Publications

2023

• Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression
  
  • Mancini Laure
  • Guirao Boris
  • Ortica Sara
  • Labusch Miriam
  • Cheysson Felix
  • Bonnet Valentin
  • Phan Minh Son
  • Herbert Sébastien
  • Mahou Pierre
  • Menant Emilie
  • Bedu Sébastien
  • Tinevez Jean-Yves
  • Baroud Charles
  • Beaurepaire Emmanuel
  • Bellaiche Yohanns
  • Bally-Cuif Laure
  • Dray Nicolas
  Science Advances, American Association for the Advancement of Science (AAAS), 2023, 9 (35), pp.eadg7519. The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA -negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaA neg daughter, which regains the size and behavior of its mother, and a neurogenic deltaA pos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaA neg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations. (10.1126/sciadv.adg7519)
  DOI : 10.1126/sciadv.adg7519
• Last Year At Marienbad: Unusual Nucleic Acid structures
  
  • Mergny Jean-Louis
  • Trantírek Lukáš
  • Capranico Giovanni
  Biochimie, Elsevier, 2023, 214, pp.1-4. (10.1016/j.biochi.2023.09.022)
  DOI : 10.1016/j.biochi.2023.09.022
• Color TSFG microscopy of red blood cells and oxygenation
  
  • Ferrer Ortas Júlia
  • Mahou Pierre
  • Escot Sophie
  • Stringari Chiara
  • David Nicolas B
  • Bally-Cuif Laure
  • Dray Nicolas
  • Négrerie Michel
  • Supatto Willy
  • Beaurepaire Emmanuel
  Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering, 2023, 12630, pp.1. We present color third-order sum-frequency generation (color TSFG) microscopy, a multiphoton imaging strategy based on the simultaneous detection of several third-order coherent signals produced by two synchronized femtosecond pulse trains. We demonstrate that it can be used to obtain red blood cell (RBC)specific label-free contrast in live zebrafish and is a promising tool for probing RBC oxygenation. (10.1117/12.2670229)
  DOI : 10.1117/12.2670229
• G-quadruplex forming sequences in the genes coding for cytochrome P450 enzymes and their potential roles in drug metabolism
  
  • Saad Mona
  • Zhang Rongxin
  • Cucchiarini Anne
  • Mehawej Cybel
  • Mergny Jean-Louis
  • Mroueh Mohamad
  • Faour Wissam
  Biochimie, Elsevier, 2023, 214, pp.45-56. The majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in cytochrome genes regulation, and emphasize the importance of G-quadruplexes in drug metabolism. (10.1016/j.biochi.2023.08.014)
  DOI : 10.1016/j.biochi.2023.08.014
• Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy
  
  • Raoux Clothilde
  • Chessel Anatole
  • Mahou Pierre
  • Latour Gaël
  • Schanne-Klein Marie-Claire
  Light: Science and Applications, Nature Publishing Group, 2023, 12 (1), pp.190. A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues. (10.1038/s41377-023-01224-0)
  DOI : 10.1038/s41377-023-01224-0
• Understanding DNA replication dynamics in Haloferax volcanii using superresolution microscopy
  
  • Noury Dorian
  • Olivier Nicolas
  • Lestini Roxane
  , 2023.
• High-pulse-energy frequency comb in the MIR
  
  • Bournet Quentin
  • Jonusas M.
  • Natile Michele
  • Guichard François
  • Bonvalet A.
  • Joffre M.
  • Druon Frédéric
  • Hanna Marc
  • Georges Patrick
  , 2023.
• The H-NOX protein structure adapts to different mechanisms in sensors interacting with nitric oxide
  
  • Yoo Byung-Kuk
  • Kruglik Sergei
  • Lambry Jean-Christophe
  • Lamarre Isabelle
  • Raman C.S.
  • Nioche Pierre
  • Négrerie Michel
  Chemical Science, The Royal Society of Chemistry, 2023, 14 (31), pp.8408-8420. Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (∼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation. (10.1039/d3sc01685d)
  DOI : 10.1039/d3sc01685d
• Insight into the manufacturing and conservation state of skin-based collections by nonlinear optical microscopy
  
  • Robinet Laurianne
  • Schmeltz Margaux
  • Heu-Thao Sylvie
  • Galante Giulia
  • Schanne-Klein Marie-Claire
  • Latour Gael
  , 2023.
• G-quadruplexes in the evolution of hepatitis B virus
  
  • Brázda Václav
  • Dobrovolná Michaela
  • Bohálová Natália
  • Mergny Jean-Louis
  Nucleic Acids Research, Oxford University Press, 2023, 51. Abstract Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of ‘genetic camouflage’ to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material. (10.1093/nar/gkad556)
  DOI : 10.1093/nar/gkad556
• Tunable ultrafast mid-IR source intensity noise properties
  
  • Bournet Quentin
  • Natile Michele
  • Guichard François
  • Zaouter Yoann
  • Jonusas M
  • Bonvalet A.
  • Joffre M.
  • Druon Frédéric
  • Hanna Marc
  • Georges Patrick
  , 2023.
• Objective characterisation of corneal transparency by analysis of clinical SD-OCT images
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  , 2023, 12632. (10.1117/12.2670743)
  DOI : 10.1117/12.2670743
• Classification of healthy and pathological human corneas by the analysis of clinical SD-OCT images using machine learning
  
  • Vilbert Maëlle
  • Soubeiran Corentin
  • Memmi Benjamin
  • Georgeon Cristina
  • Borderie Vincent
  • Chessel Anatole
  • Plamann Karsten
  , 2023.
• Time-resolved vibrational spectroscopy using Chirped-Pulse Upconversion of mid-infrared pulses generated with a 250-kHz Yb fiber laser by intrapulse difference frequency mixing
  
  • Jonusas M.
  • Bournet Quentin
  • Natile Michele
  • Guichard François
  • Zaouter Yoann
  • Bonvalet A.
  • Druon Frédéric
  • Hanna Marc
  • Georges Patrick
  • Joffre M.
  , 2023.
• DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence
  
  • Lista María José
  • Jousset Anne-Caroline
  • Cheng Mingpan
  • Saint-André Violaine
  • Perrot Elouan
  • Rodrigues Melissa
  • Di Primo Carmelo
  • Gadelle Danielle
  • Toccafondi Elenia
  • Segeral Emmanuel
  • Berlioz-Torrent Clarisse
  • Emiliani Stéphane
  • Mergny Jean-Louis
  • Lavigne Marc
  Retrovirology, BioMed Central, 2023, 20 (1), pp.10. Background: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. Results: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. Conclusions: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies. (10.1186/s12977-023-00625-8)
  DOI : 10.1186/s12977-023-00625-8
• Third harmonic imaging contrast from tubular structures in the presence of index discontinuity
  
  • Morizet Joséphine
  • Olivier Nicolas
  • Mahou Pierre
  • Boutillon Arthur
  • Stringari Chiara
  • Beaurepaire Emmanuel
  Scientific Reports, Nature Publishing Group, 2023, 13, pp.7850. Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2 ′-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarizationresolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries. (10.1038/s41598-023-34528-7)
  DOI : 10.1038/s41598-023-34528-7
• The Mappa Mundi of Albi - Materials identification of an 8th century manuscript
  
  • Robinet Laurianne
  • Heu-Thao Sylvie
  • Tournié Aurélie
  • Michelin Anne
  • Latour Gael
  • Andraud Christine
  • Schanne-Klein Marie-Claire
  • Deschaux Jocelyne
  , 2023. The Mappa mundi of Albi is one of the oldest examples of representation of the occidental world (Figure 1). The non-symbolic and non-abstract map was drawn on parchment and was probably made in the 8th century in the south of France or northern Spain. The Mappa mundi with the associated Index of winds and seas on the adjacent page, is included in a 77 folio manuscript (Ms29) conserved at the médiathèque Pierre Amalric in Albi (Tarn, France). Because it is a document of exceptional importance for global cartographic history and more widely for the history of the representation of space, the map together with the index were recorded in the UNESCO Memory of the World Register in 2015. A detailed study was performed on the map to examine the structure of the manuscript in which it is inserted, and to characterize the different constitutive materials. Observations and physico-chemical analyses were performed from the micro to the macroscale, using mostly non-invasive analytical techniques, in order to increase the knowledge on the manufacturing technique of this document and its conservation state. The observations evidenced the presence of three main parts in the manuscript which quires differ in their assembly and parchment quality. Hyperspectral imaging in the visible and near-infrared ranges combined with statistical data treatments, helped visualize the spatial distribution of the different components and revealed peculiar areas. Punctual spectroscopic analyses combining X-ray fluorescence, Fiber Optical Reflectance Spectroscopy (FORS), reflectance infrared and micro-Raman spectroscopies allowed the characterization of the ink, the colouring materials and organic compounds presents. For the parchment, the animal species of the skin was identified by proteomic analyses, and its conservation state examined in-situ at the micrometer scale using nonlinear optical microscopy. The results obtained from these analyses will be put in perspective with the studies led by historians on this map, as well as the existing material studies carried out on manuscripts produced in that period in Europe.
• G-Quadruplex Structures Are Key Modulators of Somatic Structural Variants in Cancers
  
  • Zhang Rongxin
  • Shu Huiling
  • Wang Yuqi
  • Tao Tiantong
  • Tu Jing
  • Wang Cheng
  • Mergny Jean-Louis
  • Sun Xiao
  Cancer Research, American Association for Cancer Research, 2023, 83 (8), pp.1234-1248. Abstract G-quadruplexes (G4) are noncanonical secondary genome structures. Aberrant formation of G4s can impair genome integrity. Investigation of the relationship between G4s and somatic structural variants (SV) in cancers could provide a better understanding of the role of G4 formation in cancer development and progression. In this study, we combined bioinformatic approaches and multiomics data to investigate the connection between G4s and the somatic SVs. Somatic SV breakpoints were significantly enriched in G4 regions, regardless of SV subtypes. This enrichment was only observed in regions demonstrated to form G4s in cells (“active quadruplexes”), rather than in regions with a sequence compatible with G4 formation but without confirmed G4 formation (“potential quadruplexes”). Several genomic features affected the connection between G4s and SVs, with the enrichment being notably strengthened at the boundary of topologically associated domains. Somatic breakpoints were also preferentially associated with G4 regions with earlier replication timing and open chromatin status. In patients with cancer with homologous recombination repair defects, G4s and somatic breakpoints were substantially more strongly associated. Machine learning models were constructed that showed that G4 propensity is a potent feature for predicting the density of SV breakpoints. Altogether, these findings suggest that the G4 structures play a critical role in modulating the production of somatic SVs in cancers. Significance: G-quadruplex structure formation constitutes a critical step in the production of somatic structural variants in cancers, suggesting G-quadruplex structures as potential targets for future cancer prevention and treatment strategies. (10.1158/0008-5472.CAN-22-3089)
  DOI : 10.1158/0008-5472.CAN-22-3089
• Birth, cell fate and behavior of progenitors at the origin of the cardiac mitral valve
  
  • Farhat Batoul
  • Bordeu Ignacio
  • Jagla Bernd
  • Blanc Hugo
  • Loulier Karine
  • Simons Benjamin
  • Beaurepaire Emmanuel
  • Livet Jean
  • Pucéat Michel
  , 2023. Mitral valve prolapse is often associated with several congenital heart malformations. During development, endocardial cells of the atrioventricular canal undergo endothelial to mesenchymal transition (EndMT) to give rise to the different mitral valvular cells. However, our understanding of the identity and fate decisions of these endocardial cells during development is lacking. Here, using single-cell RNA sequencing (scRNA-seq) of genetically labeled AVC endocardial cells at E9.5 in murine heart, we uncovered and genetically characterized a restricted population of pro-EndMT cells and further distinct populations of valve progenitors that together contribute to different endothelial and interstitial valvular cells in E16.5 embryonic and P0 postnatal murine mitral valve. Moreover, by genetically labeling endocardial cells using CAG ERT2Cre+/- / Brainbow +/- mice at embryonic stage E8.5, prior to EndMT, we observed specific modes of growth of endocardial derived clones in E16.5 embryonic mitral valve. Collectively, our data reveal the identity of specified endocardial cells and the distinct types of clonal contribution of these cells to the formation of the mitral valve. They further open the path towards finding new cell targets for a therapeutic approach of congenital and acquired valve diseases. (10.1101/2022.08.06.503022)
  DOI : 10.1101/2022.08.06.503022
• DNA i-motif formation at neutral pH is driven by kinetic partitioning
  
  • Školáková Petra
  • Gajarský Martin
  • Palacký Jan
  • Šubert Denis
  • Renčiuk Daniel
  • Trantírek Lukáš
  • Mergny Jean-Louis
  • Vorlíčková Michaela
  Nucleic Acids Research, Oxford University Press, 2023, 51 (6), pp.2950-2962. Abstract Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms. (10.1093/nar/gkad119)
  DOI : 10.1093/nar/gkad119
• Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase
  
  • Samire Poutoum
  • Zhuang Bo
  • Légeret Bertrand
  • Baca-Porcel Ángel
  • Peltier Gilles
  • Sorigué Damien
  • Aleksandrov Alexey
  • Beisson Frédéric
  • Müller Pavel
  Science Advances, American Association for the Advancement of Science (AAAS), 2023, 9 (13). Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP ( Cv FAP) can convert n -octanoic acid in vitro four times faster than n -hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a Cv FAP-based production rate over 10-fold higher for n -heptane than for n -pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that Cv FAP’s high catalytic activity on n -octanoic acid is, in part, due to an autocatalytic effect of its n -heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons. (10.1126/sciadv.adg3881)
  DOI : 10.1126/sciadv.adg3881
• Intensity noise in difference frequency generation-based tunable femtosecond MIR sources
  
  • Bournet Q
  • Natile M
  • Jonusas M
  • Guichard F
  • Zaouter Y
  • Joffre M
  • Bonvalet A
  • Druon F
  • Hanna Marc
  • Georges P
  Optics Express, Optical Society of America - OSA Publishing, 2023, 31 (8), pp.12693-12702. We characterize the intensity noise of two mid-infrared (MIR) ultrafast tunable (3.5-11 µm) sources based on difference frequency generation (DFG). While both sources are pumped by a high repetition rate Yb-doped amplifier delivering 200 µJ 300 fs at a central wavelength of 1030 nm, the first is based on intrapulse DFG (intraDFG), and the second on DFG at the output of an optical parametric amplifier (OPA). The noise properties are assessed through measurement of the relative intensity noise (RIN) power spectral density and pulse-to-pulse stability. The noise transfer mechanisms from the pump to the MIR beam is empirically demonstrated. As an example, improving the pump laser noise performance allows reduction of the integrated RIN (IRIN) of one of the MIR source from 2.7% RMS down to 0.4% RMS. The intensity noise is also measured at various stages and in several wavelength ranges in both laser system architectures, allowing us to identify the physical origin of their variation. This study presents numerical values for the pulse to pulse stability, and analyze the frequency content of the RINs of particular importance for the design of low-noise high repetition rate tunable MIR sources and future high performance time-resolved molecular spectroscopy experiments. (10.1364/oe.486509)
  DOI : 10.1364/oe.486509
• G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing
  
  • Esnault Cyril
  • Garcia-Oliver Encar
  • El Aabidine Amal Zine
  • Robert Marie-Cécile
  • Magat Talha
  • Gawron Kevin
  • Basyuk Eugénia
  • Karpinska Magda
  • Pigeot Alexia
  • Cucchiarini Anne
  • Luo Yu
  • Verga Daniele
  • Mourad Raphael
  • Radulescu Ovidiu
  • Mergny Jean-Louis
  • Bertrand Edouard
  • Andrau Jean-Christophe
  , 2023. Despite their central role in transcription, it has been difficult to define universal sequences associated to eukaryotic promoters. Within chromatin context, recruitment of the transcriptional machinery requires opening of the promoter but how DNA elements could contribute to this process has remained elusive. Here, we show that G-quadruplex (G4) secondary structures are highly enriched mammalian core promoter elements. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes both in vivo and in vitro and display a strong positioning potential. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected transition to effective RNA production in live imaging experiments. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause release by the RNA Polymerase II. (10.1101/2023.02.24.529838)
  DOI : 10.1101/2023.02.24.529838
• Characterizing G-quadruplex formation : development and integration of novel assays
  
  • Luo Yu
  , 2023. Guanine-rich nucleic acid sequences can generate four-stranded, noncanonical secondary structures called G-quadruplexes (G4). G4 structures have been identified in the genomes of different species and are involved in physiological processes. Numerous putative G4 sequences (PQS) have been mined by G4 predicting algorithms and G4 chromatin immunoprecipitation sequencing (ChIP-seq). However, finding a sequence relatively rich in guanines does not necessarily mean it can form a G4 structure. The main aim of this PhD thesis is to develop novel in vitro high-throughput G4 characterization assays, suitable for validating a vast amount of PQS identified by in silico G4 prediction methods and ChIP-seq. Förster Resonance Energy Transfer (FRET) is a powerful tool in characterizing intramolecular G4 structures that were dual-labeled by a chromophore (i.e. FAM) and a corresponding quencher (i.e. Tamra): at the low temperature, the two ends of the G4 are in close proximity and the FAM fluorescence is quenched via a FRET mechanism; with the temperature increasing, G4 unfolds and two ends are split apart, restoring gradually FAM fluorescence. Melting temperature (Tm) of a G4 structure could be calculated based on the FRET-melting curve. In this context, a new methodology called the FRET-melting competition (FRET-MC) assay has been developed, which allows to follow 48 duplicated samples within 2 hours. FRET-MC is based on the competitive binding of a selective G4 ligand (PhenDC3) between a labeled G4 reporter (FAM-Tel21-TAMRA, F21T) and an unknown competitor present in a large excess. PhenDC3 stabilizes the G4 structure of F21T and leads to an increase of the Tm of F21T of about 23 °C. An excess of G4 competitors can trap PhenDC3, leading to a decrease of the Tm of F21T back to the Tm value of F21T alone. In contrast non-G4 competitors have little influence on the F21T-PhenDC3 interaction. FRET-MC works well in most cases, but cannot be used to pick up G4s with low thermal stability, as these weak G4 behave as ssDNA at the temperature where F21T starts to melt. For this reason, an alternative isothermal FRET assay compatible with weakly stable G4s was developed. Iso-FRET exploits two labeled RNA probe strands: one of them 37Q (37-quencher) form a G4 thanks to the binding of PhenDC3, and the second one F22 (FAM-22) is partially complementary to 37Q. To this system an unlabeled competitor sequence is added. When the competitor is not a G4, PhenDC3 remains bound to 37Q and stabilizes its G4 structure, preventing duplex formation between 37Q and F22, allowing a high fluorescence signal of F22. On the contrary, an excess of a G4 competitor traps PhenDC3, which is no longer available to bind to 37Q, which in turns allows this oligonucleotide to hybridize to F22 resulting in fluorescence quenching. Iso-FRET can be performed at 25 °C and 37 °C (physiological temperature), which are acceptable to thermolabile G4 competitors. In parallel, a study focusing on the conformation of G4-prone GC-rich sequences which are rich both in cytosines and guanines was conducted. The structures of GC-rich sequences were dependent on cytosine content and [K⁺] / [Na⁺] ratio in buffer. Spectroscopic results shown that the G4 structure (CEB25) tolerates two three continuous cytosines tracks in potassium / sodium mixed buffers containing 40 mM or higher KCl concentration, implying that cytosine contained G4-forming sequences can still adopt a G4 structure in the intracellular environment. UV-melting results evidenced that the presence of cytosines in G4 loops does not decrease G4s stability, but rather increases the probability of forming a competing structure, either a hairpin or a duplex. As bioinformatics analysis indicates that the majority of cytosine-contained PQS located at pre-mRNAs, it will be interesting to transpose our experiments on DNA sequences to RNA motifs, in order to determine if the latter is more or less prone to hairpin / G4 competition.
• Chimeric Biocatalyst Combining Peptidic and Nucleic Acid Components Overcomes the Performance and Limitations of the Native Horseradish Peroxidase
  
  • Zhang Xiaobo
  • Qiu Dehui
  • Chen Jielin
  • Zhang Yue
  • Wang Jiawei
  • Chen Desheng
  • Liu Yuan
  • Cheng Mingpan
  • Monchaud David
  • Mergny Jean-Louis
  • Ju Huangxian
  • Zhou Jun
  Journal of the American Chemical Society, American Chemical Society, 2023, 145 (8), pp.4517-4526. Chimeric peptide-DNAzyme (CPDzyme) is a novel design of an artificial peroxidase that relies on the covalent assembly of DNA (quadruplex-DNA, or G4), peptides and an enzyme cofactor (hemin) in a single scaffold. An accurate control of the assembly of these different partners allows for the design of the efficient CPDzyme prototype G4-Hemin-KHRRH, found to be >2,000-fold more active (in terms of conversion number kcat) than the corresponding but non-covalent complex and, more importantly, >1.5-fold active than the corresponding native peroxidase (horseradish peroxidase, or HRP) when considering a single catalytic center. This unique performance originates in a series of improvements gradually made thanks to an accurate selection and arrangement of the different components of the CPDzyme, in order to benefit from synergistic interactions between them. The optimized prototype G4-Hemin-KHRRH is efficient and robust as it can be used under a wide range of non-physiologically relevant conditions (organic solvents, high temperature (95°C), in a wide range of pH (from 2 to 10)), thus compensating for the shortcomings of natural enzymes. Our approach thus opens broad prospects for the design of ever more efficient artificial enzymes. (10.1021/jacs.2c11318)
  DOI : 10.1021/jacs.2c11318