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Transmission spectroscopy provides insight into the atmospheric properties and consequently the formation history, physics, and chemistry of transiting exoplanets. However, obtaining precise inferences of atmospheric properties from transmission spectra requires simultaneously measuring the strength and shape of multiple spectral absorption features from a wide range of chemical species. This has been challenging given the precision and wavelength coverage of previous observatories. Here, we present the transmission spectrum of the Saturn-mass exoplanet WASP-39b obtained using the SOSS mode of the NIRISS instrument on the JWST. This spectrum spans $0.6 - 2.8 \mu$m in wavelength and reveals multiple water absorption bands, the potassium resonance doublet, as well as signatures of clouds. The precision and broad wavelength coverage of NIRISS-SOSS allows us to break model degeneracies between cloud properties and the atmospheric composition of WASP-39b, favoring a heavy element enhancement ("metallicity") of $\sim 10 - 30 \times$ the solar value, a sub-solar carbon-to-oxygen (C/O) ratio, and a solar-to-super-solar potassium-to-oxygen (K/O) ratio. The observations are best explained by wavelength-dependent, non-gray clouds with inhomogeneous coverage of the planet's terminator., Comment: 48 pages, 12 figures, 2 tables. Under review at Nature

The newly operational JWST offers the potential to study the atmospheres of distant worlds with precision that has not been achieved before. One of the first exoplanets observed by JWST in the summer of 2022 was WASP-96 b, a hot-Saturn orbiting a G8 star. As part of the Early Release Observations program, one transit of WASP-96 b was observed with NIRISS/SOSS to capture its transmission spectrum from 0.6-2.85 microns. In this work, we utilise four retrieval frameworks to report precise and robust measurements of WASP-96 b's atmospheric composition. We constrain the logarithmic volume mixing ratios of multiple chemical species in its atmosphere, including: H$_2$O = $-3.59 ^{+ 0.35 }_{- 0.35 }$, CO$_2$ = $-4.38 ^{+ 0.47 }_{- 0.57 }$ and K = $-8.04 ^{+ 1.22 }_{- 1.71 }$. Notably, our results offer a first abundance constraint on potassium in WASP-96 b's atmosphere, and important inferences on carbon-bearing species such as CO$_2$ and CO. Our short wavelength NIRISS/SOSS data are best explained by the presence of an enhanced Rayleigh scattering slope, despite previous inferences of a clear atmosphere - although we find no evidence for a grey cloud deck. Finally, we explore the data resolution required to appropriately interpret observations using NIRISS/SOSS. We find that our inferences are robust against different binning schemes. That is, from low $R = 125$ to the native resolution of the instrument, the bulk atmospheric properties of the planet are consistent. Our systematic analysis of these exquisite observations demonstrates the power of NIRISS/SOSS to detect and constrain multiple molecular and atomic species in the atmospheres of hot giant planets., Comment: 12 pages, 5 Figures. Accepted for publication in MNRAS. Companion paper to Radica et al., 2023

The SOSS mode of the NIRISS instrument is poised to be one of the workhorse modes for exoplanet atmosphere observations with the newly launched James Webb Space Telescope. One of the challenges of the SOSS mode, however, is the physical overlap of the first two diffraction orders of the G700XD grism on the detector. Recently, the ATOCA algorithm was developed and implemented as an option in the official JWST pipeline, as a method to extract SOSS spectra by decontaminating the detector -- that is, separating the first and second orders. Here, we present APPLESOSS (A Producer of ProfiLEs for SOSS), which generates the spatial profiles for each diffraction order upon which ATOCA relies. We validate APPLESOSS using simulated SOSS time series observations of WASP-52\,b, and compare it to ATOCA extractions using two other spatial profiles (a best and worst case scenario on-sky), as well as a simple box extraction performed without taking into account the order contamination. We demonstrate that APPLESOSS profiles retain a high degree of fidelity to the true underlying spatial profiles, and therefore yield accurate extracted spectra. We further confirm that the effects of the order contamination for relative measurements (e.g., exoplanet transmission or emission observations) is small -- the transmission spectrum obtained from each of our four tests, including the contaminated box extraction, is consistent at the $\sim$1$\sigma$ level with the atmosphere model input into our noiseless simulations. We further confirm via a retrieval analysis that the atmosphere parameters (metallicity and C/O) obtained from each transmission spectrum are consistent with the true underlying values., Comment: 11 pages, 10 figures. Published in PASP

The present-day envelope of gaseous planets is a relic of how these giant planets originated and evolved. Measuring their elemental composition therefore presents a powerful opportunity to answer long-standing questions regarding planet formation. Obtaining precise observational constraints on the elemental inventory of giant exoplanets has, however, remained challenging due to the limited simultaneous wavelength coverage of current space-based instruments. Here, we present thermal emission observations of the non-transiting hot Jupiter $\tau$ Boo b using the new wide wavelength coverage (0.95$-$2.50$\,\mu$m) and high spectral resolution ($R=70\,000$) SPIRou spectrograph. By combining a total of 20 hours of SPIRou data obtained over five nights in a full atmospheric retrieval framework designed for high-resolution data, we constrain the abundances of all the major oxygen- and carbon-bearing molecules and recover a non-inverted temperature structure using a new free-shape, nonparametric TP profile retrieval approach. We find a volume mixing ratio of log(CO)$\,\,=-2.46_{-0.29}^{+0.25}$ and a highly depleted water abundance of less than $0.0072$ times the value expected for a solar composition envelope. Combined with upper limits on the abundances of CH$_4$, CO$_2$, HCN, TiO, and C$_2$H$_2$, this results in a gas-phase C/H ratio of 5.85$_{-2.82}^{+4.44}\times\,$solar, consistent with the value of Jupiter, and an envelope C/O ratio robustly greater than 0.60, even when taking into account the oxygen that may be sequestered out of the gas-phase. Combined, the inferred super-solar C/H, O/H, and C/O ratios on $\tau$ Boo b support a formation scenario beyond the water snowline in a disk enriched in CO due to pebble drift., Comment: 27 pages, 14 figures, 3 tables, Accepted for publication in The Astronomical Journal

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DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2–7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2–7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired., DNA replication is initiated at defined genomic sites called origins of replication following ORC pre-replicative complex assembly. Here the authors identify a protein ubiquitylating ORC that is involved in origin activation and may act as a selector of origins to be fired.

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6(lacZ)) showed that inactivation of ERK3 in Mapk6(lacZ) mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive (Mapk6(KD)) or conditional (Mapk6(Δ)) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6(lacZ) mice but not in Mapk6(KD) or Mapk6(Δ) mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6(lacZ) mutant mice.

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation., Origins of replications are associated with potential G quadruplexes forming structures (G4s). Here the authors reveal the functional role of G4 elements in DNA replication initiation.

The physiological functions of the atypical MAP kinase ERK3 remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6lacZ) showed that inactivation of ERK3 in Mapk6lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically-inactive (Mapk6KD) or conditional (Mapk6Δ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is ncessary for optimal post-natal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that expression of several Mapk6 neighboring genes is deregulated in Mapk6lacZ mice, but not in Mapk6KD or Mapk6Δ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are likely to be responsible for the perinatal lethality phenotype of Mapk6lacZ mutant mice.

International audience; DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects DNA polymerase errors during replication to maintain genomic integrity. In E. coli, the DNA helicase UvrD is implicated in MMR, yet an analogous helicase activity has not been identified in eukaryotes. Here, we show that mammalian MCM9, a protein involved in replication and homologous recombination, forms a complex with MMR initiation proteins (MSH2, MSH3, MLH1, PMS1, and the clamp loader RFC) and is essential for MMR. Mcm9−/− cells display microsatellite instability and MMR deficiency. The MCM9 complex has a helicase activity that is required for efficient MMR since wild-type but not helicase-dead MCM9 restores MMR activity in Mcm9−/− cells. Moreover, MCM9 loading onto chromatin is MSH2-dependent, and in turn MCM9 stimulates the recruitment of MLH1 to chromatin. Our results reveal a role for MCM9 and its helicase activity in mammalian MMR.

DNA replication in metazoan cells initiates at multiple discrete chromosomal sites called replication origins. Recent genome-wide studies have mapped thousands of origins in animal and plant cells, but without yielding a distinct and universal consensus sequence. However, origin-associated regions with particular base composition features have been identified, such as the G-rich OGRE motif, predicted to form G-quadruplexes. Epigenetic marks such as histone modifications that promote open chromatin also favor origin formation.

International audience; We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.

International audience; To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile.

DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1−/− mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IκB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.

International audience; As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67(phox). We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage.

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G1 cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G1 phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27Kip1 expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27Kip1 protein. The time course of p27Kip1 decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27Kip1 synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-β1 by aortic SMC. These results identify p27Kip1 as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.

Les enzymes ubiquitines ligases au cours du cycle cellulaire Le cycle cellulaire est l’ensemble des evenements qui permettent a une cellule d’engendrer deux cellules filles. La division cellulaire commence par la duplication des composants de la cellule, incluant la replication fidele des chromosomes lors de la phase de synthese d’ADN (phase S). Ces composants sont ensuite repartis (le plus souvent egalement) entre deux cellules filles lors de la mitose (phase M). La phosphorylation et la proteolyse selectives des proteines sont deux mecanismes qui coordonnent la progression au cours des phases du cycle cellulaire. Ainsi, les proteine kinases Cdk (cyclin-dependent kinases) favorisent la reproduction cellulaire en stimulant l’entree des cellules en phase S et en mitose. Leur activite au cours du cycle cellulaire est regulee par l’expression de leurs sous-unites activatrices (cyclines) et inhibitrices (CKI, Cdk inhibitor). L’abondance de ces regulateurs depend d’une machinerie proteolytique tres conservee dans l’evolution : le systeme ubiquitine-proteasome [1]. L’attachement de molecules d’ubiquitine aux proteines cibles qui sont ensuite degradees par le proteasome est catalyse par les enzymes ubiquitine ligases ou E3. Les complexes APC (anaphase-promoting complex) et SCF (Skp1/Cullin 1/F-box) sont les deux E3 essentielles aux transitions du cycle cellulaire [1]. L’activite de l’APC est importante pour la progression en mitose et le maintien de la cellule en phase G1 jusqu’a la phase S (Figure 1A). L’APC reconnait ses divers substrats en se liant a deux sous-unites activatrices : Cdc20 (active en mitose) et Cdh1 (active en fin de mitose et en phase G11). Un des substrats importants de l’APCCdh1 est la proteine a Un nouvel axe du mal tumoral ?

In the genome of eukaryotic cells, DNA synthesis is initiated at multiple sites called origins of DNA replication. Origins must fire only once per cell cycle and how this is achieved is now well understood. However, little is known about the mechanisms that determine when and where replication initiates in a given cell. A large body of evidence indicates that origins are not equal in terms of efficiency and timing of activation. Origin usage also changes concomitantly with the different cell differentiation programs. As DNA replication occurs in the context of chromatin, initiation could be influenced by multiple parameters, such as nucleosome positioning, histone modifications, and three-dimensional (3D) organization of the nucleus. This view is supported by recent genome-wide studies showing that DNA replication profiles are shaped by genetic and epigenetic processes that act both at the local and global levels to regulate origin function in eukaryotic cells.

International audience; Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps.

Skp2 is the substrate binding subunit of the SCF(Skp2) ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APC(Cdh1), which targets Skp2 for degradation in early G(1) and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCF(Skp2) ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.

International audience; During each cell cycle, thousands of DNA replication origins are activated in each cell of a metazoan organism. Although they appear site-specific, their usage and organization are rather plastic. Moreover, no strict sequence specificity has been observed in contrast to bacterial or Saccharomyces cerevisiae DNA replication origins. Epigenetic regulation linked to chromatin structure, chromosome organization, and transcription has been suggested to explain how DNA replication origins are selected and recognized by replication initiation factors. In this paper, we review these epigenetic features and discuss how, during the previous mitosis, chromosomal architecture might prepare DNA replication origins for a new cell cycle.

Mitogen-activated protein (MAP) kinases are typical examples of protein kinases whose enzymatic activity is mainly controlled by activation loop phosphorylation. The classical MAP kinases ERK1/ERK2, JNK, p38 and ERK5 all contain the conserved Thr-Xxx-Tyr motif in their activation loop that is dually phosphorylated by members of the MAP kinase kinases family. Much less is known about the regulation of the atypical MAP kinases ERK3 and ERK4. These kinases display structural features that distinguish them from other MAP kinases, notably the presence of a single phospho-acceptor site (Ser-Glu-Gly) in the activation loop. Here, we show that ERK3 and ERK4 are phosphorylated in their activation loop in vivo. This phosphorylation is exerted, at least in part, in trans by an upstream cellular kinase. Contrary to classical MAP kinases, activation loop phosphorylation of ERK3 and ERK4 is detected in resting cells and is not further stimulated by strong mitogenic or stress stimuli. However, phosphorylation can be modulated indirectly by interaction with the substrate MAP kinase-activated protein kinase 5 (MK5). Importantly, we found that activation loop phosphorylation of ERK3 and ERK4 stimulates their intrinsic catalytic activity and is required for the formation of stable active complexes with MK5 and, consequently, for efficient cytoplasmic redistribution of ERK3/ERK4-MK5 complexes. Our results demonstrate the importance of activation loop phosphorylation in the regulation of ERK3/ERK4 function and highlight differences in the regulation of atypical MAP kinases as compared to classical family members.

The Polycomb group gene Bmi1 is essential for the proliferation of neural and hematopoietic stem cells. Much remains to be learned about the pathways involved in the severe hematopoietic phenotype observed in Bmi1 homozygous mutant mice except for the fact that loss of p53 or concomitant loss of p16Ink4a and p19Arf functions achieves only a partial rescue. Here we report the identification of E4F1, an inhibitor of cellular proliferation, as a novel BMI1-interacting partner in hematopoietic cells. We provide evidence that Bmi1 and E4f1 genetically interact in the hematopoietic compartment to regulate cellular proliferation. Most importantly, we demonstrate that reduction of E4f1 levels through RNA interference mediated knockdown is sufficient to rescue the clonogenic and repopulating ability of Bmi1−/− hematopoietic cells up to 3 mo post-transplantation. Using cell lines and MEF, we also demonstrate that INK4A/ARF and p53 are not essential for functional interaction between Bmi1 and E4f1. Together, these findings identify E4F1 as a key modulator of BMI1 activity in primitive hematopoietic cells.

This paper focuses on an investigation of the dynamic characteristics of a spherical aerostat on a single tether. A portable test facility was constructed to gather experimental data required to characterize the system. All experiments were in the supercritical range, at Reynolds numbers greater than 3.7 × 10. The balloon’s drag coefficient was determined based on position measurements. The balloon’s large oscillations and surface roughness, combined with the wind turbulence, resulted in a substantial increase in the drag coefficient. To further study the system, a numerical simulation was developed. The aerostat is modeled as a rigid body attached to a tether, and is subject to buoyancy, aerodynamic drag and gravity. The tether is modeled using a lumped-mass approach, while includes stiffness and damping. The dynamics simulation of the system is obtained by formulating the equations of motion of the aerostat and the cable nodes in 3D space and integrating them numerically. The simulation is then validated by comparing its results with experimental data. Finally, a modal analysis of the natural modes of the system is performed.

Cell cycle progression is negatively regulated by the pocket proteins pRb, p107, and p130. However, the mechanisms responsible for this inhibition are not fully understood. Here, we show that overexpression of p107 in fibroblasts inhibits Cdk2 activation and delays S phase entry. The inhibition of Cdk2 activity is correlated with the accumulation of p27, consequent to a decreased degradation of the protein, with no change of Thr187 phosphorylation. Instead, we observed a marked decrease in the abundance of the F-box receptor Skp2 in p107-overexpressing cells. Reciprocally, Skp2 accumulates to higher levels in p107−/− embryonic fibroblasts. Ectopic expression of Skp2 restores p27 down-regulation and DNA synthesis to the levels observed in parental cells, whereas inactivation of Skp2 abrogates the inhibitory effect of p107 on S phase entry. We further show that the serum-dependent increase in Skp2 half-life observed during G1 progression is impaired in cells overexpressing p107. We propose that p107, in addition to its interaction with E2F, inhibits cell proliferation through the control of Skp2 expression and the resulting stabilization of p27.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.

Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G(1) arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.

Accumulating evidence suggests that angiotensin II type II (AT(2)) receptor subtype negatively regulates cell proliferation in pathophysiological conditions associated with tissue remodeling. However, the mechanisms through which AT(2) receptor achieves this effect remain poorly understood. In this study, we demonstrate that expression of AT(2) receptor inhibits the proliferation of rat fibroblasts in a ligand-independent manner. The antiproliferative action of AT(2) is dependent on the density of surface receptors. We show that AT(2) receptor expression negatively regulates G1 phase progression in both cycling cells and G0-arrested cells stimulated to re-enter the cell cycle, but has no detectable effect on apoptosis. The delay in cell-cycle progression of AT(2)-expressing cells is associated with downregulation of cyclin E expression, decreased assembly of cyclin E-Cdk2 complexes, and the resulting attenuation of Cdk2 activation. The induction of Cdk4 expression and activity is also markedly attenuated, which likely contributes to the inhibition of cyclin E expression. Ectopic expression of Cdk4 alleviates the proliferation defect of AT(2)-expressing cells. These findings suggest that the growth-inhibitory effects of the AT(2) receptor are attributable in part to its spontaneous inhibitory action on the cell cycle machinery.

The ability to express and purify proteins in large amounts through recombinant DNA technology has enabled significant advances in the biomedical sciences. Several commercially available or home-made vectors allow expression and easy purification of recombinant proteins in Escherichia coli. Generally, such vectors are designed to produce fusion proteins containing a molecular tag at the N-terminal end. Calmodulin, streptavidin, maltose binding protein, thioredoxin, hexahistidine ðHis6Þ, and glutathione S-transferase (GST) are examples of tags chosen for their high binding affinity toward specific ligands [7]. However, this procedure also has inherent limitations. In some cases, proteins overexpressed in E. coli form insoluble aggregates that are difficult to solubilize or are found mainly as truncated forms [1]. Purification of recombinant fusion proteins sometimes requires several chromatography steps and renaturation protocols. These problems are particularly evident for large proteins, for which yields of purification are frequently very low and preclude further analysis or use of the recombinant protein. Although several procedures have been developed to improve protein solubility in E. coli [1,2,6,8], less progress has been made to overcome the problem of truncated protein expression due to proteolysis, intrinsic instability, or premature translation termination caused by codon usage bias [4]. Here we describe the construction of novel dual-tag prokaryotic expression vectors that allow for enrichment in full-length recombinant proteins and facilitate their subsequent purification by sequential chromatography on metal and glutathione affinity columns. Extracellular signal-regulated kinase 3 (ERK3) is a member of the MAP kinase family of serine/threonine kinases [5]. In the course of our studies on this enzyme, we experienced problems in producing good yields of full-length GST fusion constructs of ERK3 using conventional prokaryotic expression systems. To circumvent these problems, we created a novel set of dual-tag prokaryotic expression vectors, pHGST.1 and pHGST. 2T, that were engineered to produce N-terminal His6and C-terminal GST-tagged fusion proteins (Fig. 1). In theory, the sequential purification of recombinant proteins on glutathione and metal affinity resins should: (1) allow for enrichment in full-length protein and (2) facilitate the purification process and increase the purity of the final product. pHGST vectors were constructed by first subcloning the two annealed oligonucleotides 50 CTA GCA TGA ATT CGG GAT CCA TGG GTC GAC TCG AGC TCG GAA 30 and 50 AGC TTT CCG AGC TCG AGT CGA CCC ATG GAT CCC GAA TTC ATG 30 into the NheI/HindIII sites of pRSET-A (Invitrogen Life Technologies, Burlington, Canada) to generate pRSET-MCS. The GST coding sequence was amplified by PCR using pGEX-KG [3] as template with the following oligonucleotides: 50 GCC GAG CTC TCC CCT ATA CTA GGT TAT TGG 30 and 50 CCC AAG CTT TTA ATC CGA TTT TGG AGG ATG GTC 30. The amplicon was then digested with SstI/ HindIII and subcloned into the SstI/HindIII sites of pRSET-MCS to yield pHGST.1. The pHGST.2T vector was obtained by ligation of the following annealed oligonucleotides into NcoI/XhoI-digested pHGST.1: 50 CAT GGC GGC CGC CTC GAG TCT GGT TCC Analytical Biochemistry 310 (2002) 219–222

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin–Cdk2 complexes.

Cell division is one of the most complex processes a cell undergoes. For this to happen properly, the genetic material stored in a cell must be faithfully copied or replicated. During this process, DNA replication is initiated at pre-defined sites in the genome, called "origins of replication". The activation of these origins is highly regulated, as a dysfunction in origin activity is linked to several human pathologies. Several proteins have been found at replication origins, but none of them explain how to be activated origins are recognized and selected. Our research group aims to understand how DNA replication origins are regulated in metazoan cells, to this aim, a proteomic approach was performed to define the interactome of human replication origins. Our goal was to identify new factors that could be involved in replication origin regulation. Using this methodology, a novel E3 ubiquitin ligase, named OBI1 (for ORC-ubiquitin-ligase-1), was identified prior to my arrival in the laboratory. OBI1 binds the origin recognition complex (ORC complex) and my project aimed at further characterizing the role of this new protein in DNA replication. Our experimental strategy used two different model systems: an in-vivo model based on human cells in culture, and an in-vitro DNA replication system derived from Xenopus eggs.Our analyses in human cells revealed that OBI1 was a crucial gene involved in cellular proliferation, this observation was later attributed to OBI1’s role in DNA replication and more specifically, to replication origin activation. Indeed, OBI1 knockdown resulted in a deficient origin firing and a decrease in the chromatin recruitment of factors involved in origin firing. A further functional analysis showed that OBI1 multiubiquitylates two subunits of the ORC complex, ORC3 and ORC5. This ubiquitylation was directly linked to OBI1’s role in origin firing, after the over-expression of non-ubiquitylable ORC3/5 mutants yielded similar results to OBI1’s knock down. Altogether, our results demonstrated that OBI1 encoded for a protein essential for origin activation, and allowed us to propose its main role: by multiubiquitylating a subset of the ORC complex, OBI1 could select the replication origins to be activated amongst all the potential replication origins set in G1 phase of the cell cycle. After this set of experiments, now published, we wanted to address the mechanistic impact of the multiubiquitylation of ORC on origin activation. Our preliminary experiments suggest a role of the histone acetyl-transferase (HAT) GCN5/KAT2A in the “OBI1 pathway”In the second part of my project, we used the in vitro DNA replication system, based on Xenopus laevis egg extracts, to study the role of OBI1 and ubiquitylation in origin activation. Our in-vitro analyses confirmed the conservation of OBI1 in Xenopus Laevis and its recruitment to the chromatin during DNA replication. We showed that de novo ubiquitylation takes place on chromatin during origin activation. Moreover, using E1 inhibitors, we found that active ubiquitylation is important for efficient origin firing. Interestingly, our loss of function experiments suggested that OBI1’s impact on origin activation could defer in early development when compared to somatic-like conditions.Taken together, the discovery of this new replication initiation factor provided key information on the role of ubiquitylation in general and OBI1 in particular on origin activation and selection. Such selection could participate as well in the regulation of the timing of DNA replication.; La division cellulaire est l’un des processus cellulaires les plus complexes. Pour que cette division se déroule correctement, la cellule doit répliquer de manière fiable l’intégralité de son génome. Durant ce processus, la réplication de l’ADN est initiée a des sites prédéfinis du génome, appelés « origines de réplication ». Vu qu’un dysfonctionnement de l'activité des origines est lié à plusieurs pathologies humaines, leur activation doit être hautement régulée. Plusieurs protéines ont été trouvées aux origines de la réplication, mais aucune n’explique comment ces origines sont reconnues et sélectionnées pour l’activation. Notre groupe de recherche vise à comprendre comment les origines de réplication sont régulées dans les cellules de métazoaires. Dans ce but, une approche protéomique a été réalisée pour définir l'interactome des origines de réplication humaine, dans l’objectif d'identifier de nouveaux facteurs qui pourraient être impliqués dans la régulation des origines. À l'aide de cette approche, une nouvelle ubiquitine ligase, nommée OBI1 (ORC-ubiquitine-ligase-1), a été identifiée avant mon arrivée au laboratoire. OBI1 se lie au complexe de reconnaissance des origines (complexe ORC) et mon projet vise à mieux caractériser le rôle de cette nouvelle protéine dans la réplication de l'ADN. Notre stratégie expérimentale est basée sur deux modèles différents: un modèle in vivo de cellules humaines en culture et un système de réplication de l'ADN in vitro dérivé d'œufs de Xénope.Nos analyses sur des cellules humaines ont d’abord révélé qu’OBI1 était crucial pour la prolifération cellulaire. Cette observation a été ensuite attribuée à son rôle dans la réplication de l’ADN et plus précisément dans l’activation des origines de réplication. En effet, la déplétion d’OBI1 a montré une diminution de recrutement à la chromatine de facteurs impliqués dans l’activation des origines. De plus, une analyse fonctionnelle a montré qu'OBI1 multiubiquitine ORC3 et ORC5, deux sous-unités du complexe ORC. Cette ubiquitination a été ensuite liée au rôle d’OBI1 dans l’activation des origines de réplication, après que la surexpression des mutants ORC3 / 5 non-ubiquitinables ait donné des résultats similaires à ceux observés lors de la déplétion d’OBI1. Dans l’ensemble, nos résultats ont démontré qu’OBI1 est une protéine essentielle à l’activation des origines et nous ont permis de mettre en place une hypothèse suggérant qu’en ubiquitinant ORC3/5, OBI1 pourrait jouer un rôle dans la sélection des origines destinées à l’activation, parmi toutes les origines définies antérieurement. Après cette étude, maintenant publiée, nous avons voulu aborder le rôle de la multiubiquitination des ORC dans l’activation des origines. Nos expériences préliminaires suggèrent un rôle de l'histone acétyl-transférase (HAT) GCN5 / KAT2A.Dans la deuxième partie de mon projet, nous avons utilisé le système in vitro, basé sur des extraits d'œufs de xénope, pour étudier le rôle de l'OBI1 et de l'ubiquitination dans l'activation des origines de réplication. Nos analyses ont confirmé la conservation d’OBI1 chez Xenopus Laevis et son recrutement a la chromatine lors de la réplication. Nous avons montré que l'ubiquitination se produit sur la chromatine lors de l'activation de l'origine. De plus, en utilisant des inhibiteurs de E1, nous avons constaté que l’ubiquitination est importante pour l’activation des origines. De façon intéressante, la déplétion de OBI1 dans ce système embryonnaire a suggéré un rôle diffèrent d’OBI1 dans l’activation des origines dans le système embryonnaire comparé aux conditions plus somatiques.Finalement, la découverte de ce nouveau facteur d'initiation a fourni des informations essentielles sur le rôle de l'ubiquitination et d’OBI1 dans l'activation et la sélection des origines de réplication. Une telle sélection pourrait également participer à la régulation du « timing » de la réplication de l'ADN.