Global hyperactivation of enhancers stabilizes human and mouse naïve pluripotency through inhibition of CDK8/19 Mediator kinases

Pluripotent stem cells (PSCs) transition between cell states in vitro and reflect developmental changes in the early embryo. PSCs can be stabilized in the naïve state by blocking extracellular differentiation stimuli, particularly FGF-MEK signaling. Here, we report that multiple features of the naïve state in human and mouse PSCs can be recapitulated without affecting FGF-MEK-signaling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 kinases removes their ability to repress the Mediator complex at enhancers. Thus CDK8/19 inhibition increases Mediator-driven recruitment of RNA Pol II to promoters and enhancers. This efficiently stabilizes the naïve transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naïve pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naïve pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity

The asymmetric distribution of RNA polymerase II and nucleosomes on replicated daughter genomes is caused by differences in replication timing between the lagging and the leading strand

International audienceChromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or whether chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription, and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We found a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNA polymerase II (RNAPII) bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: If transcription and replication are codirectional, the leading strand replicates first; if they are counterdirectional, the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 that impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. We propose that active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state

Mechanics of DNA Replication and Transcription Guide the Asymmetric Distribution of RNAPol2 and New Nucleosomes on Replicated Daughter Genomes

Replication of the eukaryotic genome occurs in the context of chromatin. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. In order to better understand the transmission of gene expression states from one cell generation to the next we have developed a method for following chromatin structure dynamics during replication-ChIP-NChAP-Chromatin Immuno-Precipitation-Nascent Chromatin Avidin Pulldown-which we used to monitor RNAPol2 and new nucleosome binding to newly-replicated daughter genomes in S. Cerevisiae. The strand specificity of our libraries allowed us to uncover the inherently asymmetric distribution of RNAPol2 and H3K56ac-a mark of new histones-on daughter chromatids after replication. Our results show a range of distributions on thousands of genes from symmetric to asymmetric with enrichment shifts from one replicated strand to the other throughout S-phase. We propose a two-step model of chromatin assembly on nascent DNAwhich provides a mechanistic framework for the regulation of asymmetric segregation of maternal histones, and discuss our model for chromatin assembly in the context of a mechanism for gene expression buffering without a direct role for H3K56ac

L'amphioxus ou comment devient-on un vertébré

L'évo-dévo est une jeune discipline dont le but est d'essayer d'expliquer l'évolution morphologique des organismes par la modification des mécanismes développementaux et des réseaux de gènes. Une des questions majeures de cette discipline est l'origine des vertébrés. Il semble désormais admis que les vertébrés sont issus d'un ancêtre chordé invertébré, et plusieurs modèles au sein des représentants vivants des chordés sont utilisés actuellement pour répondre à cette question. Le petit monde de l'évo-dévo s'intéressant à l'apparition des vertébrés est actuellement en pleine ébullition avec l'arrivée des séquences complètes de plusieurs génomes permettant des analyses comparatives de plus en plus fiables, et avec le développement de modèles "non classiques" auxquels il est désormais possible d'appliquer les techniques nécessaires à l'étude fine du développement embryonnaire. L'un de ces modèles est l'amphioxus (genre Branchiostoma) dont nous allons décrire ici les caractéristiques faisant de lui un sympathique "filet d'anchois éclairant l'évolution des chordés" (Garcia-Fernandez, 2006a, b)

The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20.

The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin

Development of a semi-closed aquaculture system for monitoring of individual amphioxus (Branchiostoma lanceolatum), with high survivorship

The European amphioxus Branchiostoma lanceolatum is becoming an important model for developmental studies, and as such requires more study both in the field and in the laboratory. We present an experimental set-up with temperature and flow rate control that allows easy care and monitoring of individual amphioxus. Over the course of several months, 98/100 individuals in two size categories and experiencing different levels of handling stress survived. Flow cytometry and gut contents indicate that the system meets the nutritional needs of the amphioxus. This simple and effective system for separate aquaculture of individual amphioxus prevents infections due to crowding. It should be particularly useful for future breeding, genetic, behavioural and life history studies, and can be easily adapted to other marine organisms

Atypical Regulation of a Green Lineage-Specific B-Type Cyclin-Dependent Kinase

Cyclin-dependent kinases (CDKs) are the main regulators of cell cycle progression in eukaryotes. The role and regulation of canonical CDKs, such as the yeast (Saccharomyces cerevisiae) Cdc2 or plant CDKA, have been extensively characterized. However, the function of the plant-specific CDKB is not as well understood. Besides being involved in cell cycle control, Arabidopsis (Arabidopsis thaliana) CDKB would integrate developmental processes to cell cycle progression. We investigated the role of CDKB in Ostreococcus (Ostreococcus tauri), a unicellular green algae with a minimal set of cell cycle genes. In this primitive alga, at the basis of the green lineage, CDKB has integrated two levels of regulations: It is regulated by Tyr phosphorylation like cdc2/CDKA and at the level of synthesis-like B-type CDKs. Furthermore, Ostreococcus CDKB/cyclin B accounts for the main peak of mitotic activity, and CDKB is able to rescue a yeast cdc28(ts) mutant. By contrast, Ostreococcus CDKA is not regulated by Tyr phosphorylation, and it exhibits a low and steady-state activity from DNA replication to exit of mitosis. This suggests that from a major role in the control of mitosis in green algae, CDKB has evolved in higher plants to assume other functions outside the cell cycle