dataesr/scientific-paragraphs-categorization huggingface dataset:
We therefore compared expression changes between U2OS and U2OS Fl-SUPT3H for genes with either a TATA-less or a TATA-box in their promoters, as defined by the Eukaryotic Promoter Database (Material and Methods) (Figure  2D ). We observed no significant difference between the two gene classes, suggesting that TATAbox containing gene promoters do not show a stronger sensitivity to SUPT3H re-expression than TATA-less promoters in human U2OS cells. In conclusion, SUPT3H appears to regulate the transcription of only a few genes in U2OS Fl-SUPT3H cells, independently of the presence a TATA-box element in their promoter. This observation is in marked contrast with the strong, global decrease of nascent transcript levels observed in S. cerevisiaeSPT3 deletion mutants.
On the contrary, as described earlier, the loss of ATXN7L3, a critical subunit of the SAGA DUB module lead to the stabilization of H2Bub1 (Supplementary Figure  S3B )  (47). These results confirm that, as observed in human U2OS cells, mouse SUPT3H has no major role in SAGA integrity and HAT/DUB activities in mESCs. Thus, these data also suggest that the SAGA central HF module can assemble with a hexameric nucleosome-like structure without the intramolecular HF pair of SUPT3H.
Finally, we compared transcription changes between genes possessing TATA-less and TATA-box containing promoters (Figure  5F ). We observed no difference between the two gene classes suggesting that TATA-box containing promoters do not show a stronger sensitivity to the loss of Supt3 than TATA-less promoters in mESCs. These data together show that the mouse SAGA complex lacking SUPT3H, as observed in human U2OS cells, affects the expression of a small subset of genes in FCS + LIF + 2i medium.
ACKNOWLEDGEMENTS We thank all members of the  Tora lab  for protocols, thoughtful discussions and suggestions,  A. Ben Shem  for comments and critically reading the original version of the manuscript, the  GenomEast platform [France Génomique consortium  ( ANR-10-INBS-0009 )] for library preparation and sequencing;  C. Ebel  and  M. Philipps  for help with FACS sorting,  B. Reina San Martin  and  N. Jung  for help with the CRIPR/Cas9 Supt3 KO experiment, designing and establishing the corresponding vectors, and the IGBMC cell culture facility for help with  U2OS ,  HeLa  and  E14  ES cells.
FUNDING Agence Nationale de la Recherche (ANR)  [ ANR-19-CE11-0003-02 ,  ANR-PRCI-19-CE12-0029-01 ,  ANR-20-CE12-0017-03 ];  NIH [MIRA  grant  R35GM139564 ];  NSF  [ NSF   1933344  to L. T. ,  ANR-18-CE12-0026  to  D. D. ,  ANR-20-CE12-0014-02  to D. H. and D. D. ];  IdEx-University of Strasbourg PhD program ; ' Fondation pour la Recherche Médicale ' (FRM) association [ FDT201904008368  to V. F. ]; [ ANR-10-LABX-0030-INRT ], under the  frame program Investissements d'Avenir  [ ANR-10-IDEX-0002-02 ]. Funding for open access charge:  Agence National de la Recherche (ANR).
DATA AVAILABILITY The 4sU-seq data generated for this publication have been deposited in NCBI's Gene Expression Omnibus  (57)  and are accessible through GEO Series accession numbers GSE175901 and GSE202823. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (58) partner repository with the dataset identifier PXD026991. BED and BigWIG datasets used in Supplementary Figure  S1D  were downloaded from GEO repository: GSM1901948 (U2OS; Pol II ChIP), GSM4194653 (U2OS; H3K4me3), GSM4194660 (U2OS; H3K27ac ChIP), GSM803533 (HeLa; Pol II ChIP), GSM733682 (HeLa; H3K4me3 ChIP) and GSM733684 (HeLa; H3K27ac ChIP)  (59) (60) (61) (62).
Whole-genome sequencing (WGS) significantly increases our ability to infer phylogenetic relationships between isolates. By sequencing 50 ST410 CP-Ec isolates received by the F-NRC between 2011 and 2015  (10) , we showed that 36 (72%) belonged to a newly described ST410 lineage producing OXA-181  (11). We showed that this clade is characterized by mutations in the two porin genes ompC and ompF leading to a decreased outer membrane permeability to certain b-lactams and by a four-codon duplication (YRIN) in the ftsI gene encoding the penicillin binding protein 3 (PBP3) leading to a decreased susceptibility to b-lactams targeting this PBP  (10). After a thorough analysis of CP-Ec genome sequences from public databases for mutations in these three genes, we proposed that CP-Ec followed three different evolutionary trajectories.
Strikingly, among the 28 isolates of the G2 clade, 24 were collected in France and four in the Netherlands, suggesting at this time a more regional dissemination. In none of the isolates of the four clades could an IncL plasmid, that generally carries bla OXA-48  (13) , be identified by PlasmidFinder  (14). It suggests a chromosomal integration as previously shown among ST38 isolates collected in the United Kingdom  (15). Analysis of representative isolates of the G1 and G3 cluster (G1, GCA_005886035. 1; G3, GCA_004759025. 1), whose genome sequences were completely  FIG 6  Core genome phylogeny of ST38 isolates. ML Phylogeny was based on genome sequences of 92 CP-Ec from the F-NRC and 464 genome sequences retrieved from EnteroBase and from the NCBI database, including 331 carrying a carbapenemase gene.
ST131 evolution has been thoroughly analyzed, and four main lineages A, B, C1, and C2 have been described  (21). We identified only 17 ST131 CP-Ec isolates received by the F-NRC (Fig. 2 ), 10 belonged to lineage A, while the others were scattered in the three other lineages (Fig. S3B ). The main carbapenemase associated with these isolates was OXA-48 (n = 10). We also did not observe any association with the carriage of bla CTX-M genes, as only four ST131 CP-Ec isolates carried a bla CTX-M-15 gene. These results are in accordance with the United Kingdom survey (8) and our previous observations from sequences retrieved from public databases  (10). ST940 (phylogroup B1) and ST1284 (phylogroup A) represented 2. 4% (n = 17) and 1. 9% (n = 14) of the sequenced isolates, while they were poorly represented in sequence databases.
De novo assemblies were generated from the trimmed reads with SPAdes v3. 12. 0 (34), using k-mer sizes of 51, 71, 81, and 91, the coverage cutoff option was set to "auto" and the "careful" option was activated. QUAST v2. 2  (35)  was used to assess the assembly quality, and contigs shorter than 500 bp were filtered out for phylogenetic analyses. The ST38 isolates CNR65D6 and CNR85I8 were sequenced to completion by using the long-read Pacbio technology. PacBio reads were assembled with the RS_HGAP_Assembly. 3 protocol from the SMRT analysis toolkit v2. 3  (36)  and with Canu (37), polished with Quiver  (36) , and manually corrected by mapping Illumina reads using Breseq  (38). Assembled genomes were annotated with Prokka v1. 9  (39). To analyze the F-NRC CP-Ec isolates belonging to the main ST recovered during the analysis in a more global context, their genome sequences were combined to the assembled genome sequences from the same ST retrieved from the NCBI database (July 2019) and in some STs from EnteroBase (http://enterobase.
warwick. ac. uk/, April 2021). Retrieved genomes were annotated in the same way as the genome sequences of the F-NRC isolates (Table  S2 ). To generate a core genome phylogeny tree of all isolates, the best reference genome was first selected among a set of 18 genomes analyzed by Touchon et al. (40)  using the software refRank  (41)  and three subsets of 100 randomly selected sequences from our study as input. This led to the selection of the genome sequence of strain MG1655-K12 (NC_000913. 3) as the reference for read mapping and SNP identification. Sequence reads for the 17 reference genomes  (40)  as well as for the genome sequence of Escherichia fergusonii strain ATCC 35469 (NC_011740. 1), used as an outgroup, were simulated with ART  (42). Trimmed sequencing reads were mapped against the MG1655-K12 genome with BWA-MEM algorithm of the BWA v0.
7. 4 package  (43). For SNP calling, the Genome Analysis Toolkit (GATK) v3. 6. 0 (44) was used with the following criteria, a minimum depth coverage (DP) of 10, a quality by depth (QD) bigger than 2, a fisher strain bias (FS) below 60, a root mean square of the mapping quality (MQ) above 40, and the mapping quality rank sum test (MQRankSum) and the read position rank sum test (ReadPosRankSum) greater than 212. 5 and 28, respectively. A maximum likelihood tree was estimated with RAxML v8. 28 (45) using core genome SNPs after removing positions in the accessory genome identified with the filter_BSR_variome. py script from the LS-BSR pipeline  (46). For STs with more than 10 CP-Ec isolates from the F-NRC, a core genome alignment was generated with Parsnp (47), by using a finished genome sequence as reference.
A closely related isolate outside the ST lineage was selected from the global phylogenetic tree, including all F-NRC isolates and used as an outgroup to root the ST phylogenetic trees. Maximum likelihood (ML) trees were generated with RAxML v8. 28  (45)  after removing regions of recombination with Gubbins  (48). All graphic representations were performed by using ITOL  (49). MLST type, phylogroup, resistance gene, and plasmid replicon identification. Sequence type was assigned to each assembly through a python script that relies on BLAST  (10). Antibiotic resistance genes (ARGs) and mutations in gyrA, parC, and parE quinolone resistance determining regions (QRDRs) were identified with Resfinder 4. 0 and PointFinder (50) run locally, respectively. The scripts and database (retrieved 4 January 2021) were downloaded from the repositories of the Centre for Genomic Epidemiology (https://bitbucket.
ACKNOWLEDGMENTS This work was supported by grants from the  French National Research Agency  ( ANR-10-LABX-62-IBEID ,  ANR-10-LABX-33 ,  INCEPTION  project  [PIA/ANR-16-CONV-0005 ]) and from the  European Union 's  Horizon 2020 research and Innovation Program  under grant agreement  773830  (Project  ARDIG ,  One Health  EJP). Pengdbamba Dieudonné Zongo  is a scholar of Ed525 CDV,  Sorbonne Université, Paris.
The apparent molecular weight of the observed band is given to the left of each row of squares. Data are organized by species (abbreviated to the left of each collection of squares) and by substrate (top). Two sets of four time points (D3, D5, D7, and D10, noted above each column of squares) represent two biological replicates measured for each substrate species combination (See figure on next page. ) (i. e. trypsin, streptavidin) were generally found to have TMT ratios close to 1, indicating that a TMT 127/126 ratio close to 1 is a robust basis on which to exclude background signals (Fig. 3 ). In all cases, the strongest hits from ABP-Cel were putative cellulases or xylanases from families GH7, GH5_5, GH10, and GH12. The detected enzymes represent a majority of the total predicted GH5_5 (85% of the annotated genes across all 10 fungi) and GH7 (83% of annotated genes) cellulases annotated in the genomes of each fungus (Table  1 ), indicating that this method is suitable for the broadly specific detection of core cellulases.
However, such non-specific reactivity is not in line with general epoxide reactivity, which favours cysteine residues  [60]. Larger datasets are needed to explore the significance and consistency of the marginal detections observed in pulldown experiments using ABP-Cel. Comparing the predicted molecular weights (MWs) of proteomic hits with observed bands on SDS-PAGE presents a challenge due to the known tendency of fungi to glycosylate or proteolyse secreted protein and the complexity of the band patterns on each gel. However, we attempted some inference considering both expected correlations between band intensity and spectral count (SC), and between theoretical and apparent MWs. Considering the case of the P. sanguineus wheat straw secretome, we observed minor bands at 25, 32 and 41 kDa and a strong broad band at 49 kDa.
Funding The authors thank the  Natural Sciences and Engineering Research Council of (Post-Doctoral  Fellowship to NGSM), the  Royal Society  ( Ken Murray Research Professorship  to GJD), the  Biotechnology and Biological Sciences Research Council (BBSRC)  (grant  BB/R001162/1  to GJD), the  French National Research Agency  ( ANR-13-BIME-0002  to JGB), the  Netherlands Organization.
YhcN displays structural similarities with RBM domains To get structural insights into the soluble domain of YhcN, we produced a recombinant construct encompassing residues Ala24 to Glu189 (Fig. 3A ) as a His 6 -SUMO fusion (HS-YhcN A24-E189 ) and purified it to homogeneity (see the Methods section). The purified YhcN A24-E189 protein formed needle-shaped crystals (Fig. 3B ) that diffracted to a resolution of 1. 77 Å (see Table  1  for data collection and refinement statistics). To solve this structure by molecular replacement, templates were selected according to structural homology predictions performed through the HHpred server. As none of the closest predicted homologues (including SctJ RBM2 from Salmonella typhimurium, SctJ RBM2 from Escherichia coli EPEC, SpoIIIAH from B. subtilis and MamB from Desulfamplus magnetovallimortis) yielded a structure solution, we performed ab initio phasing.
Ab initio phase determination was achieved by locating three helices of 14 residues using the ARCIMBOLDO_LITE program (http://chango. ibmb. csic. es/) (Sammito et al. , 2013). The high quality of the first model obtained with this strategy allowed building a YhcN model from residue Arg79 to Pro186 but no electron density was observed from Ala24 to Asp78. Mass spectrometry analysis of dissolved crystals provided an experimental molecular weight of 18,703 Da (theoretical value of 18,702 Da), indicating that the N-terminal region of YhcN had not suffered from proteolytic degradation. We therefore deduce that the absence of electron density for the region encompassing residues Ala24 to Asp78, which is not predicted to contain any secondary structure (prediction performed using the Jpred 4 server, https://www.
compbio. dundee. ac. uk/jpred/), is due to flexibility in the crystal. YhcN crystal structure contains a three-stranded β-sheet (β1β2β3) packed against two α-helices (α1α2) on one side and two C-terminal α-helices (α3α4) on the other side (Fig. 3C ). Its overall fold resembles RBMs displaying the αββαβ topology. Consistent with this, the DALI server detected structural similarities between YhcN and RBMs found in inner membrane components of various secretion systems  (Morlot and Rodrigues, 2018; Spreter et al. , 2009). The α1β1β2α2β3 region of YhcN aligns best with the RBM domain of SpoIIIAH from B. subtilis (PDB entry 3UZ0, RMSD (root-mean-square deviation) of 1. 78 Å, 15% sequence identity, 32% sequence similarity over 64 residues) (Fig. 3D ). It superimposes to a lesser extent onto the RBM2 of SctJ from S.
typhimurium (PDB entry 5TCP, RMSD of 2. 41 Å, 10% sequence identity, 30% sequence similarity over 64 residues) (Fig. 3E ), the RBM2 of FliF from S. typhimurium (PDB entry 6SD2, RMSD of 2. 42 Å, 21% sequence identity, 33% sequence similarity over 69 residues) (Fig. S4A ), the RBM2 of SctD from S. typhimurium (PDB entry 5TCP, RMSD of 2. 51 Å, 7% sequence identity, 32% sequence similarity over 63 residues) (Fig. S4B ), the RBM core of B. subtilis SpoIIIAG (PDB entry 5WC3, RMSD of 2. 64 Å, 13% sequence identity, 31% sequence similarity over 64 residues) (Fig. S4C ) and SpoIIIAF (PDB entry 6DCS, RMSD of 3. 89 Å, 7% sequence identity, 21% sequence similarity over 65 residues) (Fig. S4D ). The structural similarities between YhcN and ring-forming domains found in secretion systems suggest that YhcN might be a component of a new transport system, or a ring-forming protein that might have evolved toward a new function (see the Discussion section).
More intriguingly, the DALI server also detected structural similarities between YhcN and the C-terminal metal-binding domain of MamM from Magnetospirillum gryphiswaldense (PDB entry 6H8A, r. m. s. d. of 3. 03   Å, 12% sequence identity, 35% sequence similarity over 695 residues) (Fig. S4E ), which belongs to the family of cation diffusion facilitators that control metal cation homeostasis  (Cotrim et al. , 2019). However, YhcN does not possess a transmembrane transport domain as found in MamM, no metal ion was detected in the crystal structure of YhcN and none of the MamM metal-binding residues are conserved in YhcN. It thus appears unlikely that YhcN is a homologue of MamM C-terminal domain.
After extensive wash with PBS, the slides were mounted using Vectashield antifade mounting medium (Vectorlabs). Samples were observed with a two-deck Olympus IX83 optical microscope equipped with a UPFLN 100X O-2PH/1. 3 objective and an ORCA-Flash4. 0 Digital sCMOS camera from Hamamatsu. Images were acquired using the Volocity software package.
Mass spectrometry analyses Protein samples were denaturated and diluted to 5 µM in 0. 03% TFA. Four µL of cooled samples (10 • C) were first desalted using a reverse phase-C8 cartridge (Zorbax 300SB-C8, 5um, 300 µm ID × 5 mm, Agilent Technologies) and eluted in 67% acetonitrile, 0. 03% TFA, for mass spectrometry detection. Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI-MS) analysis was performed using a 6210 LC/ESI-TOF mass spectrometer interfaced with an HPLC binary pump system (Agilent Technologies). Mass spectra were recorded in the 300-3200 m/z range, in the positive ion mode with spectra in the profile mode. The MS spectra were acquired and the data processed with the MassHunter workstation software (v. B. 02. 00, Agilent Technologies) and with the GPMAW software (v.
NMR analyses Conventional 1 H 1D NMR and 1D HET-SOFAST experiments were performed on a 700 MHz spectrometer (Bruker Avance III HD Liquid) equipped with a triple resonance cryo-probe. Quantitative compactness measurements  (Schanda et al. , 2006)  were obtained recording two 1D HET-SOFAST data sets with and without a band-selective inversion pulse covering the aliphatic region. Data were acquired at 25 • C and processed using the Topspin3. 5 software (Bruker, Inc. ) and the NMRlib library  (Favier and Brutscher, 2019). The time domain data were multiplied with a 90 • phase-shifted sine-bell apodization function and zero-filled to 8192 complex data points prior to Fourier transformation. The reference and saturated intensities were obtained by integrating the spectra from 8. 5 to 9. 5 ppm.
SEC-MALLS analyses SEC-MALLS analyses were performed using ENrich TM SEC650 (Biorad) or Superdex 75 (Pharmacia) 10x300 gel-filtration columns connected to an analytic system including a L2130 pump (Hitachi), an Elite LaChrom L-2400 UV detector (Hitachi), an Optilab T-rEX refractometer (Wyatt technologies) and a DAWN HELEOS-II multi angle laser light scattering detector (Wyatt technologies). The column and the systems were equilibrated in 10 column volumes of 25 mM Tris-HCl pH 8. 0, 150 mM NaCl. Fifty μl of protein samples concentrated at a minimum of 2 mg⋅mL -1 were injected with a flow rate of 0. 5 mL⋅min -1. Protein concentration was quantified online by measuring the differential refractive index and using an averaged refractive index increment dn/dc of 0. 185 mL⋅g -1. Accurate weight-averaged molar mass determination was performed with the Astra 6 software (Wyatt Technologies) and curves were represented with Excel (Microsoft office 2013).
Protein crystallization and X-ray data collection High-throughput crystallization trials were performed with a Cartesian PixSys 4200 crystallization robot (Genomic Solutions, U. K. ). Hanging drops containing 100 nl of protein (48, 24 or 12 mg⋅mL -1 ) and 100 nl of reservoir solution were set up in 96-well Crystal Quick plates (Greiner) and incubated at 20 • C. Initial crystal hits were refined manually by setting up hanging drops containing 1 µL of purified YhcN A24-E189 sample (48 or 24 mg/ml) and 1 µL of reservoir solution in 24-well plates (Molecular Dimensions) incubated at 20 • C. Needleshaped crystals (dimensions of about 20 × 20 × 100 µm) were finally obtained within 2 days in 100 mM HEPES pH 7. 5, 20% (w/v) PEG 8,000. Before flash freezing into liquid nitrogen, crystals were soaked for 5 to 30 sec in a cryo-protecting solution containing 100 mM HEPES pH 7.
5, 21% (w/v) PEG 8,000 and 15% (v/v) glycerol. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France), on the ID30A-3 (MASSIF-3) beamline. The experimental beamline parameters and data quality of the collected images were monitored with MxCuBE  (Gabadinho et al. , 2010). Statistics on data collection and refinement are summarized in Table  1.
Structure determination and refinement Following indexation and integration of the diffraction data using the XDS program suite  (Kabsch, 2010)  (see Table  1 ), ab initio phase determination was achieved using the ARCIMBOLDO_LITE program  (Sammito et al. , 2013)  to combine the location of α-helices with PHASER  (McCoy et al. , 2007)  and density modification with SHELXE  (Thorn and Sheldrick, 2013). Based on secondary structure predictions performed by the JPRED4 server (http://www. compbio. dundee. ac. uk/jpred/), we searched for three α-helices containing 14 residues in coiled mode, using 27 cores of the Cobalt cluster at the CCRT (http://www-hpc. cea. fr/e n/complexe/ccrt. htm. ). This strategy yielded a model of high quality, allowing automated building of the YhcN model (from R79 until P186) using Phenix  (Terwilliger et al.
, 2008). The structure of YhcN was completed by cycles of manual building with COOT  (Emsley and Cowtan, 2004) , addition of water molecules with ARP/wARP  (Langer et al. , 2008)  and refinement with REFMAC  (Murshudov et al. , 2011) , as implemented in the CCP4 program suite. Stereochemical verification was performed with PROCHECK  (Laskowski et al. , 1993) , secondary structures were assigned with DSSP  (Kabsch and Sander, 1983 ) and figures were generated with PyMol (http://www. pymol. org). Coordinates of the final refined model were deposited at the Protein Data Bank (PDB, http://www. rcsb. org) and were assigned PDB entry code.
Acknowledgements We thank members of the  Vernet and Rodrigues laboratories  for advice and encouragement. We thank  Juan Fontecilla-Camps  for discussions regarding the fold of RBM domains,  Jacques-Philippe Colletier  and  Eric Faudry  for advising the thesis work of B. L. , and  Andrea Dessen  for access to the crystallography processing computers. This work is supported by the  French Research Agency  in the framework of the " Investissements d'Avenir" programme to CBH Graduate School  ( ANR-17-EURE-0003 ) and GRAL Labex, by a  China scholarship Council  grant to B. L. , and by grant  DP190100793  awarded to C. M. and C. D. A. R. , from the  Australian Research Council  (https://www. arc. gov. au). This work used the platforms of the Grenoble Instruct-ERIC centre (ISBG;  UMS 3518 CNRS-CEA-UGA-EMBL ) within the  Grenoble Partnership for Structural Biology  (PSB), supported by  FRISBI  ( ANR-10-INBS-05-02 ) and GRAL, financed within the  University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS  ( ANR-17-EURE-0003 ).
, 2022). Now, in eLife, Diego del Alamo, Davide Sala, Hassane Mchaourab and Jens Meiler report how AF2 can also predict different conformations of membrane proteins  (Del Alamo et al. , 2022). To do so, the researchers -based at Vanderbilt University and Leipzig University -used a set of eight membrane proteins representing different structural classes and mechanisms of action. This included five unique transporters (LAT1, ZnT8, MCT1, STP10, and ASCT2), whose structures had been previously experimentally determined in both inward-and outward-facing conformations (Figure  1 ), and three representative G-proteincoupled receptors (CGRPR, PTH1R, and FZD7), whose structures had been solved experimentally in active and inactive states. None of these proteins were part of the original AF2 training set, which included structures located in the Protein Data Bank (PDB).