dataesr/scientific-paragraphs-categorization huggingface dataset:
We found that F1 and F3 differential peaks are significantly enriched in several GO terms (Fig. 5A ). The common features that we identified in F1 and F3 males were the over-representation of genes associated with transcription factor activities (Isl1, Nkx6-1, Tgif2, Wwc1, Insm1, Tshz3, Trim24) (Fig. 5A  and Supplementary Tables  3  and 4 ) Next, we used ChEA (Chip Enrichment Analysis) to compare the differential regions with published datasets. The ChEA analysis revealed that the targets of POU5F1 and ZFP57 were significantly enriched both in F1 and F3 differential peaks (Fig. 5B ). For example, master regulator of pluripotent genes, POU5F1 has 50 targets in F1 generation (p-value = 2. 09E-06, adj. p-value = 3. 35E-04), out of the 559 targets identified in mouse mesoderm cells  44  (Supplementary Table  5 ).
Red indicates high and blue indicates low expression. genes of ZFP57 with recently available datasets of sperm histone H3K4me3 containing fraction  10. We found that 692 out of 1088 (63. 6%) of all known targets of ZFP57 found in mouse genome retain their histones in sperm (Supplementary Fig. 6A ) and 47. 8% are preserved in paternal histone fraction in 8-cell embryo (Supplementary Fig. 6B )  10. To determine whether Zfp57 is differentially expressed at the time of imprint establishment during embryonic stage, we compared Zfp57 expression in control and CD-exposed E15. 5 testis by RT-qPCR. We found that Zfp57 expression was significantly reduced by 25% compared to control testis (Fig. 1C ). In addition, we asked whether target genes of ZFP57 are differentially expressed in embryonic testes.
To examine the relationship between differential peaks in F1 and F3 generations, we intersected the two datasets. Out of 761 regions, we found 54 peaks (7. 1%) of altered H3K4me3 occupancy in F3 progeny that could be detected in F1 males as well. While the number of overlapping peaks is relatively low, they represented 76 genes with important cellular functions (Supplementary Table  13 ). The common peaks identified have either decreased or increased H3K4me3 occupancies. For example, genes Nkx6-1 and Prkg1 have decreased and increased peak occupancies in both generations, respectively (Fig. 5C ). The overall pattern of differential peak locations relative to TSS is similar in both generations (Supplementary Fig. 9 ). In summary, our data suggest that multiple altered H3K4me3 peaks are found in the F1 generation of CD-exposed males and that a limited number of these changes is preserved in non-exposed F3 generation, with ZFP57 being a strong candidate for mediating the transgenerational inheritance.
We found a significant increase in ESR1 binding in regions of Pigo, Nlrc5 genes. In general, the change in ESR1 binding positively correlates with H3K4me3 occupancy. In aggregate, our data strongly support the hypothesis that CD effects are, at least partially, mediated through altered ESR1 binding. Histone H3K27me3 and H4K5Ac occupancies are altered in F3 males. We also asked whether other epigenetic histone marks are affected upon CD exposure. We performed ChIP-qPCR analysis of H3K27me3 and H4K5Ac using whole testes of F3 males. We chose the common regions with altered H3K4me3 occupancy in both F1 and F3 males and regions with ESR1-binding sites for qPCR target analysis. We found that changes in H3K4me3 occupancies in F3 males have stronger overlap with changes in H3K27me3 when compared to H4K5Ac occupancies (9 out 16 sites were changed in H4K27me3 occupancy (e.
Instead, it was argued that histone marks provide memory of recent transcriptional activity and that marks not coupled with active transcription are involved in transcriptional regulation processes (reviewed in  47  ). Nonetheless, H3K4me3 marks are lineage-specific and are highly conserved in mammals, similar to the binding sites for transcription factors (which are determined by these marks) that are also organized in conserved domains  48, 49. It is, thus, conceivable that in some cases histone marks could inform gene expression. To explore the correlation between histone marks and DEGs, we identified peaks overlapping DEGs. We found that 25 genes out of 377 DEGs were present in both datasets. These genes are associated with cell division (Prim2, Rb1), ATP binding and transcriptional regulation (Nkx6-1, Zfp735) (Supplementary Fig.
Recent studies that examined the dynamics of histone epigenetic marks in early zygotes provided evidence that H3K4me3 proteins transmit the memory of paternal histones  9, 10. The enrichment of targets for ZFP57 in altered peaks in both F1 and F3 generations implicates ZFP57 as a potential key mediator of transgenerational inheritance. ZFP57 is a master regulator involved in the establishment and maintenance of the imprinting marks (  55, 62  ; reviewed in  63  ). ZFP57 can also bind to non-imprinted genes at poised enhancers and can repress cis-acting regulatory elements whose activity has to be silenced during development  64. The genes encoding the adhesion molecules are over-represented in both F1 and F3 datasets. Given the role of cell adhesion molecules in controlling cell division (e.
g. 65 ), it is possible that their increased expression in CD-lineage can hamper this process. The H3K4me3-containing peaks that overlap between F1 and F3 generations are likely to be the best candidates for conferring the epigenetic memory of CD effects even though they represent less than 10% of all altered peaks. This data lends further support to the idea that modifications imposed during developmental exposure to CD could be transmitted to next generations through ZFP57-mediated epigenetic regulation. We suggest that a decrease in ZFP57 (a repressor) could lead to increased expression of its targets (for example, expression of cadherin genes), as observed. However, further work is required to establish whether change in ZFP57 activity directly mediates the transgenerational effects. A ChIP with an anti-ZFP57 antibody could be used to identify its targets in embryonic CD-treated progeny.
The nuclear receptors (RARB and THRB) that are known to cooperate with ESR1 signalling  73, 74  also harbours the binding motifs for ESR1 within differential peaks, suggesting the potential role for these receptors in mediating the CD effects. In summary, our data suggest that the interplay between POU5F1, ZFP57 and ESR1 network is important for transgenerational inheritance of epigenetic changes caused by endocrine disrupting chemicals (e. g. , CD). Does CD promote inheritable changes in germline?. It stands to reason that a large number of altered peaks that we observed are secondary to a limited number of heritable primary changes in the genome, as most of the histone marks disappear during the histone-to-protamine transition. Since altered H3K4me3 peaks are located proximal to the binding sites of important transcription factor (TF) regulators, perturbed TF binding could globally affect the germ cell gene expression program, and eventually, all cells types that originate from SG  75.
A limitation of this study was the use of the whole testis for identifying the epigenetic and expression changes in F1 and F3 generations. While changes in the F3 generation can only arise through the germline, we cannot currently attribute specific F1 changes to any single testis cell type. To better assign and directly relate changes between generations, ChIP in sperm fraction of mouse genome would be required. Sperm contains up to 10% of the histones  5  , and the recent data on human and bovine sperm histone distribution show that sperm contains a limited number of histone-associated promoters including genes associated with RNA and protein processing functions  8. We compared the genes, which are retained in human histone containing fraction and the genes located in our differential peaks.
For example, POU5F1 has fewer targets in F3 compared to F1, suggesting that epigenetic changes are erased with every generation. It is also possible that some of the changes we observed could be accounted for by other epigenetic mechanisms such as noncoding RNA. The analysis of our RNA-seq data showed that expression of several noncoding RNAs was modified (Supplementary Table  16 ) and these (or additional RNA species that we have not examined) could also contribute to the observed phenotype in testis or other organs. It has been shown that sperm contains a large number of noncoding RNAs  76  , which potentially could contribute to transgenerational inheritance. For example, long non-coding RNA 1700001G01Rik which was up-regulated in F3 testis was found in spermatozoa fraction of RNA in a previous study (72).
Animal experimentation. Outbred Swiss mice were used for all experiments. After breeding, the day of vaginal plug detection was considered embryonic day 0. 5 (E0. 5). CD (Santa Cruz, Dallas, Texas 75220, U. S. A) was suspended in olive oil and administered at 100 μg/kg/day in a volume of 150 μl for each mouse. CD was administered via oral gavage to 8-week-old pregnant mice from E6. 5 to E15. 5. The control mice were treated with the same volume of oil. These control and CD-treated mice are called F0. Male progeny of F0 females are called F1 and were crossed with non-littermate and untreated female to give rise to F2 generation. Male progeny of F2 were crossed with non-littermate and untreated female to give rise to F3 generation. Animals from at least three cofounders from independent F0 pregnancies were used for most data points.
Meiotic surface spreads. The surface spreads were prepared in 5 independent experiments from F3 generation control or CD progeny as described in a previous study  79. For synapsing defects analysis, images of at least 30 randomly chosen cells in the pachytene stage of prophase I of meiosis were taken for each sample. We used Chi-squared test to assess the statistical significance. The slides and images were independently reanalysed by two researchers. For DNA repair efficiency analysis, surface spreads cells were co-immunostained against DMC1 and SYCP3 antibodies. Minimum 20 cells in pachytene stages were randomly taken with detectable DMC1 foci and counted. The data were averaged and presented as number of DMC1 foci per cell. We used t-test to assess the statistical significance. RNA extraction, reverse transcription.
RNA-seq library sequencing and data processing. A strand-specific library preparation protocol for RNA sequencing was performed using the sequencing technological platform at IGBMC, Strasbourg, France. We used three biological replicates for these experiments. The sequencing was performed in massive parallel sequencing paired-end mode, and the size of the sequencing tag was 50 bp. Reads in FASTQ format were processed for quality control using the FastQC tool (http://www. bioinformatics. babraham. ac. uk/projects/fastqc/). To analyse differentially expressed genes, the quality-checked reads for each condition were processed using TopHat version 2. 2. 7  80. The reads were mapped to the reference genome [Mus musculus Ensembl mm10 sequence], and the alignment files were generated as BAM files.
These files were used as the input for Cufflinks, a complementary method used to generate assembled transcripts for each condition, and the abundance was evaluated using read data. The assembled transcripts were compared and annotated using Cuffmerge against the Ensembl (mm10) gene annotations  81. These assemblies were used in Cuffquant and Cuffnorm in the Cufflinks 2. 2. 1 package to calculate expression levels. To identify transcripts that were differentially expressed between chlordecone-and control-derived samples, we first selected the cases for which we obtained values greater than the 50th centile of all values in at least one condition, and we then filtered the transcripts for which there was a more than a 2-fold difference between the CD-and control-derived samples. Finally, we used a statistical test implemented in the R package, Limma, with the false-discovery rate set at less than 10%  82.
RNA-Seq data were visualized using the Integrated Genomics Viewer (version 2. 3. 36)  83. Chromatin immunoprecipitation and high-throughput sequencing. Chromatin immunoprecipitation in adult tissue was performed as previously described  84. We used whole testis from one animal for each biological replicate. For embryonic ChIP-qPCR, we pooled 30 testes from E15. 5 of F1 foetuses. The protocol was similar to immunoprecipitation in adult tissue except that chromatin was not dialysed after sonication but instead diluted 6 times with ChIP-dilution buffer. We performed ChIP using antibody against ESR1. The immunoprecipitated DNA was purified, quantitated, and equal amount of ChIP and Input DNA were taken for qPCR. We used as negative control, the region of Gapdh gene located far from TSS. We normalized ESR1 chip data for Gapdh and found the enrichment as ChIP to input ratio for each target gene and for each sample.
Peak calling and identification of differential peaks. Approximately 115 million tags were derived from the anti-H3K4me3 ChIP. The resulting sequences were mapped back to the mouse mm10/Ensembl genome using Bowtie 2. 2. 7 with a seed length of 20. Only tags that passed the quality filter and mapped uniquely to the genome were used. ChIP enrichment was further verified using CHANCE  85. H3K4me3 mark peaks were identified using MACS 2. 1. 1  86  with two biological replicate samples, including the corresponding input, using a shift-size window of 73 bp, no model, and a p-value threshold <10E-5. The number of mapped reads was multiplied by a scale factor to normalize the total number of reads in different samples. To compare the H3K4me3 ChIP datasets of the CD-treated and control samples, differential peak calling was performed using the following steps.
First, from all of the peaks called using the protocol described above, we retained only those peaks with average values above the 50th quintile. Second, we selected peaks with fold changes above 1. 5 or 2 and FDR < 10%. Statistical significance was calculated using a Limma test  82. The annotation of significantly differential peaks was performed using CEAS  87. The results were visualized using the Integrative genomics viewer IGV version 2. 4. 2  83. Gene Ontology (GO) term analyses. Gene Ontology (GO) term analyses were performed using DAVID v6. 7 with default parameters  88. Functional annotation of ChIP-seq data. Functional annotation was performed using GREAT version 3. 0. 0 89 or EnrichR  90, 91  with default parameters. We used ChEA to functionally annotate differential peaks. ChEA is a database of genes potentially regulated by specific transcription factors, where the data were extracted manually from different ChIP-seq experiments for each transcription factor  92.
Acknowledgements The authors would like to thank the  Microarray and Sequencing platform at Strasbourg , a member of the ' France Génomique' consortium  ( ANR-10-INBS-0009 ). We also thank the  GenOuest bioinformatics core facility  for providing computing resources and assistance. We are indebted to  Pierre-Yves Kernanec  for animal care and treatment in addition to help with dissections and  Madina Ahmad Ibrahim  and  Tuyen Ha Nguyen  for help during cell quantification. This work was supported by the  Chair of Excellence programme of the European University of Brittany ,  Atip-Avenir program  ( R13139NS ) from  INSERM  to F. S. C. H. was supported by  Shandong Provincial Natural Science Foundation, China  ( ZR2017LB018 ). L. L. is supported by a fellowship from  Inserm/ARED.
Our data show that CC-mediated dimerisation of TRIM25 is unperturbed in these complexes, as is its ability to synthesise unanchored K63-linked poly-ubiquitin chains. Instead, a comparison with the structure of a TRIM25 CC-PRYSPRY fragment, also reported here, shows that NS1 binding displaces the PRYSPRY domain from the CC, thereby preventing RIG-I ubiquitination. Furthermore, our study suggests that the ability of NS1 to self-associate into higher order oligomers and crosslink different molecules of TRIM25, is important for NS1 function.
2b ). Complex formation between TRIM25-CC and NS1-ED does not induce major conformational changes in either protein and the structures of the bound and unbound domains overlap with RMSD values of 2. 41 Å (for 156 residues) for TRIM25-CC (PDB:4LTB) and 0. 56 Å (for 117 residues) for NS1-ED and apo-NS1-ED (PDB:3O9T) (Supplementary Fig. 1b-d )  32, 37. The biggest difference between apo TRIM25-CC and the NS1-bound state is seen in the linker connecting helix α2 to the central helix α3, which is pushed from its position in the apo TRIM25-CC structure towards helix α1 due to contacts by NS1-ED from interface A. This copy of NS1-ED makes contacts with both TRIM25 chains within the CC dimer via a highly conserved motif of NS1 that includes a short α helix comprising residues 95-99 (Fig. 2c ). Previous mutational analysis identified E96 and E97 in this motif as crucial for TRIM25 binding and inhibition of interferon production  27.
To gain insight into this arrangement we crystallised a complex between TRIM25-CC and the triple mutant NS1-FL (R38A/K41A/W187A) and solved the structure by molecular replacement at 4. 26 Å resolution (R/R free = 0. 27/0. 31) (Table  1 ). Electron density is observed for two TRIM25 CC dimers, one complete NS1-FL dimer and two additional NS1-ED domains (Fig. 3a ; Supplementary Fig. 2a ). This structure is the first complex between full-length NS1 and a target protein, and provides crucial insight into the spatial arrangements of the RBD and ED during target binding. The RBDs of the NS1 dimer form the canonical homodimeric 6helical bundle as observed in the structures of unbound NS1-FL (PDB: 3F5T and 4OPH), the RBD alone (PDB: 1AIL) and an RBD/dsRNA complex (PDB: 2ZKO)  29, 3035, 38. Interestingly, the two NS1 monomers within the homodimer are not symmetrical; while both monomers align well on the NS1-RBD (RMSD of 0.
These results raise the question as to how complex formation with NS1 interferes with the ability of TRIM25 to ubiquitinate RIG-I. To investigate this further, we aimed to gain further structural insight into the functional architecture of TRIM25 by determining the position of the substrate-binding PRYSPRY domain. The structure of TRIM25 CC-PRYSPRY. The structure of a human TRIM25 CC-PRYSPRY construct (189-630) was determined at 3. 6 Å resolution (Table  1 ). The structure was solved by molecular replacement using the CC structure (PDB:4LTB) and a de novo determined structure of the human PRYSPRY domain (residues 434-630) at 2. 0 Å resolution (Supplementary Table  1 ). The human PRYSPRY domain structure is very similar to that of mouse TRIM25 (residues 440-634, PDB:4B8E)  22  with an RMSD of 0.
64 Å for 189 matched Cα atoms and sequence identity of 81. 5% (Supplementary Fig. 3 ). The CC-PRYSPRY structure has one and a half CC dimers in the asymmetric unit with a crystallographic twofold completing the second dimer (Supplementary Fig. 4a-c ). Each bow-shaped CC dimer has two PRYSPRY domains bound towards the ends, maintaining the twofold symmetry (Fig. 4a ). The CC structure with the PRYSPRY is only slightly distorted from that of the CC alone (PDB:4LTB, RMSD 1. 16 Å for 169 aligned Cα). The 73-residue long linker (residues 361-433) connecting the CC and PRYSPRY domain is not visible in the electron density so the exact connectivity is uncertain (Fig. 4a ). The footprint of the PRYSPRY domain on the CC, which corresponds to a total buried surface area of 1433 Å 2 , spans residues 269-287 on helix α1 and residues 320-326 on the extended connection between α2 and α3, all on the same monomer (Fig.
Given that overexpression of NS1 suppresses IFN production in luciferase reporter assays, this implies that unanchored K63 chains are not sufficient to activate the RIG-I/MAVS pathway under these conditions. Comparison of our NS1/TRIM25 structure with the NS1-ED/ p85β structure (PDB: 3L4Q) reveals remarkable similarities in the manner in which the NS1-ED recognises its targets  43, 44. p85β, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) | DOI: 10. 1038/s41467-018-04214-8 | www. nature. com/naturecommunications complex, is a multi-domain protein containing a rigid elongated CC domain (β-iSH2) which is targeted by IAV NS1 to stimulate PI3K activity. Intriguingly, NS1-ED binds β-iSH2 through the same interface observed in our complex with TRIM25-CC, involving the short NS1 α-helix comprising residues 95-99 that packs against the target CC region (Supplementary Fig.
However, structural characterisation of the CC domains of a number of TRIM family proteins have shown that they all form anti-parallel dimers of ~170 Å in length, which based on sequence conservation likely constitutes a general property of this protein family  37  (Supplementary Fig. 8 ). The crystal structure of a CC-PRYSPRY fragment of TRIM20 shows that the two PRYSPRY domains are positioned close to the dimer twofold axis with a centre of mass separation of only ~45 Å (PDB:4CG4). This is a consequence of the short helical linker between the end of the helical CC and the PRYSPRY domain in TRIM20 (Supplementary Fig. 8b ). Even so, the linker can bend leading to some variability in the position of the PRYSPRY domain, which does not itself make any stabilising contact with the CC. In contrast, the TRIM25 linker is much longer, although of variable length and sequence between species, and is unstructured, allowing the PRYSPRY domain to form a transient interaction nearer the CC ends (centre of mass separation ~110 Å) that is important for substrate ubiquitination (Fig.
5, 100 mM NaCl and 500 μM TCEP. TRIM25 coiled-coil-PRY-SPRY was identified as a dimer (elution volume Ve = 12 ml) confirmed by MALLS (data not shown). 1 mM TCEP was added to the sample and the protein was concentrated using an AMICON spin concentrator. Full-length TRIM25 was inserted into pIEX-Bac3 vector (Merck Millipore) with an N-terminal His 10 -tag and an HRV 3 C protease cleavage site using ligation independent cloning (LIC). Protein was expressed in SF9 insect cells (ThermoFisher Scientific 1149015), using a protocol described previously  51. Cells from 2. 1 L of culture were resuspended in lysis buffer (100 mM HEPES, pH 7. 0, 500 mM NaCl, 5% (v/v) glycerol, 1 mM TCEP, 20 mM Imidazole, 10 mM MgCl 2 ), supplemented with 1 mM PMSF, 2 tablets of EDTA-free protease inhibitor cocktail tablets (Roche), and 25 units/ml Benzonase nuclease (Sigma).
Fujita, Kyoto University, Japan) and 25 ng pRL-TK (Promega) using Lipofectamine 2000. Luciferase activity was measured 18-20 h later using the dual-luciferase assay reporter system (Promega). Firefly luciferase values were divided by Renilla luciferase values to normalise for transfection efficiency. All data are shown as fold increase relative to mocktransfected cells. SEC-MALLS. Analytical SEC-coupled to multi angle laser light scattering (MALLS) profiles were recorded at 16 angles using a DAWN-HELEOS-II laser photometer (Wyatt Technology) and differential refractometer (Optilab TrEX) equipped with a Peltier temperature-regulated flow cell maintained at 25 °C (Wyatt Technology). Samples of purified proteins at different concentrations (100 μl volume) were applied to a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.
5, 150 mM NaCl, 0. 5 mM TCEP and 3 mM NaN 3 at a flowrate of 0. 5 ml/min. The data were analysed using ASTRA 6. 1. Binding studies by biolayer interferometry. The binding of NS1 constructs to immobilised TRIM25-CC was measured on an Octet RED biolayer interferometer (Pall ForteBio Corp. , Menlo Park, CA, USA). His-tagged TRIM25-CC was immobilised on Ni-NTA biosensors (Pall ForteBio Corp. , Menlo Park, CA, USA) using a concentration of ~12 μg/ml. The binding of the NS1 constructs (at 1-55 µM) to immobilised TRIM25-CC was measured at 25 °C with a 200 s association step followed by a 200 s dissociation step. The buffer was 50 mM HEPES (pH 7. 5), 150 mM NaCl, 1 mM DTT and 0. 005% Tween-20. The equilibrium dissociation constant was determined by analysing the variation of the response with construct concentration using non-linear least-squares methods and assuming a simple 1:1 binding model.