Children of the moon cannot be exposed to the sun because of their deficiency in DNA repair proteins including XPC, the factor that recognizes ultraviolet damage. However, this failure is not enough to explain all their symptoms. In this study, researchers from Frédéric Coin's team, including Nicolas Le May, (CNRS / Inserm / University of Strasbourg) showed that the XPC factor was also involved in a fundamental mechanism of gene expression, transcription. These results are published on July 4, 2018 in the journal Nature Communications.
Xeroderma pigmentosum is a genetic disease that can result from a mutation in the gene coding for the XPC protein, a DNA damage sensor that recognizes lesions produced by ultraviolet rays. Patients who are very sensitive to the sun have an increased risk of skin cancer but also develop neurological or ocular disorders. These symptoms, originally associated with defects in DNA repair, may also be related to disturbances in transcription, a fundamental mechanism of gene expression. Frédéric Coin's team therefore sought to establish the link between the XPC factor and transcription.
In normal cells, the researchers observed the presence of XPC on nearly 500 genes. The XPC occupancy sites on DNA coincide precisely with those of RNA polymerase II, the enzyme that catalyzes transcription. On the other hand, in cells derived from patients in whom XPC is defective, they showed that the expression of these genes was deregulated and that RNA polymerase II was no longer recruited correctly on their promoter, thus highlighting the link between the XPC factor and transcription.
The researchers then analyzed histone modifications that are essential to the development of an ideal chromatin environment for gene expression. They were particularly interested in the acetylation of histone H3, mediated by two major transcription complexes, SAGA and ATAC. The researchers observed that the acetylation of histone H3 in the 500 genes targeted by XPC was deficient in its absence. This is explained by the fact that XPC interacts with the ATAC complex and allows its recruitment on genes. Finally, they showed that the XPC protein was specifically recruited from the 500 genes through its interaction with the E2F1 protein, a transcription factor recognizing particular DNA sequences present upstream of the promoters of the 500 genes. Thus a succession of events, initiated by the recruitment of the transcription factor E2F1 followed by the arrival of XPC and the recruitment of ATAC on specific genes leads to the remodeling of chromatin and the expression of these genes.
These results establish that in addition to repairing DNA, XPC regulates transcription, and provide a better understanding of the molecular basis of defects in patients with Xeroderma pigmentosum.
This study was funded by ANR, the Cancer Research Association, Korea's National Research Foundation, the National League Against Cancer, the Foundation for Medical Research, and the South Province Research Incentive Award in New Caledonia.
The transcription is an essential mechanism for gene expression and is regulated by many protein factors. In humans, the TFIID factor, composed of the TATA box binding protein and 13 associated factors (TAFs: TBP-associated factors), is crucial to the initiation of transcription by RNA polymerase II. In this study, Laszlo Tora’s team at the IGBMC (CNRS, Inserm and University of Strasbourg) associated with American, British and Australian collaborators studied the consequences of a mutation in the gene coding for one of the associated factors, the TAF8 protein, in a child with an intellectual disability. Although this mutation causes an almost complete loss of TAF8, the researchers showed that it did not affect the overall transcription, contrary to what is observed after total inactivation of the TAF8 gene in mice. These results were published on April 13 in the journal Human Molecular Genetics.
The expression of genes coding for proteins requires the assembly of many molecules on a sequence promoting transcription. The first complex to bind to the promoter is the general transcription factor, TFIID, composed of the TATA box binding protein (TBP) and 13 associated factors (TAFs). Current studies suggested that the assembly of an entire TFIID complex was essential for the initiation of transcription and survival of eukaryotic cells.
In a child with an intellectual disability and a major developmental delay, Laszlo Tora’s team showed that a mutation of the TAF8 gene, one of the associated factors, results in a shift in the genetic code reading frame and in the production of an extremely unstable mutant protein, undetectable in cells derived from the patient. In addition, immunoprecipitation and proteomic analyses show that, in these cells, the formation of the TFIID complex is strongly altered and that only partial complexes are identified. Eventually, the researchers showed that this disorganization of the TFIID complex does not affect the overall transcription by RNA polymerase II.
These observations are much unexpected because they contrast with what is observed with complete inactivation of the TAF8 gene in mice. Indeed, the total loss of the TAF8 protein causes extremely early embryonic death and, in mouse embryonic stem cells, an overall decrease in transcription by RNA polymerase II associated with cell death.
The transcription by RNA polymerase II is thus extremely resistant in humans: a small residual quantity of TAF8 proteins is sufficient for the patient's survival. Nevertheless, the alterations of the TFIID complex are probably at the origin of its major developmental delay.
This study was funded by the ANR and the ERC.
Several rare myopathies are caused by alterations of proteins involved in muscle cell structure. These cause non-degraded toxic aggregates in the muscle. An earlier study by Dr. Jocelyn Laporte's team at IGBMC revealed an interaction between desmin, an intermediate filament of the cell cytoskeleton, and myotubularin (MTM1) in the dysfunctional degradation process related to two myopathies. In this new study recently published in Nature Cell Biology and conducted in synergy with Dr. Isabela Sumara's team at IGBMC, the degradation mechanism of defective intermediate filaments is revealed, opening up new therapeutic research perspectives.
The accumulation of aberrant proteins in the striated muscle is a characteristic of a group of genetic pathologies called proteinopathies. More particulary, myofibrillary myopathies are due to alterations in the proteins of the muscular contractile apparatus such as the intermediate filaments of desmin. An accumulation of polymers of misfolded desmin causes the progressive formation of toxic protein aggregates leading to skeletal muscle atrophy.
The mechanism used by the muscle cell to degrade these misfolded proteins is still not well known. Dr Jocelyn Laporte’s team in collaboration with Dr Izabela Sumara’s team, both from IGBMC, has demonstrated a molecular mechanism used by the skeletal muscle to detect and degrade these aberrant proteins. This process involves a protein complex containing myotubularin (MTM1), a protein whose deficiency causes a severe myopathy called myotubular myopathy, and another protein called ubiquilin-2 (UBQLN2) involved in protein recognition and degradation.
The discovery of this mechanism opens up a new field of investigation: exploring these molecular machines in other proteinopathies could indeed allow to identify more specific therapeutic targets in these pathologies. This project, developed under the supervision of Dr. Hnia, in Dr Jocelyn Laporte’s team, was completed at the Institute of Metabolic and Cardiovascular Diseases in Toulouse (I2MC, INSERM-UMR-1048).
This study was in part-financed by AFM (Association Française contre les Myopathies) and ANR (Agence Nationale pour la Recherche), IDEX and LABEX INRT.
Visualize without disturbing structures and processes taking place in the living cell nucleus is now possible thanks to an innovative technique developed by Laszlo Tora’s team at the IGBMC (CNRS/Inserm/Unistra) in close collaboration with Etienne Weiss at the ESBS Research Institute (CNRS/Unistra). This method allows to introduce, efficiently and without damaging the cells, antibodies labelled with small fluorescent molecules. In the cytoplasm of cells, these fluorescent antibodies bind to their antigens or targets. When the target is a nuclear protein, antibodies are transported with them into the nucleus, allowing researchers to locate and track the movements of this nuclear protein with high accuracy and in real time. The results of this study were published on February 12th, 2018 in the Journal of Cell Biology.
Protein detection by immunofluorescence is a technique that has been widely used for many years, where proteins or their post translational modifications are identified with antibodies coupled to microscopically detectable fluorescent compounds. The disadvantage of this process is that the cells need to be fixed and permeabilized, which may damage them, to allow the entry of labelled antibodies. Furthermore, the need to fix cells does not allow the observation of the progression of a biological mechanism over time.
In this new study, researchers from Laszlo Tora’s and Etienne Weiss's teams have developed an ingenious method that allows them to observe chromatin-based mechanisms in the nucleus of living cells, without altering the observed processes. This technique allows the introduction of any antibody labelled with fluorescent compounds that bind specifically to nuclear factors into the cells by using an electric micro-shock that does not modify cells physiology. When these antibodies are in the cytoplasm, they bind to the nuclear factors produced in this cell compartment. Since these factors are naturally transported to the nucleus to exert their function, the nuclear factors linked antibodies are taken to the nucleus. "The antibodies, which bind to these newly synthesized factors, are transported in the nucleus of the cell like small backpacks and, as they are marked with fluorochromes of any color, they will send a signal which allows us to observe the nuclear factors to which they are linked", explains Laszlo Tora. In addition, this visualization of nuclear factors in living cells is very effective because each antibody can be labelled with five to seven fluorescent molecules. The treated cells were observed with a confocal microscope and, in collaboration with the Basel Imaging Centre, with a very high-resolution microscope, the 3D-SIM. Precise observation of proteins that have nuclear activity during the cell cycle allows to see their dynamics and motion in the nucleus of living cells. Researchers have also shown that this strategy makes it possible to detect in real time the appearance and management of DNA damages in the nucleus by detecting a post translational modifications. By using an antibody fragment that has the same properties as a complete antibody and targets a major actor in triggering DNA repair (gammaH2AX), researchers were able to reveal areas in the nucleus where cell DNA is damaged by a substance causing genome changes, called genotoxic agent.
Given the large number of antibodies available on the market, this simple and innovative method will undoubtedly provide new information about nuclear proteins and a better understanding of their behavior in transmitting and maintaining the integrity of genetic information.
This study was funded by the ANR, ERC and the Regional League Against Cancer.
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Ribosomes play a vital role in living beings: they are protein production stations in all cell types. These large complexes are made up of two kinds of molecules: proteins and ribosomal RNA (rRNA). Through structural studies, Bruno Klaholz's team at the IGBMC revealed more than 130 chemical modifications of the rRNAs. Published in the journal Nature on November 15 2017, these results not only provide a better understanding of the structural and functional roles of human ribosome modifications, but also open up new therapeutic pathways for the treatment of pathologies such as cancer or certain genetic diseases in which dysregulation of protein synthesis is involved.
Genuine molecular machines, ribosomes decipher the information contained in a copy of DNA, called messenger RNA, to synthesize proteins. During ribosomes biogenesis, chemical modifications occur at precise locations of rRNAs. The alteration of these modifications is sometimes associated with disruptions in protein synthesis. Moreover, these modifications in the rRNA structure seem to modulate the activity of antibiotics, indicating that they can influence the ribosome interaction with this kind of drugs.
Chemical modifications of rRNA have been known for decades, particularly in microbes, but their observation remained difficult. In this study, Bruno Klaholz's team at the IGBMC determined the localization and three-dimensional structure of 136 various types of modifications (methylations, acetylations, etc.) of human ribosome rRNAs. The researchers have thus uncovered the role of these chemical modifications which consists in stabilizing the rRNA structure.
The researchers also analyzed the structure of three antibiotics-binding sites, revealing that these molecules are either in direct contact or very close to the rRNAs modifications sites, implying a role of these modifications in the mechanism of action and specificity of these inhibitors. Moreover, the unexpected presence of certain rRNA modifications suggests that rRNA modifications in human ribosomes could vary between different cell types, especially between normal cells and cancer cells, and could thus constitute a signature for cancer states.
This study represents a major result made possible by cryo electron microscopy, a revolutionary method for the observation of macromolecules coupled with image processing and 3D reconstruction, by which the researchers were able to construct a detailed atomic model of the human ribosome. Recognized recently by the Nobel Prize in Chemistry 2017, this technique provides the double advantage of being able to study biological samples without altering their properties and also allows to fix the orientation of the objects to enable a 3D reconstruction. This work was carried out using a high-resolution electron microscope (Titan Krios) within the French and European infrastructures, FRISBI and Instruct, hosted at the Centre for Integrative Biology at IGBMC.
Beyond mapping at the atomic level, the visualization of rRNA chemical modifications provides unprecedented insights into their role in the stabilization of rRNA structure, in the translation process and in diseases related to ribosome dysfunctions, paving the way for the development of new drug.
This study was supported by the INCa, the Ligue, the ANR, the CPER 2007-2013 (the Greater Region, the Eurometropole of Strasbourg, the General Council of the Bas-Rhin, the Ministry of National Education, higher education and research), FRM, Inserm, CNRS, FRISBI and Instruct-ERIC.
The Sweden Nobel Prize in Chemistry 2017 was awarded to Switzerland's Jacques Dubochet, the German-born American Joachim Frank and Britain's Richard Henderson for developing electronic cryo-microscopy, a revolutionary molecular observation method coupled with 3D reconstruction. This technique allows not only to study biological samples without altering their properties, as it is the case with dyes, but also to produce high-resolution three-dimensional structures of biomolecules which represents a revolution in the field of structural biology.
FRISBI open access to national and international scientific, academic and industrial scientific communities to cryo-microscopy devices of high technology: Polara and Titan Krios, see our platforms catalogue http://frisbi.eu/platforms-catalogue/
At a Royal Society awards ceremony celebrating the prestigious new European Commission-approved status for structural biology Instruct Research Infrastructure, UK Minister for Universities, Science, Research and Innovation Jo Johnson recognised the value and relevance of collaborative work between the UK and European scientists.
The defense of an organism depends on its ability to recognize danger signals. In mammals including humans, macrophages represent a predominant cellular system that acts against pathogens and removes cellular debris. Activation of macrophages represents a crucial immune defense mechanism and its deregulation contributes to uncontrolled inflammation and autoimmune diseases. Romeo Ricci’s team at the IGBMC has identified a new signaling pathway involved in the intracellular innate immune defense system. For the first time, they provided evidence that the Golgi apparatus, an important cellular organelle, is involved in the activation of the inflammasome, an intracellular multiprotein complex capable of sensing tissue damage and infectious agents. They also showed that pharmacologic interference with this newly identified mechanism can be used to mitigate inflammation. This study was published on 17 July 2017 in the Journal of Experimental Medecine (JEM).
The macrophage harbors two defense systems against dangers signals: on the one hand, specific receptors on their membrane surface and, on the other hand, intracellular complexes including the the various subsets of inflammasomes that are subjects of this study.
Romeo Ricci’s teamwas particularly interested in the NLRP3 inflammasome subunit activation mechanism. NLRP3 has been recently shown to directly bind to mitochondria-associated endoplasmic reticulum membranes (MAMs) allowing for inflammasome activation. This work showed for the first time that the Golgi apparatus activates Protein Kinase D (PKD) leading to NLRP3 phosphorylation. This phosphorylation was essential to release NLRP3 from MAMs resulting in recruitment of additional components and assembly of the mature NLRP3 inflammasome in the cytoplasm triggering an inflammatory response.
Patients affected by a rare genetic disease called CAPS, in which the gene encoding for the NLRP3 protein is mutated, the NLRP3 inflammasome is constitutively activated resulting in auto-inflammation. Using immune cells isolated from these patients, they demonstrated that inhibition of PKD blocked NLP3 inflammasome activation in vitro and mitigated release of pro-inflammatory factors providing a solid basis for new treatment options in NLRP3-related inflammatory diseases.
The study was funded by a USIAS fellow grant from the University of Strasbourg and the European Research Council (ERC).
The European Commission adopted the Instruct-ERIC decision on the 4th July 2017. Instruct is the 17th ERIC to be adopted.
Instruct was initiated in 2008 as a European-funded Preparatory Phase Project 211252 in the ESFRI Programme. In 2011, Instruct entered a transitional phase and in 2012 was operational, providing the first access to Instruct infrastructure through its Centres. Instruct has grown to provide new infrastructure, now modes of service delivery, excellent training courses and internships and has supported scientists through its R&D Pilot programme. Instruct-ERIC will provide the stability and sustainability for Instruct services to the structural biology community and to other life sciences communities within Europe.
Instruct was awarded an EU-funded project 'Instruct-ULTRA' in 2017 to further expand Instruct membership and services. Instruct-ULTRA will enable Instruct to work with new communities and extend and evolve the Instruct infrastructure to meet the demand of researchers wanting high quality structural technologies and methods.
In the nine years of Instruct development, there have been many people who have contributed and this success is thanks to them. Congratulations to all who played their part and we look forward to a bright and productive future as Instruct-ERIC.
See more at https://www.structuralbiology.eu
Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs.
This work was carried out using a high resolution electronic cryo-microscope (Titan Krios) within the French and European infrastructures, FRISBI and Instruc-ERIC