; Les variantes H3 réplicative et H3.3 non réplicative, étroitement liées, présentent des besoins spécifiques au cours du développement chez les vertébrés. On ne sait pas encore s'il s'agit d'un mode de dépôt distinct ou de rôles uniques une fois incorporés dans la chromatine. Pour démêler ces deux aspects, nous avons tiré parti du développement précoce de Xenopus combiné à des tests de chromatine. Nos travaux antérieurs ont montré que chez le Xénopus, l'épuisement du variant non réplicatif H3.3 entrave le développement à la gastrulation, sans compensation par la fourniture du variant réplicatif H3.2. Nous avons systématiquement muté H3.3 à chaque quatre résidus qui diffèrent de H3.2 et avons testé leur capacité à remédier aux défauts de développement. De manière surprenante, toutes les variantes mutées H3.3 ont fonctionnellement complété le H3.3 endogène, quelle que soit leur voie d'incorporation, à l'exception d'un résidu. Ce résidu particulier, la sérine en position 31 dans H3.3, est phosphorylé sur la chromatine de manière dépendante du cycle cellulaire. Bien que la substitution par l'alanine n'ait pas permis de remédier à l'appauvrissement de H3.3, un résidu phosphomimique a suffi. Nous concluons que le moment de la gastrulation révèle une importance critique du résidu H3.3S31 indépendamment de la voie d'incorporation du variant. Nous discutons de la façon dont ce résidu unique conservé au cours de l'évolution confère une propriété unique à cette variante chez les vertébrés pendant le cycle cellulaire et l'engagement du destin cellulaire.

International audience; mRNA translation is coupled to multiprotein complex assembly in the cytoplasm, or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially-segregated modes of co-translational interactions foster the ordered assembly of NPC subunits, and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

International audience; Anti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.; La fonction anti-silencing 1 (ASF1) est un chaperon d'histone H3-H4 conservé, impliqué dans la dynamique des histones pendant la réplication, la transcription et la réparation de l'ADN. Surexprimée dans les tissus en prolifération, y compris dans de nombreuses tumeurs, l'ASF1 est devenue une cible thérapeutique prometteuse. Ici, nous combinons des approches structurelles, informatiques et biochimiques pour concevoir des peptides qui inhibent l'interaction ASF1-histone. En partant de la structure du complexe ASF1-histone humain, nous avons mis au point une stratégie de conception rationnelle combinant la fixation des épitopes et l'optimisation des contacts d'interface pour identifier un puissant inhibiteur peptidique avec une constante de dissociation de 3 nM. Lorsqu'ils sont introduits dans des cellules en culture, les inhibiteurs entravent la prolifération cellulaire, perturbent la progression du cycle cellulaire et réduisent la migration et l'invasion des cellules d'une manière proportionnelle à leur affinité pour l'ASF1. Enfin, nous constatons que l'injection directe du plus puissant inhibiteur du peptide ASF1 dans les allogreffes de souris réduit la croissance des tumeurs. Nos résultats ouvrent de nouvelles voies pour utiliser les inhibiteurs de l'ASF1 comme des pistes prometteuses pour le traitement du cancer.

Mendiratta et al. review the interplay between the different regulatory layers that affect the transcription and dynamics of distinct histone H3 variants along the cell cycle., As the building blocks of chromatin, histones are central to establish and maintain particular chromatin states associated with given cell fates. Importantly, histones exist as distinct variants whose expression and incorporation into chromatin are tightly regulated during the cell cycle. During S phase, specialized replicative histone variants ensure the bulk of the chromatinization of the duplicating genome. Other non-replicative histone variants deposited throughout the cell cycle at specific loci use pathways uncoupled from DNA synthesis. Here, we review the particular dynamics of expression, cellular transit, assembly, and disassembly of replicative and non-replicative forms of the histone H3. Beyond the role of histone variants in chromatin dynamics, we review our current knowledge concerning their distinct regulation to control their expression at different levels including transcription, posttranscriptional processing, and protein stability. In light of this unique regulation, we highlight situations where perturbations in histone balance may lead to cellular dysfunction and pathologies.

International audience; Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur in vivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches ex vivo, we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues.; Les cellules souches peuvent être maintenues par des divisions cellulaires symétriques (DCS) et des divisions cellulaires asymétriques (DCA). On comprend mal comment et quand ces divisions se produisent in vivo chez les vertébrés. Ici, nous avons développé une méthode de traçage cellulaire clonogène qui démontre la distribution asymétrique des facteurs de transcription ainsi que de l'ADN ancien et nouveau dans les cellules souches de muscles de souris pendant la régénération des muscles squelettiques. En combinant le traçage d'une seule cellule et des niches artificielles ex vivo, nous montrons comment les cellules passent des ACD aux SCD, ce qui suggère qu'elles ne sont pas engagées dans un mode de division cellulaire obligatoire. En outre, nous avons généré des souris indicatrices d'histone H3 marquées par SNAP et nous avons découvert que, contrairement aux cellules souches germinales de mouches, les résultats différentiels du destin sont associés à une distribution symétrique des variantes d'histone H3.1 et H3.3 dans les cellules souches musculaires de souris. Ce système de marquage H3-SNAP polyvalent et efficace permettra d'étudier les mécanismes sous-jacents au maintien de l'identité et de la plasticité épigénomiques dans une variété de tissus.

Posté le 18 décembre 2018 sur BioRxiv; Stem cells are maintained through symmetric or asymmetric cell divisions. While various mechanisms initiate asymmetric cell fates during mitosis, possible epigenetic control of this process has emerged recently. The asymmetrical distribution of a canonical histone H3 variant during mitosis in fly germline has suggested a role for partitioning old and new nucleosomes in asymmetric cell fates. Here, we provide resources for single cell assays and show the asymmetric segregation of transcription factors along with old and new DNA in mouse muscle stem cells ex vivo and in vivo . However, these differential fate outcomes contrast with a symmetric distribution of the canonical H3.1 vertebrate variant. These findings point to different evolutionary mechanisms operating in fly germline stem cells and vertebrate somatic stem cells to mitigate epigenetic regulation of asymmetric cell fates.; Les cellules souches sont maintenues par des divisions cellulaires symétriques ou asymétriques. Alors que divers mécanismes déclenchent des destins cellulaires asymétriques pendant la mitose, un éventuel contrôle épigénétique de ce processus est apparu récemment. La distribution asymétrique d'une variante canonique de l'histone H3 pendant la mitose dans la lignée germinale de la mouche a suggéré un rôle pour la partition des anciens et des nouveaux nucléosomes dans les destins cellulaires asymétriques. Ici, nous fournissons des ressources pour les essais sur cellules individuelles et montrons la ségrégation asymétrique des facteurs de transcription avec l'ancien et le nouvel ADN dans les cellules souches musculaires de souris ex vivo et in vivo . Cependant, ces résultats de destins différentiels contrastent avec une distribution symétrique de la variante canonique des vertébrés H3.1. Ces résultats indiquent que différents mécanismes d'évolution opèrent dans les cellules souches germinales de mouches et les cellules souches somatiques de vertébrés pour atténuer la régulation épigénétique des destins cellulaires asymétriques.

International audience; Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.; Les centromères et les centrosomes sont des acteurs mitotiques essentiels. Les centromères sont des sites chromosomiques uniques caractérisés par la présence de l'histone H3-variante de la protéine centromère A (CENP-A) [1]. La CENP-A recrute la majorité des composants des centromères, collectivement appelés le réseau constitutif associé aux centromères (CCAN) [2]. Le CCAN est nécessaire à l'assemblage des cinétochores, un complexe multiprotéique qui fixe les microtubules du fuseau (MT) et est requis pour la ségrégation des chromosomes [3]. Dans la plupart des cellules animales, le site dominant pour la nucléation des MT dans la mitose sont les centrosomes, qui sont composés de deux centrioles, entourés par une matrice riche en protéines de matériel péricentriolaire dense en électrons (PCM) [4]. Le PCM est le site de nucléation de la MT pendant la mitose [5]. Même si les centromères et les centrosomes sont reliés par des MT lors de la mitose, on ne sait pas si des défauts dans l'une des deux structures ont un impact sur la fonction de l'autre. Ici, en utilisant la microscopie à haute résolution combinée à l'élimination rapide du CENP-A dans les cellules humaines, nous avons découvert que la perturbation de la fonction des centromères a un impact sur l'intégrité des pôles du fuseau mitotique. Cela inclut la libération des extrémités négatives de la MT du centrosome, ce qui entraîne une dispersion de la PCM et un mauvais positionnement du centriole au niveau des pôles du fuseau. Sur le plan mécanique, nous montrons que ces défauts résultent d'une dynamique anormale de la MT du fuseau due à des fixations cinétochores-MT défectueuses. Il est important de noter que la restauration de l'intégrité des pôles du fuseau mitotique après l'inactivation des centromères entraîne une diminution de la fréquence de la mauvaise ségrégation des chromosomes. Dans l'ensemble, notre travail identifie une relation inattendue entre les centromères et le maintien de l'intégrité du pôle mitotique nécessaire pour assurer la précision mitotique et donc pour maintenir la stabilité génétique.

Summary Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene’s ability to read positional information along the embryo’s anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies., Author summary Despite very limited time, organisms develop in reproducible ways. In the early stages of fly development the information about maternal signals is read out in a few minutes to produce steep and precise gene expression patterns. Motivated by recent live imaging experiments in fly embryos, we explore the consequences of the trade-off between a rushed but reproducible readout and a steep expression pattern on the regulatory modules of gene expression. We show that the current view of one anterior gradient morphogen binding to six binding sites is quantitatively inconsistent with the experimental data given the short readout time, suggesting other regulatory features.

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression., Author summary During development, the first thing that an embryo needs to know is the orientation of its body. We study how the head-to-tail axis forms in the fruit fly embryo. To position the axis, the embryo relies on proteins called morphogens broadcasting instructions to other genes, so that cells know, depending on where they are along the axis, what they should become. The concentration of the Bicoid morphogen is much higher in the head of the embryo and lower towards the tail. Bicoid activates hunchback, which divides the embryo in two parts. By visualizing this process in real time in living embryos, we see the hunchback gene activated very efficiently in the head part, where each nucleus expresses the gene, whereas it is not expressed at all in the tail part. Intriguingly, the boundary separating the two domains is precisely positioned and becomes very steep in no more than three minutes. We use a modeling approach to understand how this is achieved so rapidly. Given the parameters of the system, we find that although our model is able to reproduce the fast dynamics of the process, it fails to reproduce the steepness of the boundary suggesting that a more complex approach is needed to capture the additional mechanisms involved.

Epigenetic modulation of effector T cells The epigenetic states and associated chromatin dynamics underlying the initiation and maintenance of memory and effector CD8 + T cells are poorly understood. Pace et al. found that mice lacking the histone H3 lysine 9 methyltransferase Suv39h1 had markedly reduced antigen-specific effector CD8 + T cell responses to Listeria monocytogenes infection (see the Perspective by Henning et al. ). Instead, CD8 + T cells in these mice were enriched for genes associated with naïve and memory signatures and showed enhanced memory potential and increased survival capacity. Thus, Suv39h1 marks chromatin through H3K9me3 deposition and silences memory and stem cell programs during the terminal differentiation of effector CD8 + T cells. Science , this issue p. 177 ; see also p. 163

The HIRA histone chaperone complex deposits the histone variant H3.3 onto chromatin in a DNA synthesis-independent manner. It comprises three identified subunits, HIRA, UBN1 and CABIN1, however the functional oligomerization state of the complex has not been investigated. Here we use biochemical and crystallographic analysis to show that the HIRA subunit forms a stable homotrimer that binds two subunits of CABIN1 in vitro. A HIRA mutant that is defective in homotrimer formation interacts less efficiently with CABIN1, is not enriched at DNA damage sites upon UV irradiation and cannot rescue new H3.3 deposition in HIRA knockout cells. The structural homology with the homotrimeric replisome component Ctf4/AND-1 enables the drawing of parallels and discussion of the functional importance of the homotrimerization state of the HIRA subunit., The HIRA histone chaperone complex is involved in the deposition of the histone variant H3.3. Here the authors, by using biochemical and crystallographic approaches, report the homotrimerization of the HIRA subunit which is critical for the functional activity of the complex.

International audience; Centromeres, epigenetically defined by the presence of the histone H3 variant CenH3, are essential for ensuring proper chromosome segregation. In mammals , centromeric CenH3 CENP-A deposition requires its dedicated chaperone HJURP and occurs during telophase/early G1. We find that the cell-cycle-dependent recruitment of HJURP to centromeres depends on its timely phosphorylation controlled via cyclin-dependent kinases. A nonphosphorylat-able HJURP mutant localizes prematurely to centro-meres in S and G2 phase. This unregulated targeting causes a premature loading of CenH3 CENP-A at centromeres, and cell-cycle delays ensue. Once recruited to centromeres, HJURP functions to promote CenH3 CENP-A deposition by a mechanism involving a unique DNA-binding domain. With our findings, we propose a model wherein (1) the phosphorylation state of HJURP controls its centromeric recruitment in a cell-cycle-dependent manner, and (2) HJURP binding to DNA is a mechanistic determinant in CenH3 CENP-A loading.

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos., Author Summary The fly embryo provides a natural laboratory to study the dynamics of transcription and its implications for the developing organism. Using live imaging experiments we investigate the nature of transcription regulation of the hunchback gene—the first to read out the maternal Bicoid gradient. While traditional time trace analysis methods based on OFF time distributions or autocorrelation functions fail for short signals, our tailored autocorrelation function overcomes these limitations revealing bursty dynamics that is reproducible between cell cycles and embryos. The inferred rates result in a lot of variability in the readout of nuclei sensing similar Bicoid concentrations, suggesting additional readout mechanisms than a one-to-one mapping of the input onto the output.

International audience; The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A–H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

The regulation of transcription is a fundamental process underlying the determination of cell identity and its maintenance during development. In the last decades, most of the transcription factors, which have to be expressed at the right place and at the right time for the proper development of the fly embryo, have been identified. However, mostly because of the lack of methods to visualize transcription as the embryo develops, their coordinated spatiotemporal dynamics remains largely unexplored. Efforts have been made to decipher the transcription process with single molecule resolution at the single cell level. Recently, the fluorescent labeling of nascent RNA in developing fly embryos allowed the direct visualization of ongoing transcription at single loci within each nucleus. Together with powerful imaging and quantitative data analysis, these new methods provide unprecedented insights into the temporal dynamics of the transcription process and its intrinsic noise. Focusing on the Drosophila embryo, we discuss how the detection of single RNA molecules enhanced our comprehension of the transcription process and we outline the potential next steps made possible by these new imaging tools. In combination with genetics and theoretical analysis, these new imaging methods will aid the search for the mechanisms responsible for the robustness of development. WIREs Dev Biol 2016, 5:296–310. doi: 10.1002/wdev.221 For further resources related to this article, please visit the WIREs website.

International audience; Although our understanding of centromere maintenance, marked by the histone H3 variant CenH3CENP-A in most eukaryotes, has progressed, the mechanism underlying the de novo formation of centromeres remains unclear. We used a synthetic system to dissect how CenH3CENP-A contributes to the accumulation of CENP-C and CENP-T, two key components that are necessary for the formation of functional kinetochores. We find that de novo CENP-T accumulation depends on CENP-C and that recruitment of these factors requires two domains in CenH3CENP-A: the HJURP-binding region (CATD) and the CENP-C-binding region (CAC). Notably, HJURP interacts directly with CENP-C and is critical for de novo accumulation of CENP-C at synthetic centromeres. On the basis of our findings, we propose that HJURP serves a dual chaperone function in coordinating CenH3CENP-A and CENP-C recruitment.

International audience; Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal his-tones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono-and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of het-erochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-β/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription., Author summary Tissue regeneration is a fundamental process that maintains the integrity of the intestinal epithelium when faced with chemical or microbial stresses. In both healthy and diseased conditions, pro-regenerative cytokines function as central coordinators of gut renewal, linking inflammation to stem cell activity. In Drosophila, the upstream events that stimulate the production of the primary cytokine Unpaired 3 (Upd3) in response to indigenous or pathogenic microbes have yet to be elucidated. In this study, we demonstrate that upd3 expression is driven in different cell types by separate microbe-responsive enhancers. In enterocytes (ECs), cytokine induction relies on the Yki/Sd, Mad/Med, and AP-1 transcription factors (TFs). These TF complexes are activated downstream of the Hippo, TGF-β and Src-MAPK pathways, respectively. Inhibiting these pathways in ECs impairs upd3 transcription, which in turn blocks intestinal stem cell proliferation and reduces the survival rate of adult flies following enteric infections. Altogether, our study identifies the major microbe-responsive enhancers of the upd3 gene and sheds light on the complexity of the gene regulatory network required in ECs to regulate tissue homeostasis and stem cell activity in the digestive tract.

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3–H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization. DOI: http://dx.doi.org/10.7554/eLife.23474.001, eLife digest Animal and plant cells contain very long DNA molecules that are tightly packaged by being wrapped around proteins called histones to form structures known as nucleosomes. While this is a useful way to store DNA, it also makes it inaccessible to many proteins and other molecules that activate genes, copy DNA or perform other important cell processes. To enable these processes to take place, the cell can selectively disassemble particular nucleosomes and remove the histone proteins. Afterwards, the nucleosomes must reassemble to repackage the DNA. A single nucleosome contains four pairs of histones, with two pairs consisting of a H3 and a H4 histone. Histone chaperones assemble nucleosomes in a two-step process. First, two of these histone H3-H4 pairs (collectively known as a tetramer) interact with DNA to form a group or “complex”. Then, two more pairs of different histones bind to complete the nucleosome. An enzyme called CAF1 is known to attach H3-H4 tetramers onto DNA as the DNA is being copied, which allows nucleosomes to form on the newly made DNA. However, it is not known how CAF1 deposits H3-H4 tetramers onto the DNA. Sauer et al. explored how yeast CAF1 works by carrying out a series of experiments in a cell-free system. The experiments showed that each CAF1 enzyme binds to a single H3-H4 pair. When attached to their histone cargo, two CAF1 enzymes bind to DNA and attach a H3-H4 tetramer onto it. The tetramer has to form in this way for histones to be correctly delivered to DNA after the DNA has been copied. Sauer et al. also identified a new region of the CAF1 enzyme that binds to DNA. Together with another region, this enables CAF1 to bind to an extended stretch of DNA that accommodates the H3-H4 tetramer. Together, the findings explain the sequence of events that take place when CAF1 attaches H3-H4 tetramers onto DNA in the first step of nucleosome formation. Future work will be required to understand the structure of CAF1 in different situations and to find out how the cell targets this enzyme to stretches of DNA that have just been copied. DOI: http://dx.doi.org/10.7554/eLife.23474.002

International audience; HP1 enrichment at pericentric heterochromatin is essential for proper chromosome segregation. While H3K9me3 is thought to be a major contributor to HP1 enrichment at pericentric domains, in mouse cells, the SUMO-protease SENP7 is required in addition to H3K9me3. How this is achieved remains elusive. Here, we find that loss of SENP7 leads to an increased time spent in mitosis. Furthermore, we reveal that a short module comprising two consecutive HP1 interaction motifs on SENP7 is the determinant for HP1 enrichment and acts by restricting HP1 mobility at pericentric domains. We propose a mechanism for maintenance of HP1 enrichment in which this module functions on top of H3K9me3 to lock contiguous HP1 molecules already docked on H3K9me3-modified nucleosomes. H3K9me3 would thus promote HP1 enrichment only if a locking system is in place. This mechanism may apply to other nuclear domains to contribute to the control of genome plasticity and integrity.