dataesr/scientific-paragraphs-categorization huggingface dataset:
The Lokiarchaeota are indicated in light-green. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S5 Fig. ML phylogenetic tree of the concatenation of 8 Woese's proteins from the curated datasets (1,582 positions). The 8 proteins correspond to all the Woese's proteins minus the RNA polymerase subunits A'/A" and B. In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Lokiarchaeota are indicated in light-green. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100).
(PDF) S6 Fig. ML phylogenetic tree of the concatenation of 24 eocyte proteins from the curated datasets (4,225 positions). The 24 proteins correspond to all the eocyte proteins minus EF2. In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Lokiarchaeota are indicated in light-green. The scalebar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S7 Fig. ML phylogenetic tree of the concatenation of the 6 AU-relevant Woese's proteins from the curated datasets (1,857 positions). In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively.
For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Lokiarchaeota are indicated in light-green. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The lokiarchaea are indicated in lightgreen, and their position is pointed on the figure. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S20 Fig. ML phylogenetic tree of the concatenation of the 36 arCOGs from the curated datasets (8,367 positions).
The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S22 Fig. ML phylogenetic tree of the concatenation of 35 arCOGs from the curated datasets (all markers except EF2; 7,724 positions). In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Lokiarchaeota are indicated in light-green. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S23 Fig. ML phylogenetic tree of the original concatenated alignment after removal of Lokiarchaeon 3 sequences (36 arCOGs; 10,547 positions).
The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S25 Fig. ML phylogenetic tree of the concatenation of the 36 arCOGs from the curated datasets after removal of Loki 3 EF2 sequence (8,425 positions). In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Lokiarchaeota are indicated in light-green. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100). (PDF) S26 Fig. ML phylogenetic tree of the concatenation of 10 AU-relevant eocyte proteins (all AU-relevant eocyte proteins minus EF2; 2,107 positions).
The Lokiarchaeota are indicated in light-green. Values at nodes indicate the Bayesian posterior probabilities. The scale-bar represents the average number of substitutions per site. (PDF) S28 Fig. ML phylogenetic tree of the concatenation of the 34 arCOGs from the curated datasets present in the two most complete thorarchaeal genomes (8,840 positions). In this tree, bacterial and eukaryotic sequences are indicated in red and blue, respectively. For Archaea, Thaumarchaeota and Aigarchaeota are indicated in pink, Crenarchaeota in orange and Euryarchaeota in olive-green. The Thorarchaea (Candidatus Thorarchaeota archaea) are indicated in black. The scale-bar represents the average number of substitutions per site. Values at nodes represent support calculated by nonparametric bootstrap (out of 100).
(PDF) S29 Fig. Alignment of the region corresponding to the split in the RNA polymerase subunit A protein sequence. Organisms' name corresponding to Bacteria, Lokiarchaea/Thorarchaea, Archaea, and Eukarya are respectively indicated in red, brown, green, and blue. (PDF) S30 Fig. Bayesian inference phylogeny of the concatenation of the two largest RNA polymerase subunits with LG substitution model (Γ4) on the new dataset. The same number (39) of Archaea (green), Eukaryotes (blue) and Bacteria (red) were selected (1,463 positions; see S5 Table for the dataset). Values at nodes indicate the Bayesian posterior probabilities. The scale-bar represents the average number of substitutions per site. (PDF) S31 Fig. Bayesian inference phylogeny of the concatenation of the two largest RNA polymerase subunits with CAT-GTR evolution model (Γ4) on the new dataset.
The same number (39) of Archaea (green), Eukaryotes (blue) and Bacteria (red) were selected (1,463 positions; see S5 Table for the dataset). Values at nodes indicate the Bayesian posterior probabilities. The scale-bar represents the average number of substitutions per site. (PDF) polymerase subunit A (on the left, starting position around 750, on the right around 1200). b. Alignment of the region corresponding to the indel located at the end of the Kae1 protein, with archaeal and eukaryotic sequences. Organisms' names corresponding to Archaea and Eukaryotes are indicated in black and blue, respectively. The archaea presenting an indel are indicated in pink. (PDF) S39 Fig. Comparison of the lokiarchaeal contigs encoding the RNA polymerase subunit B gene in the Loki Castle metagenome assembly and in the Loki 1 genome.
VDC, MG and PF are supported by an  European Research Council (ERC)  grant from the  European Union 's  Seventh Framework Program  (FP/2007-2013)/ Project  EVOMOBIL-ERC Grant  Agreement no. 340440. AN is supported by the  Higher Education Commission, Pakistan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
DNA sequencing was carried out on an ABI 3500 machine at the molecular core facility at the American University of Beirut, followed by analysis using the data collection software from Applied Biosystems Inc. (Foster City, CA). Cell Culture. Human embryonic kidney (HEK) 293 T cells, Hela cells, and JR1 rhabdomyosarcoma cells were maintained in DMEM culture media supplemented with 10% FBS and Penicillin streptomycin (P/S) and 1% Sodium pyruvate and incubated in a humid atmosphere 5% CO2 at 37 °C as previously described  [39] [40] [41]. Immunofluorescence. Hela or JR1 cells were plated onto 12-well Costar culture plates on cover slips with 50,000 cells per well. Transient transfection was done on the second day of the seeding by polyethylenimine (PEI, Sigma) with a total of 4 μg of DNA per well.
Docking. Protein-thalidomide docking simulations were performed using the default settings in Autodock-Vina  44. Thalidomide 3D structure was docked to the T-box domain of TBX5 (pdbid: 2X6V) and TBX3 (pdbid: 1H6F) respectively, and the best docking poses were selected for further investigation. The program MODELLER  45  was used to model the 3D structure of HAND2 bHLH domain based on the structure of the Scl:E47 bHLH Heterodimer bound to DNA (pdbid: 2YPB), and GATA4 DNA binding domain based on the crystal structure of GATA transcription factor-complex 3 (pdbid: 4HC9). Protein-protein docking simulations were submitted to the online webserver ClusPro (https://cluspro. bu. edu/login. php), a versatile protein-protein docking engine based on the FFT (Fast Fourier transformation). The engine outputs ranked models based on their corresponding cluster size and those with balanced coefficients were retained since there exist no prior knowledge of what forces prevail in the model  46.
Statistics. Significance (*P < 0. 05) for Luciferase, Gel Shift, and co-immuprecipitation assays was assessed using the one-way ANOVA test. The likelihood of the p. G202V minor allele to be associated with congenital pathologies (e. g Tetralogy of Fallot) was assessed using family linkage analysis. We organized and analyzed our data using a parental transmission desiquilibrium test which incorporates phenotypes from both parents and offspring within and between nuclear families, a method initially described by Purcell et al. , and applied in familiar based genetic association studies  47.
Results Because of the similarities between the phenotypes in patients with HOS and those suffering from Thalidomide toxicity, we hypothesized that Thalidomide could directly bind to TBX5. The T-Box domain is the interface of the TBX5/Thalidomide interaction. Docking simulations using Autodock-Vina revealed a strong interaction between the T-box domain of TBX5 (pdb:2X6V) and Thalidomide. The top ranking pose was selected based on the best binding energy of ~7. 5 kcal/mol (Fig. 1 ). Interestingly this interaction holds with TBX3 (pdbid:1H6F), a related member of the T-BOX family of transcription factors (Supplementary Figure  1 ). Although this interaction showed a similar energy pattern, the interacting residues do not involve those cooperating with DNA as in TBX5, which could suggest that the binding of thalidomide to TBX3 does not perturb its interaction with DNA.
5 , and Supplementary Figure  3 ). The small indexed family (Fig. 5 ) was part of a large family with many consanguineous marriages and multiple CHDs and coronary artery diseases (CAD) (Supplementary Figure  2 ). We were able to recruit 51 individuals from this family and we genotyped all of them for this variant. In addition to the cases mentioned previously, the variant was observed in 4 patients who suffered early severe myocardial infarct that lead to death in their mid-40's (Supplementary Figure  3 ). It was also found in 6 out of 39 healthy individuals within the extended members of this family (Supplementary Table  1 ). This variant was however, neither found in healthy individuals in the ExAC exome nor in the 1000 genome databases, nor in our cohort of 300 patients with CHDs and 500 healthy controls, suggesting it could have a deleterious effect on protein function (Fig.
Membrane stripping and subsequent western blot analysis was performed with anti-HA antibody in order to detect TBX5 proteins. Quantitation of the bands showed more than 80% decrease in the interaction when the extracts were incubated in the presence of Thalidomide. (Ctr: control, WB: immunoblot, IP: immnuoprecipitation). explain the phenotypes observed in children who suffered from Thalidomide toxicity at the level of the heart and limbs whereby the phenotypes are strikingly similar to patients with HOS harboring mutations in TBX5. The fact that the in silico docking data showed less affinities for Thalidomide to TBX3 versus TBX5, though both share the same docking interface to DNA  50  , argues for a differential effect of Thalidomide on various T-box proteins which would explain the tissue-specific pattern observed in children who suffered from its effect on their mothers.
This is corroborated by the recent finding in The transcriptional activity of HAND2 was assessed in co-transfection assays using the rat NPPA promoter coupled to luciferase which showed a drastic decrease in the p. G202V mutant ability to activate its target (A). Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0. 05) was assessed using the one-way ANOVA test. HA-tagged TBX5 and Flag-tagged HAND2 (WT and G202V) proteins were extracted from transiently transfected HEK293 cells. Nuclear lysates for both HAND2 and TBX5 proteins were immune-precipitated with anti-HA antibody, and visualized with western blot via anti-flag antibody. Membrane stripping and subsequent western blot analysis was performed with anti-HA antibody in order to detect TBX5 proteins.
Ten times the quantity of proteins loaded for western blot was used for immune-precipitation. Quantitation of the bands shows up to 80% inhibition of the interaction when the HAND2 mutated protein is used instead of the wild type protein (B). Transcriptional activity was assessed by luciferase assay (C,D). TBX5 and HAND2 (WT or G202V) were transiently co-transfected with the rat NPPA promoter coupled to luciferase in HEK293 cells (C), then in the presence of Thalidomide (D). Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0. 05) was assessed using the one-way ANOVA test. (+) is for 300 ng of DNA, and (++) for 700 ng of DNA. The increasing doses start at 100 reaching up to 500 ng.
TBX5 showed nuclear localization (green color) in both conditions. (Magnification X40). Same results were obtained for Hela and Hek293 cells. Transcriptional activity was assessed by luciferase assay. Increasing doses of TBX5 (100 to 500 ng) were transiently co-transfected with the rat NPPA (C), or mouse VEGF (D) promoters coupled to luciferase in HEK293 cells. Treatment with thalidomide (4 µM) or its equivalent amount of DMSO was done after 24 hours, and cells were harvested for luciferase assay. Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0. 05) was assessed using the one-way ANOVA test.
Membrane stripping and subsequent western blot analysis was performed with anti-HA antibody in order to detect TBX5 proteins. Ten times the quantity of proteins loaded for western blot was used for immune-precipitation. Quantitation of the bands shows up to 80% inhibition of the interaction when the HAND2 mutated protein is used instead of the wild type protein (B). Transcriptional activity was assessed by luciferase assay (C,D). TBX5 and HAND2 (WT or G202V) were transiently co-transfected with the rat NPPA promoter coupled to luciferase in HEK293 cells (C), then in the presence of Thalidomide (D). Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0. 05) was assessed using the one-way ANOVA test.
Acknowledgements We thank all members of the study family for their contribution to this research. We also thank members of the  Congenital Heart Disease Genetic Program (CHDGP)  especially  Drs Nadia Jradi ,  Raya Safa ,  Akl Fahed , and  Rabih Andary  for their help in recruiting the family, and  Ms. Inaam El-Rassy  from the  Molecular Core Facility at AUB  for her technical assistance in Sanger Sequencing. This work is supported by a grant from  CEDRE  and the  University Research Board  and the  Medical Practice Plan at the American University of Beirut.
4) (23) (bw = 250, mfold = 10 and P value = 1e-5). Peaks common to the 10 operations with FDR<0. 05 were kept for further analysis. Peaks were assigned to neighbouring genes using the PAVIS annotation tool (http://manticore. niehs. nih. gov/pavis2/) with the UCSC annotation for mouse mm9 and the Ensembl annotation UMD3. 1r82 for bovine Bostau6. For annotation of the bovine peaks using the mouse genome as reference, bovine coordinates were translated to mouse mm9 coordinates, as previously described  (24) , using the UCSC LiftOver tool. Coordinates translated to mm9 where then annotated using PAVIS. The correspondence of orthologous genes between mouse and bovine were analyzed using BIOMART (http://www. ensembl. org/ biomart/martview/5761d5751336b65a1461f18d849facfe). Pathway analysis was performed using the MetaCore™ GeneGo software to map genes to a global database of known networks (https://portal.
RNA-seq library preparation, sequencing and analysis Libraries were generated using the NuGEN Mondrian Technology and the SPIA amplification methodology that specifically selects and linearly reverse transcribes nonribosomal RNA. 6pM of bar-coded libraries per lane were sequenced using the Illumina HiSeq1500 and paired-end RNA sequencing chemistry (150nt read lengths at 50 million reads per sample). All datasets were processed with the RNA-seq pipeline (http://dx. doi. org/10. 5281/zenodo. 47479) as follows. Sequencing reads were quality-filtered and adapters were removed using Trimmomatic (version 0. 30)  (28). Reads shorter than 36 bp were discarded from subsequent analyses and the remaining trimmed reads were mapped to the Mus musculus genome (Ensembl version 38. 77) using Tophat2 (version 2.
Differentially expressed genes were examined with an adjusted p value cut-off of 0. 05. For alternative splicing analysis, RNA-seq datasets were analyzed independently as follows. Paired-end raw reads were cleaned of sequencing contaminants with Scythe (https://github. com/vsbuffalo/scythe. ) and quality trimmed with Sickle (https://github. com/najoshi/sickle) to keep only reads with a minimum length of 21nt. Mouse genomic sequences (mm10) and annotations (Ensembl 75) were retrieved from the UCSC genome browser. Alignments to the mouse genome were performed with STAR v2. 4. 1c  (36)  and quantification of Ensembl isoforms with Salmon v0. 3. 0 (http://biorxiv. org/content/early/2015/10/03/021592). Both programs ran with default read-matching parameters. Differential expression was calculated between pairs of conditions using one-way ANOVA and t-tests on square-root transformed TPM data of the three replicates  (37).
Conversely, direct annotation using the BosTau6 genome assembly indicated that 62% of bovine peaks were outside the gene body (48% upstream of the TSS and 14% downstream of the TES) (Figure  1A , middle panel). However, when using the mouse genome as reference after translation of the bovine coordinates, the bovine and mouse peak distributions were comparable (Figure  1A , right panel). Cross-species comparison  (24)  of the regions bound by SOX9 in bovine and mouse foetal testes, identified 4293 peaks that were common to both ChIP-seq datasets, 75% of which (3236 peaks) overlapped by >500 bp (Figure  1B ). From these 4293 peaks, 3849 neighboring genes were annotated that showed strict orthology using Biomart. This suggests that SOX9 binds to and regulate a similar core set of genes in two evolutionarily distant mammals.
Some of these genes (for instance, Col27a1/COL27A1  (48) , Fgf9/FGF9  (49)  and Sox10/SOX10  (10) ) displayed striking similarity of peak location in the mouse and bovine ChIP-seq datasets. Conversely, the peak profile of Lmo4/LMO4 (50) was relatively different between species. Gene Ontology (GO) term enrichment analysis (Supplementary Table  S1 : Sheets GO) of genes bound by SOX9 showed that the male sex differentiation pathway was enriched in the mouse (P = 3. 454 × 10 -7 ), bovine (P = 2. 972 × 10 -7 ) and the combined mouse and bovine gene sets (P = 3. 811 × 10 -6 ). Moreover, other enriched pathways, such as FGF, NOTCH, Hedgehog and WNT signalling cascades, also contribute to male sex determination and gonad function. Most genes that have been involved in mammalian gonad determination and differentiation by genetic or functional studies were identified here as SOX9 targets (Table  1 ).
Transcriptional regulation by SOX9 in foetal testis at E13. 5 Comparison of the mouse SOX9 ChIP-seq dataset with previously published transcriptomic datasets from male and female supporting cell lineages (Sertoli and granulosa cells, respectively) in mouse E13. 5 gonads (64) enabled us to identify candidate genes that could be up-or down-regulated by SOX9 cell autonomously at this stage. Specifically, 38% (557 out of 1451) of genes that were upregulated and 44% (539 out of 1225) of genes that were repressed in Sertoli cells compared with granulosa cells at E13. 5 were bound by SOX9 (Figure  2A ; see also Supplementary Table  S1 : Sheet 'ChIP-seq mouse mm9'). Then, to better understand SOX9 transcriptional function in foetal mouse testes, we analysed by RNA-seq the transcriptome of E13. 5 gonads in which SOX9 was present or not.
Sox9 conditional knock-out in differentiating Sertoli cells was performed soon after testis commitment by crossing mice carrying the Sox9 allele (Sox9 flox ) (65) with mice in which the Cre recombinase was controlled by the Amh gene promoter  (66). In this context, a mild phenotype is observed and the effect on E13. 5 gonad transcriptome can be essentially attributed to transcriptional mis-regulation in Sertoli cells due to Sox9 loss  (67). Sox9 mRNA quantification in the RNA-seq datasets obtained using E13. 5 wild type (WT) XY and XX gonads and XY mutant gonads (subsequently named XY Sox9 / or KO in figures) showed that Sox9 transcript levels were comparable in Sox9 / XY and XX gonads (Supplementary Figure  S2A ), as previously reported  (67). Detailed analysis of the Sox9 / testis transcriptome showed the downregulation of 240 mRNAs and the upregulation of 107 transcripts compared with WT XY testes Amh /AMH 1 1 0 /1 Atrx/ATRX 1 1 1 /1 Bmp2/BMP2 1 2 1 /2 Cbx2/CBX2 1 1 1 /1 Cited2/CITED2 1 0 - Ctnnb1/CTNNB1 (ß Catenin) 1 0 - Cyp26b1/CYP26B1 0 3 - Dhh/DHH 1 1 0 /1 Dmrt1/DMRT1 1 0 - Emx2/EMX2 1 3 1 /3 Fgf9/FGF9 2 3 2 /3 Fgfr2/FGFR2 1 1 0 /1 FoxL2/FOXL2 2 2 0 /1 Fst/FST 4 1 1 /1 Gadd45g/GADD45G 1 1 1 /1 Gata4/GATA4 3 3 1 /3 Hhat/HHAT 1 1 0 /1 Igf1r/IGF1R 1 3 0 /3 Insr/INSR 1 2 1 /2 Kdm3a/KDM3A (Jmjd1a/JMJD1A) 1 1 0 /1 Lhx9/LHX9 2 5 3 /5 Lmo4/LMO4 2 3 2 /3 Map3K4/MAP3K4 0 0 - Notch1/NOTCH1 1 0 - Notch2/NOTCH2 1 1 0 /1 Notch3/NOTCH3 0 2 - Nr0b1/NR0B1(Dax1/DAX1) 1 1 1 /1 Nr5a1/NR5A1 (Sf-1/SF-1) 2 1 0 /1 Pdgfa/PDGFA 2 0 - Ptgds/PTGDS 0 1 - Rspo1/RSPO1 0 0 - Six1/SIX1 1 2 1 /1 Six4/SIX4 1 1 0 /1 Sox8/SOX8 0 1 - Sox9/SOX9 2 3 2 /3 Sox10/SOX10 1 1 1 /1 Sry/SRY 0 0 - Tcf21/TCF21 (Pod1) 1 4 0 /4 Wnt4/WNT4 1 0 - Wt1/WT1 1 2 0 /2 Wwox/WWOX 1 4 0 /4 Zfpm2/ZFPM2(Fog2/FOG2) 1 5 2 /5 The number of peaks per gene are shown in both species.
This makes of them strong candidates for positive and direct transcriptional regulation by SOX9. Comparison of the expression level of these 65 genes in the WT XY, Sox9 / XY and WT XX gonad datasets allowed their classification into three groups. The first group represented genes whose expression in the XY mutant was intermediate between WT XY and XX gonads (Figure  2C , red circles), suggesting that in the absence of SOX9, these genes are still partially active. This group included genes with an important role in sexual differentiation, such as Amh (encoding anti-M üllerian hormone) (68), Sox10  (10)  or Dhh (69) (Supplementary Figure  S2B , upper panels). The second group (Figure  2C , yellow squares) included genes that were similarly expressed in XY Sox9 / and WT XX gonads (e. g. Rnd2, Mybl1 or Gjb2) (Supplementary Figure  S2B , middle panels).