dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgments We thank  Drs. Tomohiko Sato  and  Mineo Kurokawa  ( The University of Tokyo, Tōkyō, Japan ) for providing plasmid constructs and detailed procedures for Evi1 overexpression in murine hematopoietic cells. We also thank  Dr. Joseph F. Clark  ( University of Cincinnati, Cincinnati, Ohio, USA ) for advice on the use of cyclocreatine in vivo. This research was supported with grants from the  National Cancer Institute  ( NIH   1R35 CA210030-01 ); Stand-upto-Cancer; the  Bridge  Project, a collaboration between The  Koch Institute for Integrative Cancer Research at MIT  and the  Dana-Farber/Harvard Cancer Center  ( DF/HCC ) (KS and MH) and support from the  Cubans Curing Children's Cancers (4C's Fund)  (KS) and from  Koch Institute Cancer Center Support  (grant  P30-CA14051 ,  NCI ) (MH).
AP is a recipient of support from the  ATIP/AVENIR French research program , the  EHA  research grant for Non-Clinical Advanced fellow, and supported by the S t Louis Association for leukemia research, and KS is an  LLS Scholar. AP, NF, and  IBS  were awarded the " Prix Jeune Chercheur " from the  Bettencourt Foundation  and the  Franco-American Exchange Prize  from  Philippe Foundation Inc.
U Vent polymerase; cycling: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 54 °C for 30 s, and 75 °C for 20 s; 75 °C for 5 min. Designation Species Accession Number Target Sequence shCKMT1_1 Homo Sapiens NM 020990. 3 ATCACCCGTGTATGATCCTCC shCKMT1_2 Homo Sapiens NM_020990. 3 TTTACTGGCATCTAGATCCGT shCS_1 Homo Sapiens NM 004077. 2 ATCGCGGATCAGTCTTCCTTA shCS_2 Homo Sapiens NM 004077. 2 AAGAACTGATGTTTCATAGAC shCS_3 Homo Sapiens NM_004077. 2 TGTAACAGCTGCACTGAGCTG shEVI1_1 Homo Sapiens NM 001105077. 31 NM 005241. 3| TTTCTGTGAAGTAAGCATCTT NM 001105078. 3|NM 004991. 3| NM 001163999. 1|NM 001164000. 1 shEVI1_2 Homo Sapiens NM 001105077. 31 NM 005241. 3| TTGCACTTGATTTGGGTTCTG NM 001105078. 3|NM 004991. 3| NM 001163999. 1|NM 001164000. 1 shEVI1_4 Homo Sapiens NM_001105077. 31 TTCCAGTT CAGAATGAGGCGA NM_005241.
Results YEATS2 is an essential gene amplified in NSCLC. To determine whether YEATS2 plays a role in human cancers, we first examined YEATS2 gene expression status across cancers in The Cancer Genome Atlas database via The cBioPortal for Cancer Genomics. As part of the 3q26 amplicon (Supplementary Fig. 1a ), YEATS2 is highly amplified in a variety of human cancers, including lung squamous cell carcinoma (56% amplification frequency), ovarian serous cystadenocarcinoma (27%), and head and neck squamous cell carcinoma (23%) (Fig. 1a ). Importantly, YEATS2 gene expression levels are positively correlated to its amplification status in these tumors (Supplementary Fig. 1b-d ). In human NSCLC and ovarian cancer patients, high YEATS2 expression levels are correlated with worse prognosis (Supplementary Fig.
YEATS2 controls the expression of ribosome protein genes. To determine how YEATS2 regulates cancer cell growth and survival, we performed RNA-seq analysis in YEATS2 KD cells to identify the genes regulated by YEATS2 genome-wide. We used YEATS2targeting shRNA shY2-1 since this shRNA exhibited an efficient KD (Fig. 1c  and Supplementary Fig. 2b ), and we performed RNAseq experiments in duplicates. We identified 1748 genes that were downregulated (false discovery rate (FDR) < 0. 01), whereas 3361 genes upregulated, in YEATS2 KD cells compared with the control cells (Fig. 2a  and Supplementary Data 1, 2). Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes using DAVID (Database for Annotation, Visualization, and Integrated Discovery) revealed that the dysregulated genes were involved in vital biological processes, with downregulated genes enriched in the pathways regulating ribosome biogenesis, DNA replication, cell cycle, DNA repair, and splicing, whereas upregulated genes enriched in the pathways of lysosome functions, glycan degradation, and focal adhesion, etc (Fig.
The HSQC results indicate that the residues surrounding K27ac in the H3 peptide likely also contribute to the interaction between H3 and the YEATS2 YEATS domain. Analysis of the peptide-protein interaction using LIGPLOT program also revealed that the H3 24-31 K27ac peptide is stabilized by a hydrogen bonding network and hydrophobic interactions involving a number of residues including H259, S261, Y262, W282, G283, E284, F285, and Y313 (Supplementary Fig. 4e ). Indeed, alanine mutation of the sandwich pocket residues completely disrupted the binding, highlighting their essential role for H3K27ac recognition (Fig. 3k ; Supplementary Fig. 4f  and Supplementary Table  1 ). Besides, a 2-fold drop of histone P30A mutation demonstrated the requirement of flanking amino acids of H3K27 in mediating the YEATS2-H3K27ac interaction.
The ATAC complex co-localizes with H3K27ac and H3K9ac. The in vitro binding and structural data prompted us to determine whether YEATS2 co-localizes with acetylated histone H3K27 in cells. Extensive attempts to determine YEATS2 genomic distribution using commercial YEATS2 antibodies and tagged ectopic YEATS2 failed. Since YEATS2 is a stoichiometric component of the ATAC complex  12, 13, 15  , we performed chromatin immunoprecipitation (ChIP) experiments followed by high-throughput sequencing (ChIP-seq) using a validated ChIPseq grade antibody raised against another ATAC-specific subunit, ZZZ3  18  to represent the genome-wide distribution of the ATAC complex. High-throughput sequencing of ZZZ3 ChIP experiments performed in duplicate identified 949 confident ZZZ3-bound peaks in H1299 cells (Supplementary Data 4).
The diffraction data set was collected at the beamline BL17U of the Shanghai Synchrotron Radiation Facility at 0. 9791 Å. All diffraction images were indexed, integrated, and merged using HKL2000  48. The structure was determined by molecular replacement using MOLREP  49  with the AF9 complex structure (PDB ID: 4TMP) as the search model. Structural refinement was carried out using PHENIX  50  , and iterative model building was performed with COOT  51. Detailed data collection and refinement statistics are summarized in Table  1. Structural figures were created using the PYMOL (http://www. pymol. org/) or Chimera (http://www. cgl. ucsf. edu/chimera) programs. Isothermal titration calorimetry. All calorimetric experiments of the wild type or mutant YEATS domain proteins were conducted at 15 °C using a MicroCal iTC200 instrument (GE Healthcare).
Gene expressions were calculated following normalization to GAPDH levels using the comparative Ct (cycle threshold) method. Statistic differences were calculated using a two-way unpaired Student's t-test. The primer sequences for qPCR are listed in Supplemental Table  2. RNA-seq samples were sequenced using the Illumina Hiseq 2500, and raw reads were mapped to the human reference genome (hg19) and transcriptome using the RNA-Seq unified mapper. Read counts for each transcript were calculated using HTseq v0. 6. 1 using default parameters  54. Differential gene expression analyses were performed using the "exactTest" function in edgeR v3. 0  55. Gene Ontology analysis was performed using the DAVID Bioinformatics Resource 6. 7  56. The gene expression heatmap was generated using pheatmap package in CRAN (https://cran.
r-project. org/package=pheatmap). The volcano plot was drawn by using ggplot2 package (https://cran. r-project. org/package=ggplot2) in R computing environment. Cell proliferation and colony formation assays. Cell proliferations were determined by counting live cells using hemocytometer cell counter or by CellTiter-Glo luminescent cell viability assay kit (Progema-G7572). For colony formation assays, H1299 cells were seeded in 6-well tissue culture plates (400 cells/well) and grown for 10-14 days. Colonies were fixed with glutaraldehyde (6. 0% v/v), stained with crystal violet (0. 5% w/v) and photographed. Soft agar assays were performed as described below  53. Briefly, cells (1 × 10 4 ) were suspended in 1 ml top agar medium (culture medium supplied with 0. 35% agar). The cell suspensions were then overlaid onto 1.
5 ml bottom agar medium (culture medium supplied with 0. 6% agar) in six-well tissue culture plates in triplicate. Fresh medium was added to plates every 3 days. On day 21, cells were stained with 0. 005% crystal violet blue and photographed. Colony numbers and colony diameters were measured using ImageJ software with size cutoff of 15 μm. Results were quantitated from six views per sample of at least three independent replicates. Flow cytometry cell cycle analysis. Cells were harvested and single cell suspension was prepared at 2 × 10 6 in 1 ml ice-cold PBS buffer. The cell suspension was added dropwise to 9 ml 70% ethanol for fixing. The samples were kept at least 2 h at 4 °C then washed in cold PBS twice. Then the cells were treated with 100 μg/ml RNase A in PBS for 20 mins at room temperature, followed by adding propidium iodide (PI; 50 μg/ml) for staining.
The cell cycle profiling was analyzed by flow cytometry using 488 nm excitation. FITC Annexin V apoptosis assay. Phosphatidylserine (PS) translocation from the inner to the outer leaflet of plasma membrane is one of the earliest apoptotic features. The binding of Annexin V to cell surface PS was detected with a commercially available FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen 556547). Briefly, 1 × 10 5 cells were pelleted, resuspended in 100 μl of Hepesbuffered saline, and FITC-labeled annexin V and PI were added. The cells were incubated 15 min at room temperature, then the samples were transferred to ice and the sample volume brought to 0. 5 ml. Analysis was done by flow cytometry within 1 h. The results were analyzed with FlowJo software. Annexin V positive cells were determined as described in the Kit by setting quadrants to separate viable cells from PI permeant cells, and non-apoptotic cells from those staining highly for the FITC-labeled Annexin V probe.
For ChIP-seq, ChIP experiments were carried out essentially the same as described above. Samples were sequenced using the Illumina Solexa Hiseq 2500. The raw reads were mapped to human reference genome NCBI 37 (hg19) by Solexa data processing pipeline, allowing up to 2 mismatches. The genome ChIP-seq profiles were generated using MACS 1. 3. 6  57  with only unique mapped reads. Clonal reads were automatically removed by MACS. The ChIP-seq profiles were normalized to 10,000,000 total tag numbers, and peaks were called at p ≤ 1e-8. The ChIP-seq heatmap was drawn by the seqplots R package (http://github. com/ przemol/seqplots). Tumor xenograft. All animal studies were in compliance with ethical regulations at the University of Texas MD Anderson Cancer Center. Female athymic nude mice (age 6-8 weeks) were obtained from University of Texas MD Anderson Cancer Center and animals were housed under pathogen-free conditions.
Tumor xenograft assay was performed as described below  53. Briefly, three million YEATS2 knockdown H1299 cells stably expressing control pCDH vector, wild-type YEATS2, Y262A, or W282A mutants were suspended in 100 μl of serum-free RPMI 1640 and injected subcutaneously into the mice. The growth of tumors was monitored twice a week until the largest one reached the limit of tumor burden. Tumor sizes were measured using a caliper and tumor volume was calculated according to the following equation: tumor volume (mm 3 ) = (length (mm) × width 2 (mm 2 )) × 0. 5. Representative data were obtained from all the mice per experimental group. Statistical analyses were performed with one-way repeatedmeasures analysis of variance.
Acknowledgements We thank  J. Heymach ,  J. Minna ,  J. Kurie ,  M. Bedford ,  M. G. Lee , and  X. Lu  for sharing reagents. We thank the M. D. Anderson Sequencing  and  Microarray Facility  and the  Science Park Next-Generation Sequencing Facility  ( CPRIT RP120348 ) for Solexa sequencing. We thank the staff at beamline  BL17U  of the  Shanghai Synchrotron Radiation Facility  and  Dr. S. Fan  at  Tsinghua Center for Structural Biology  for their assistance in data collection and the  China National Center for Protein Sciences Beijing  for providing facility support. We thank  B. Dennehey  for editing the manuscript. This work was supported in part by grants from  NIH/NCI  ( 1R01CA204020-01 ),  Cancer Prevention and Research Institute of Texas  ( RP160237  and  RP140323 ),  Welch Foundation  ( G1719 ), and  Texas Tobacco Settlement  to X.
DOT1L limits Wnt signalling to maintain cartilage homeostasis. To understand the mechanism by which DOT1L controls cartilage homeostasis and to identify effector signalling pathways, we performed genome-wide transcriptome analysis in healthy human articular chondrocytes treated with EPZ-5676 or vehicle (geonr: GSE77916). Microarray results and subsequent qPCR validation confirmed the induction of osteoarthritis-like gene expression changes upon DOT1L blockade (Supplementary Fig. 3a  and Supplementary Table  1 ). Comparative pathway analyses of differentially regulated transcripts after EPZ-5676 treatment revealed an enrichment in genes associated with Wnt signalling (Fig. 2a ). Additional analyses highlighted the diverse regulatory role of DOT1L and further supported the links with skeletal biology and disease (Supplementary Fig.
These mice were further bred to Tg (Col2a1. Cre)o1Bhr4 mice (Jax 003554) to generate conditional cartilage-specific Dot1l knockout mice (Dot1l fl/fl ; Col2-Cre þ / À or Dot1l Cart-KO ). Genotypes of animals were confirmed by PCR on genomic ear DNA. EPZ-5676, XAV-939 and EX527 intra-articular injections. The concentration range for the in vivo administration of DOT1L inhibitor EPZ-5676 was estimated based on reported pharmacokinetics data in mouse  50. From the range that we tested in a pilot study, we selected the minimum effective concentration that showed to inhibit DOT1L methylation (5 mg kg -1 ). DOT1L inhibitor EPZ-5676, Wnt inhibitor XAV-939 (0. 1 and 0. 5 mg kg -1 ) (ref. 51), SIRT1 inhibitor EX527 (1. 25 mg kg -1 ) (ref. 52) or vehicle was injected intra-articularly in the right knee of male 8-week-old wild-type C57Bl/6 mice four times, with an interval of 4 days.
Both cartilage damage and synovial hyperplasia were assessed based on OARSI guidelines  53. Depth of lesion (0-6) was scored on frontal knee sections. Lesion grades represent the following features; 0: surface and cartilage morphology intact, 1: small fibrillations without loss of cartilage, 2: vertical clefts below superficial layer and some loss of surface lamina, 3-6: vertical clefts/erosions to the calcified cartilage extending, 3: less than 25%, 4: 25-50%, 5: 50-75% and 6: more than 75%. The score represents the mean of the tibial scores at the medial and lateral side. For synovial hyperplasia, severity was determined on a 0-3 scale. Scoring was done by two independent readers, blinded to the genotype. Subchondral bone plate histomorphometry. Histomorphometry was performed on Safranin O stained sections using an Olympus IX83 microscope and digital image analysis using Osteomeasure software, as previously described  54.
First, a box with a fixed width (800 mm) and variable height with the upper limit at the transition of calcified cartilage to subchondral bone and the lower limit at the transition submchondral bone to growth plate was created from the digital image (referred to as subchondral bone area). Next, a new box with the upper limit matched to the first box and the lower limit at the transition of the subchondral bone plate to trabecular bone was generated (referred to as subchondral bone plate area). Subchondral bone area and subchondral bone plate area were calculated using the Osteomeasure software. To correct for section artifacts, we do not show the subchondral bone plate surface area per se, but express the subchondral bone plate thickness as a ratio of the subchondral bone plate area to the subchondral bone area.
Melting curve analysis and 1% agarose gel migration of amplicons were performed to determine the specificity of the PCR. Primers used for qPCR analysis are listed in Supplementary Table  3. Microarray hybridization and data acquisition. For the microarray, human articular chondrocytes were obtained from five non-OA hip fracture patients. The cells were treated with 3 mM EPZ-5676 or vehicle control for 4 days. Each sample was divided in two: one half was used for protein extraction and the other half for RNA extraction. Effective DOT1L inhibition by EPZ-5676 was confirmed by Western blot analysis of H3K79 methylation. RNA was isolated with Nucleospin RNA II kit (Macherey-Nagel). RNA concentration and purity were assessed with a NanoDrop Spectrophotometer (NanoDrop Technologies, Centreville, DE, USA) and integrity was determined using RNA nanochips and the Agilent 2100 Bio-analyzer (Agilent Technologies, Diegem, Belgium).
The differentially expressed genes were explored with the limma package. Genes with nominal P values o0. 05 were selected for the enrichment analysis. DAVID version 6. 7 (http://david. abcc. ncifcrf. gov/)  57 or Toppgenesuite (https:// toppgene. cchmc. org)  58 tools were used for gene network analyses including categories KEGG pathway, GO molecular function, GO biological process, human and mouse phenotypes. A minimum gene count of 12 representing 1% of the differentially regulated genes recognized by DAVID was applied in the analysis. PANTHER database version 11. 1 (http://pantherdb. org)  59 was used for graphical representation of functional categories using the 'PANTHER GO-slim' and Protein Class ontology resources. Luciferase assay. Primary human chondrocytes were treated with vehicle or 3 mM EPZ-5676 for 14 days.
Acknowledgements We are grateful to  L. Storms  for her technical support in this study and for, together with  A. Hens , taking care of the animal facility management. We also thank  F. Luyten ,  P. Tylzanowski  and  A. Liston  for critically reading our manuscript, and  Naomi Dirckx  for assistance with digital image analysis. We are indepted to the traumatology and orthopedic surgeons willing to contribute samples ( A. Sermon ,  J. P. Simon  and  S. Nys ) as well as the nursing staff of the surgical theater (in particular  M. Penninckx ). This work was supported by grants from the  Flanders Research Foundation (FWO-Vlaanderen ), by  DevRepair -Belspo   IAPVII-07  and by  Marie-Curie Intra-European postdoctoral  fellowships to S. M. and F. C.
To further support that these global transcriptional effects are directly related to SAGA function, we compared all our datasets with the previously reported "slow growth gene expression signature"  (O'Duibhir et al. , 2014)  and did not observe any relevant correlation (Figure  S7 ). For all deletion strains used in this study, we analyzed the global levels of histone marks directly regulated by SAGA (H3K9ac and H2Bub) or associated with active transcription (H3K4me3 and H3K36me3). As expected, H3K9 acetylation strongly decreased upon GCN5 deletion and to a lesser extent upon depletion of SAGA structural subunits (Figure  6F ). As previously reported, H2Bub levels slightly increased upon SPT7 or SPT20 deletion and they were dramatically increased in either the ubp8Δ or the ubp8Δgcn5Δ strains (Figure  6F )  (Henry et al.
Method Details Yeast strains-Yeast strains for ChEC-seq were generated in the BY4705 background  (Brachmann et al. , 1998). SAGA subunits were endogenously tagged with 3xFLAG-MNase by transformation with gene-specific PCR products derived from pGZ108  (Zentner et al. , 2015). Different yeast strains used in this study were also derived from the previously described FY406  (Hirschhorn et al. , 1995). Deletion mutants were generated as described previously  (Bahler et al. , 1998; Janke et al. , 2004)  or by transformation with PCR products amplified from genomic DNA of the corresponding strain obtained from the Saccharomyces Genome Deletion (SGD) strain database. The HA-TBP strain expressing N-terminal HAtagged version of TBP was obtained by inserting 3HA epitopes preceded by URA3 marker using an integrative plasmid (YIplac211-3HA-TBP)  (Eyboulet et al.
, 2015). A complete and descriptive list of the strains and plasmids used in this study can be found in Supplemental Tables  S1  and S2 , respectively. ChEC-seq-For ChEC analysis, yeast strains were grown in synthetic media to an OD 600 of 0. 5-0. 7. ChEC reactions were performed as described and quenched after 5 min  (Grunberg et al. , 2016). Sequencing library preparation, alignment, track visualization, and cleavage pattern analysis were performed as described  (Grunberg et al. , 2016; Zentner et al. , 2015). The scripts used for data processing are available at https://github. com/zentnerlab/ chec-seq. HOMER (http://homer. salk. edu) (Heinz et al. , 2010)  was used to generate reads per million (RPM)-normalized average plots. TFIID-and SAGA-dependent  (Huisinga and Pugh, 2004)  or TATA-containing or TATA-less genes  (Rhee and Pugh, 2012)  were assigned to previous TSS annotations  (Xu et al.
56µm. Raw data (. CEL Intensity files) were extracted from the scanned images using the Affymetrix GeneChip® Command Console (AGCC) version 4. 1. 2. CEL files were further processed with Affymetrix Expression Console software version 1. 4. 1 to calculate probeset signal intensities, using the Affymetrix statistics-based algorithms MAS-5. 0 with default settings and global scaling as normalization method. The trimmed mean target intensity of each chip was arbitrarily set to 100. All experiments were done using at least two independent biological replicates. Raw data were normalized to S. pombe signal and used to calculate fold-changes in total and newlysynthesized RNA levels, as represented in volcano plots. Further calculations of synthesis and decay rates, based on mathematical model as previously described, were performed using a pipeline and R/Bioconductor package publicly available  (Schwalb et al.
Genes were divided in quintiles according to their expression levels in wild-type cells and the changes in synthesis rates were plotted for each quintile (E,F). Boxes contain genes between the 25 th and the 75 th centiles, the line indicates to the median and the whiskers correspond to 5 th and 95 th centiles. Synthesis rates and decay rates were determined for each S. cerevisiae gene in gcn5Δ (A), spt3Δ (B), ubp8Δgcn5Δ (C) and spt3Δgcn5Δ (D). Changes (calculated as the Log2 of the ratio between mutant and wild-type) in synthesis rates were plotted against changes in decay rates. (E) Box plots summarizing the extent of changes in mRNA synthesis for all the analyzed deletion strains. For each strain, cDTA data were obtained from at least two independent biological replicates. (F) Whole cell extracts from wild-type and the indicated deletion strains were revealed with the antibodies corresponding to histones marks regulated by SAGA (H3K9ac, H2Bub) or associated with active transcription (H3K4me3, H3K36me3) or with antibodies specific to the C-terminal domain of Rpb1 phosphorylated on serine 5 (pSer5 RNA Pol II) or on serine 3 (pSer5 RNA Pol II), as indicated.
Acknowledgements We are grateful to  Violaine Alunni  and  Christelle Thibault  ( IGBMC ) for array hybridization. We thank  Vincent Géli  and  Pierre Luciano  ( CRCM, Marseille, France ) for advices and discussion,  Gabe Zentner  ( Indiana University, USA ) for advice on ChEC-seq data analysis and P. Anthony Weil  for antibodies. We thank  Farrah El Saafin  for critically reading the manuscript. T. B. was supported by a  Marie Curie-ITN  fellowship (EU- FP7 PEOPLE-2013 program , PITN-GA-2013-606806, NR-NET) and  ARC  fellowship. This work was supported by  NIH  grant  GM053451  and  GM075114  (to S. H. ), funds from the  Agence Nationale de la Recherche  ( ANR-15-CE11-0022 SAGA2  to D. D. ;  ANR-13-BSV8-0021-03  DiscoverIID, to L. T. ) and the  European Research Council (ERC)   Advanced grant  ( ERC-2013-340551 ,  Birtoaction , to L.
T. ). This study was also supported by  ANR-10-LABX-0030-INRT , a  French State  fund managed by the  Agence Nationale de la Recherche  under the  frame program Investissements d'Avenir   ANR-10-IDEX-0002-02. N. M. is a student in the  Magistère de Génétique Graduate Program at Université Paris Diderot, Sorbonne, Paris Cité.