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Background: Several fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis. Methods: We created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules. Results: We showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo. Conclusions: Our study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy. [ABSTRACT FROM AUTHOR]

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin‐like chromatin at sub‐telomeric chromosomal regions. SIR‐mediated repressive chromatin limits DNA double‐strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non‐homologous end joining (NHEJ) or HR, we asked whether SIR‐mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin‐promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2‐dependent functions of the MRX (Mre11‐Rad50‐Xrs2) complex, thereby limiting Mre11‐mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ. Synopsis: During homologous recombination repair, the Sae2 protein stimulates MRX complex‐dependent initiation of DNA double‐strand break (DSB) resection. Here, the budding yeast heterochromatin protein Sir3 is found to directly bind Sae2 and inhibit its resection function at DSBs both in heterochromatin and euchromatin. The Sae2 C‐terminus physically interacts with the N‐terminal part of the Sir3 AAA+ domain (Sir3SaID).Sae2 and Sir4 compete for Sir3 binding.The sir3‐T557I mutation impairs Sir3‐Sae2 interaction.Sir3‐Sae2 interaction is sufficient to inhibit Sae2‐mediated stimulation of Mre11 endonuclease activity. [ABSTRACT FROM AUTHOR]

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection. Different proteins localised at telomeres ensure chromosome end stability to prevent double strand-end break recognition. Here the authors provide new insight into how in S. cerevisiae the interaction between Rif2 and Rad50 inhibits MRX functions at telomeres. [ABSTRACT FROM AUTHOR]

Interferon β (IFN-β) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-β binding motif, prevents binding of C/EBP-β, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-β expression in myeloid cells. Author summary: Genome-wide association studies identify multiple genetic variants associated with higher order phenotypes. Pinpointing the causative variant and understanding its molecular mode of action is a complex task. Using a murine model of interferon-β transcriptional deregulation, we characterize a super-enhancer controlling Ifnb1 expression in myeloid cells. The most active enhancer of this locus is conserved in humans, but presents a frequent variant found in around 30% of the population worldwide. This variant prevents binding of the C/EBP-β transcription factor, and is associated with decreased expression of IFNB1 in activated monocytes. When mimicked in the murine enhancer, it abolishes its inducible enhancer activity. Our results describe the molecular link between a point mutation and a cellular phenotype that could influence clinical situations. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks. [Display omitted] • 53BP1 is an RNA-binding protein that directly interacts with RNA-DNA chimeric structures • Perturbation of Okazaki fragment processing changes the proximity of 53BP1 with nascent DNA • Depletion of 53BP1 perturbs the maturation of Okazaki fragments Leriche et al. show that 53BP1, distinct from its canonical mode of recruitment to chromatin, is an RNA-binding protein that interacts with RNA-DNA chimeric structures constituting Okazaki fragments. 53BP1 acts as a binder of Okazaki fragments and plays a role during unstressed DNA replication. [ABSTRACT FROM AUTHOR]

The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site. [ABSTRACT FROM AUTHOR]

Background: Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. Results: We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. Conclusions: Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates. [ABSTRACT FROM AUTHOR]

Background: Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. Results: We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. Conclusions: Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates. [ABSTRACT FROM AUTHOR]

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells. [ABSTRACT FROM AUTHOR]

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale ( i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization. [ABSTRACT FROM AUTHOR]

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARα is phosphorylated at $77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of $77 governing the transcriptional activity of RARα depends on cyclin H binding at a RARa region that encompasses loop 8-9 and the N-terminal tip of helix 9 of the AF-2 domain. We propose a model in which the structural constraints of this region control the architecture of the RAR/RXR/TFIIH complex and therefore the efficiency of RARα phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex. [ABSTRACT FROM AUTHOR]

Structural genomics programs are distributed worldwide and funded by large institutions such as the NIH in United-States, the RIKEN in Japan or the European Commission through the SPINE network in Europe. Such initiatives, essentially managed by large consortia, led to technology and method developments at the different steps required to produce biological samples compatible with structural studies. Besides specific applications, method developments resulted mainly upon miniaturization and parallelization. The challenge that academic laboratories faces to pursue structural genomics programs is to produce, at a higher rate, protein samples. The Structural Biology and Genomics Department (IGBMC – Illkirch – France) is implicated in a structural genomics program of high eukaryotes whose goal is solving crystal structures of proteins and their complexes (including large complexes) related to human health and biotechnology. To achieve such a challenging goal, the Department has established a medium-throughput pipeline for producing protein samples suitable for structural biology studies. Here, we describe the setting up of our initiative from cloning to crystallization and we demonstrate that structural genomics may be manageable by academic laboratories by strategic investments in robotic and by adapting classical bench protocols and new developments, in particular in the field of protein expression, to parallelization. [ABSTRACT FROM AUTHOR]

For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25° and 30°). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations. Abbreviations IPTG — isopropyl β-d-1 thiogalactopyranoside; Mr — molecular mass; ORF — open reading frame; PCR — polymerase chain reaction; TBST — Tris-buffered saline Tween; Tris — tris(hydroxymethyl)aminomethane. [ABSTRACT FROM AUTHOR]

Investigates the structure of a hypothetical protein AQ_1354 from the hyperthermophilic bacteria Aquifex aeolicus using X-ray crystallography. Overall fold of the protein; Structure-based homology studies; Resemblance of the protein structure to metal-dependent proteinases.

The crystal structure of human cyclin H refined at 2.6 Å resolution is compared with that of cyclin A. The core of the molecule consists of two repeats containing five helices each and forming the canonical cyclin fold also observed in TFIIB. One hundred and thirty-two out of the 217 Cα atoms from the cyclin fold can be superposed with a root-mean-square difference of 1.8 Å. The structural homology is even higher for the residues at the interface with the kinase, which is of functional significance, as shown by our observation that cyclin H binds to cyclin-dependent kinase 2 (cdk2) and that cyclin A is able to activate cdk7 in the presence of MAT1. Based on this superposition, a new signature sequence for cyclins was found. The specificity of the cyclin H molecule is provided mainly by two long helices which extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite to the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H. [ABSTRACT FROM AUTHOR]

Human glioblastoma (GBM) is the most common primary malignant brain tumor. A minor subpopulation of cancer cells, known as glioma stem-like cells (GSCs), are thought to play a major role in tumor relapse due to their stem cell-like properties, their high resistance to conventional treatments and their high invasion capacity. We show that ionizing radiation specifically enhances the motility and invasiveness of human GSCs through the stabilization and nuclear accumulation of the hypoxia-inducible factor 1α (HIF1α), which in turn transcriptionally activates the Junction-mediating and regulatory protein (JMY). Finally, JMY accumulates in the cytoplasm where it stimulates GSC migration via its actin nucleation-promoting activity. Targeting JMY could thus open the way to the development of new therapeutic strategies to improve the efficacy of radiotherapy and prevent glioma recurrence. [ABSTRACT FROM AUTHOR]

Helicobacter pylori chronically colonises half of the world's human population and is the main cause of ulcers and gastric cancers. Its prevalence and the increase in antibiotic resistance observed recently reflect the high genetic adaptability of this pathogen. Together with high mutation rates and an efficient DNA recombination system, horizontal gene transfer through natural competence makes of H. pylori one of the most genetically diverse bacteria. We show here that transformation capacity is enhanced in strains defective for recN, extending previous work with other homologous recombination genes. However, inactivation of either mutY or polA has no effect on DNA transformation, suggesting that natural competence can be boosted in H. pylori by the persistence of DNA breaks but not by enhanced mutagenesis. The transformation efficiency of the different DNA repair impaired strains correlates with the number of transforming DNA foci formed on the cell surface and with the expression of comB8 and comB10 competence genes. Overexpression of the comB6-B10 operon is sufficient to increase the transformation capacity of a wild type strain, indicating that the ComB complex, present in the bacterial wall and essential for DNA uptake, can be a limiting factor for transformation efficiency. [ABSTRACT FROM AUTHOR]