Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.Cardiac glycosides are approved for the treatment of heart failure as NaProscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3  and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.The online version of this article (10.1186/s13046-019-1242-8) contains supplementary material, which is available to authorized users. MYC (c-MYC) transcription factor is a driver of oncogenic programs. It contributes to gene deregulation in cancer by promoting expression of genes involved in cell proliferation . High MYMYC stability is regulated by post-translational modifications and MYC acetylation increases its stability . The dep+/K+ pump inhibitors and approved for heart failure treatment, exhibit significant epigenetic and anticancer effects . A nM Fig. d. LikewiThe repurposing potential of cardiac glycosides in oncology has been suggested several decades ago and is currently under intense clinical investigation either alone  or in combination with chemotherapy  . HoweverProscillaridin A induced a rapid loss of MYC protein expression in MYC-driven leukemia cells through the downregulation of series of KATs  known to be involved in MYC protein stability (by lysine acetylation) and activation of MYC transcriptional programs –41, 51. Proscillaridin A treatment also resulted in a significant loss of H3 acetylation levels in MYC overexpressing cells. This effect was associated with the loss of epigenetic active marks (including H3K27ac) in promoters of MYC-dependent genes associated with cell proliferation programs. Interestingly, MYC-driven leukemic cells started to differentiate during treatment and after drug removal, suggesting the onset of a stable epigenetic reprogramming. The contribution of global histone acetylation loss to the anticancer effects of proscillaridin A remains to be better understood since loss of histone acetylation was observed in some proscillaridin A-resistant cells, suggesting that loss of H3 acetylation may not be sufficient to modulate cell viability in these low MYC expressing cells.Here, we demonstrated that proscillaridin A treatment in MYC overexpressing leukemia cells, led to the downregulation of several KATs, induced significant acetylation loss, produced MYC degradation, and induced persistent epigenetic effects and leukemia cell differentiation, which was maintained for several days after drug removal. Therefore, this study supports a strategy of simultaneously targeting several KATs to reduce efficiently acetylation in histone and in non-histone proteins, to overcome the redundant activity of KATs. The mechanism implicated in proscillaridin A-induced KATs downregulation is under investigation. Overall, we conclude that proscillaridin A has a promising potential to be repurposed as an epigenetic drug in personalized oncology, particularly in MYC overexpressing leukemia.Additional file 1:Supplementary Materials and Methods. (DOCX 24 kb)Additional file 2:Figure S1. MYC Expression Correlates with Proscillaridin A Anticancer Efficacy. A Upper panel, half maximal inhibitory concentration (IC50) after a 24h proscillaridin A treatment (ranging from 1 nM to 100 μM) in a panel of human cancer cell lines (n=4). Lower panel, MYC protein level in each untreated cell line, assessed by western blotting. ACTIN was used as a loading control (n ≥ 3). B Graph showing MYC expression (relative to ACTIN) compared to proscillaridin A IC50 (24h) in 14 cancer cell lines. Correlation was evaluated by linear regression analysis; P-value is shown on the graph (n=3). C Representative pictures of transformed primary human fibroblasts before and after transduction with RASV12, MYC and RASV12/MYC were taken by light microscopy (400X magnification). D and E MYC expression was assessed by Western blotting in WT and MYC-transduced MOLT-4 cells (D) and REH cells (E). MYC expression was calculated as a ratio over ACTIN levels . IC50 values after 24h proscillaridin A treatment (ranging from 0.1 nM to 1 μM) in MOLT-4 cells (D) and REH cells (E) (n ≥ 3). F Time course experiment in NALM-6 cells treated with 5 nM for up to 96h. MYC expression was calculated as a ratio over ACTIN levels . (PDF 1640 kb)Additional file 3:Figure S2. Transcriptomic Analysis In MOLT-4 Cells Treated with Proscillaridin A . A Heat map representing RPKM similarities between triplicates of untreated (U) and Proscillaridin A-treated  MOLT-4 cells (n = 3). Red color corresponds to the highest similarity and yellow corresponds to the lowest similarity. B Proscillaridin A  induced gene expression reprogramming of MOLT-4 cells. Volcano plots of gene expression changes in MOLT-4 cells in untreated versus treated samples. Black dots correspond to genes with P-value adjusted > 0.5. Grey dots correspond to genes with P-value adjusted < 0.5 but without significant fold change expression difference between untreated and treated cells (-0.5 < FC < 1). Downregulated genes with P-value adjusted < 0.5 and FC < -0.5 are shown in green. Upregulated genes with P-value adjusted < 0.5 and FC > 1 are shown in red. Numbers of downregulated and upregulated genes are shown on the graphs. C Metascape analysis of genes downregulated by proscillaridin A treatment . D Cell cycle analysis after BrdU staining in MOLT-4 and NALM-6 cell lines exposed to proscillaridin A . Cell fluorescence was measured by flow cytometry . E Metascape analysis of genes upregulated by proscillaridin A treatment . (PDF 905 kb)Additional file 4:Figure S3. Proscillaridin A Induced Histone 3 Acetylation Loss In MOLT-4 And NALM-6 Cells. A MOLT-4 cells were treated with proscillaridin A (5 nM) and histones were acid-extracted after 8, 16, 24, 48, 72 and 96 hours. H3 acetylation levels were quantified and expressed as a percentage of untreated cells . B Ratio of chromatin immunoprecipitation (ChIP) of H3K27 acetylation in MOLT-4 cells before and after proscillaridin A treatment  . C NALM-6 cells were treated with proscillaridin A (5 nM) and histones were acid-extracted after 8, 16, 24, 48, 72 and 96 hours. H3 acetylation levels were quantified and expressed as a percentage of untreated cells . D MOLT-4 and E NALM-6 cells were treated with proscillaridin A (5 nM) and histones were acid-extracted after 8, 16, 24, 48, 72 and 96 hours. Histone 4 acetylation levels were assessed using antibodies against K5ac, K8ac, K16ac, K20ac, and total histone 4 acetylation. H4 was used as loading control. H4 acetylation levels were quantified and expressed as a percentage of untreated cells . (PDF 567 kb)Additional file 5:Figure S4. Histone Methylation Is Not Significantly Altered After Proscillaridin A Treatment On Histone H3. MOLT-4 (A) and NALM-6 (B) cells were treated with proscillaridin A (5 nM) and histones were acid-extracted after 8, 16, 24, 48, 72 and 96 hours. Histone 3 methylation levels were assessed using antibodies against K4me3, K9me3, and K27me3. H3 was used as loading control. H3 methylation levels were quantified and expressed as a percentage of untreated cells . C Confocal microscopy (60X) of MOLT-4 cells stained with DAPI revealed heterochromatin modulation after proscillaridin A treatment . White arrows indicate loss of heterochromatin regions. (PDF 1592 kb)Additional file 6:Figure S5. H3K27 Acetylation DNA Occupancy Is Lost After Proscillaridin A Treatment In MOLT-4 Cells. Metascape analysis of A downregulated genes and B upregulated genes after proscillaridin A treatment  marked by H3K27ac in their promoter regions (-500 bp / +500 bp). (PDF 657 kb)Additional file 7:Figure S6. Proscillaridin A Treatment Downregulated MYC Target Genes That Are Marked By H3K27ac In Promoter Regions. Map of A co-expression pathways and B protein-protein physical interactions of MYC target genes marked by H3K27ac in untreated MOLT-4 cells. (PDF 1418 kb)Additional file 8:Figure S7. Loss of Acetylation In MYC Protein And MYC After Proscillaridin A Treatment In High MYC Expressing Cells. A Mass spectrometry analysis on 2 MYC peptides (LVSEK(ac)LASYQAAR) after proscillaridin A treatment  in MOLT-4. Log2 normalized intensity is shown . B Map of co-expression pathways of the 28 proteins that lost acetylation after proscillaridin A treatment  in MOLT-4 cells. (PDF 890 kb)Additional file 9:Figure S8. MYC Inhibition Induced By Proscillaridin A Is Regulated By KAT Activities. A MOLT-4 cells were treated with proscillaridin A (5 nM) and KAT3A, KAT3B, KAT5, KAT2A, KAT2B, KAT6A and KAT7 expression levels were assessed by western blotting. ACTIN was used as loading control. B KAT2B and KAT7 expression levels were quantified and expressed as percentage of untreated cells (n=3). C and D Class I HDAC (C) and KAT (D) expression transcripts (RPKM) expression after proscillaridin A treatment  in RNA-sequencing data set (*indicates Log2 FC<-0.5 and $ indicates Log2 FC > 1). E MOLT-4 cells were treated with KAT3B/A inhibitor C646 (10 μM) and with proscillaridin A (5 nM) and histones were acid-extracted after 48 hours. Histone 3 acetylation levels were assessed using antibodies against K14ac, K18ac, K27ac, and pan histone 3 acetylation. H3 was used as loading control. F Left panel, MOLT-4 cells were treated with KAT3B/A inhibitor C646 (10 μM) and with Proscillaridin A (5 nM) and KAT5, KAT3A and KAT3B expression levels were assessed by western blotting. ACTIN was used as loading control. Right panel, KAT5, KAT3A and KAT3B levels were quantified and expressed as a percentage of untreated cells . (PDF 1077 kb)Additional file 10:Figure S9. Proscillaridin A Induces KAT Downregulation Specifically In High MYC Expressing Cells. A-C MOLT-4, NALM-6, SW48 and A549 cell lines were treated with proscillaridin A  and fibroblasts transduced with RASV12, MYC and RASV12/MYC were treated with proscillaridin A . A KAT3A (CBP), KAT3B (P300), KAT5 (TIP60), KAT2A (GCN5), KAT2B (PCAF), KAT6A (MOZ) and KAT7 (HBO1) expression levels were assessed by western blotting. ACTIN was used as loading control. B Histone 3 acetylation levels were assessed by using antibodies against K9ac, K14ac, K18ac, K27ac, and pan histone 3 acetylation. H3 total was used as loading control. C Histone 3 acetylation levels were quantified and expressed as percentage of control . (PDF 2285 kb)


COL1A1 and COL1A2. It has been well established to identify the classical variants as well as consensus splicing-site-variants in these genes in our previous studies. However, how atypical variants affect splicing in OI patients remains unclear. From a cohort of 867 OI patients, we collected blood samples from 34 probands which contain 29 variants that are located close to splice donor/acceptor sites in either COL1A1 or COL1A2. By conducting minigene assay and sequencing analysis, we found that 17 out of 29 variants led to aberrant splicing effects, while no remarkable aberrant splicing effect was observed in the remaining 12 variants. Among the 17 variants that affect splicing, 14 variants led to single splicing influence: 9 led to exon skipping, 2 resulted in truncated exon, and 3 caused intron retention. There were three complicated cases showing more than one mutant transcript caused by recognition of several different splice sites. This functional study expands our knowledge of atypical splicing variants, and emphasizes the importance of clarifying the splicing effect for variants near exon/intron boundaries in OI.Osteogenesis Imperfecta (OI) is a rare inherited bone dysplasia, which is mainly caused by mutations in genes encoding type I collagen including  Osteogenesis imperfecta (OI), also known as brittle bone disease, is an inherited skeletal dysplasia characterized by frequent fractures, blue sclerae, bone deformity, and relaxation of skin and ligament. OI is considered as a rare bone disease and its prevalence is reported to be 1 in 15,000 live births . Based oCOL1A1 (MIM# 120150) and COL1A2 (MIM# 120160)  . Mutatiod MBTPS2 . NeverthTypical mutation spectrum of OI includes missense, nonsense, frameshift, and splice site mutations. Despite of these classical mutations, it was shown that a large portion of DNA variants disrupted splicing in cancer-related diseases . HoweverCOL1A1/COL1A2 in peripheral blood, RNAs from the tissue of OI patients would be ideal for examining whether the variants can affect RNA splicing. However, the availability of the tissue of OI patients is limited. Therefore, a minigene assay, which is based on patients’ genomic DNA, represents a valid and powerful approach to study the splicing pattern . The genomic DNA and cDNA sequences of COL1A1 (NC_000017.11) and COL1A2 (NC_000007.14) were obtained from National Center for Biotechnology Information (NCBI) reference sequence and University of California, Santa Cruz (UCSC) Genome browser database (http://genome.ucsc.edu/). The altered proteins were named based on the sequencing of mutant transcripts.The variants of COL1A1 or COL1A2 variants close to the exon/intron boundaries were enrolled for minigene splicing assay. All variants identified in this study have been submitted to the Osteogenesis Imperfecta Variant Database (http://oi.gene.le.ac.uk/). A total number of 867 patients (from 489 families) diagnosed as OI were recruited for this study from 2014 to 2018. Information of their phenotypes, including number of fractures, blue sclerae, affected skeletal location, and bone deformity were recorded after obtaining patients’ informed consent. Tissue samples, including peripheral blood and/or skin, were collected to detect the variants. After sequence analysis, 34 probands from different families carrying Online software ESE Finder 3.0 and Human Splicing Finder (version 3.1) were used to predict the splicing effect of each of the variants. Analysis of ESE Finder was performed to detect exonic splicing enhancers for SR proteins as well as alterations in splice sites. SRProteins matrix library was used to analyze the variants located in exons and SpliceSites matrix library was used for variants in introns. All analyses were performed with default threshold values.Genomic DNA was extracted from the peripheral blood, and 1–3 μg genomic DNA was used for WES as described previously . SequencCOL1A1 and COL1A2 after WES, and to verify the splicing variants after minigene assay. The process was described previously (http://primer3.ut.ee/). Sequencing was conducted in Applied Biosystems 3730xl DNA Analyzer . Result of Sanger sequencing was analyzed using CodonCode Aligner . The sequence results were aligned to reference sequences COL1A1 (NC_000017.11) and COL1A2 (NC_000007.14) and DNA alignment was conducted using DNAman .Sanger sequencing was employed to verify the variants in eviously . BrieflyCOL1A1 and COL1A2 from 34 probands were selected for the minigene splicing assay (http://primer3.ut.ee/) , followed by sequencing verification. Both the purified constructs of wild type and mutant type were transferred into HEK293T cells using Invitrogen Lipofectamine 3000 Transfection Kit (Thermo Fisher Scientific). HEK293T cell line was selected to eliminate endogenous interference for its low expression of type I collagen. After 24 h incubation, RNA was isolated using Trizol reagent (Invitrogen). One microgram total RNA was used for RT-PCR using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). PCR products were separated on 1% agarose gel containing ethidium bromide. The target DNA bands were purified using GeneJET Gel Extraction Kit , followed by DNA sequencing with ABI3730xl . The procedure was summarized in the schematic map  containing 15% FCS  and 1% antibiotics (Sigma)]. RNA was isolated using Trizol reagent (Invitrogen) when dermal fibroblasts were cultured for 3 passages. After reverse transcription, PCR products were separated on 1% agarose gel followed by sequencing confirmation.Skin samples were collected from probands PUMC-253, 371, 98, 401, and 216 following the skin biopsy process or surgical operation. Cleaned dermal tissues were cut into small pieces of 1 mmCOL1A1 and COL1A2  carried 22 different classical splicing mutations (with gt/ag mutations) in d COL1A2 Table S2COL1A1, c.1089+6T > G in COL1A2, c.1197+5G > A in COL1A2, c.2026-1_2042dup in COL1A2, c.792G > A in COL1A2) (in silico tools: Human Splicing Finder (version 3.1) and ESE Finder 3.0  in the exon 16 displayed the exon skipping effect as well  led to the activation of cryptic donor site in the exon (COL1A1 (PUMC-401) also resulted in the utilization of an alternative donor site (c.617_618GT) and generated truncated exon 8  was observed that an AG site (c.660_661AG) in exon 9 in COL1A1 was utilized as the splicing acceptor (COL1A1 (PUMC-276) and c.4249-3_4249-2del in COL1A1 (PUMC-290) which showed the same splicing effects , one mutant transcript with alternative donor site in intron 7 was recognized  were found with intron retention caused by alternative acceptor site in intron in this study Table 1.COL1A1 (PUMC-401), c.3036_3045+2del in COL1A1 (PUMC-480), and c.2404G > A in COL1A2 (PUMC-296)  Table 1.COL1A1 formed four mutant splicing isoforms (1 (c.588+1_588+2gt) and ag1 (c.643-2_643-1ag) generated transcript 1 with skipping of exon 8 (2 (c.588+1_588+2gt) and ag2 (c.660_661AG) formed transcript 2 with deletion of exon 8 and partial exon 9 (3 (c.589-62_589-61gt) and ag3 (c.643-2_643-1ag) generated transcript 3 with deletion of exon 8 and insertion of partial intron 7 (4 (c.588+1_588+2gt) and ag4 (c.589-2_589-1ag), together with gt4 (c.617_618GT) and ag4 (c.643-2_643-1ag) with the effect of truncated exon 8  and ag1 (c.3046-2_3046-1ag) generated transcript 1 with exon 41 skipping (33%); Utilizing gt2 (c.3029-3030 GT) and ag2 (c.3046-2_3046-1ag) generated transcript 2 with deletion of partial exon 41 (67%). Another patient, PUMC-296 (c.2404G > A in COL1A2) showed a missense variant at the first nucleotide in exon 40. Two transcripts were found by minigene analysis: one with a missense variant  and the other with an insertion of 49 bp (40%) by recognition of alternative 3′ splice site (c.2404-51_2404-50ag) in intron 39  carried the variants in introns without aberrant splicing, and they were excluded from the pathogenic variants. In particular, variant c.2026-1_2042dup in COL1A2 (PUMC-253) should be highlighted. This variant may cause aberrant splicing because the duplication covered the 3′ boundary of intron 33 to the 5′ partial exon 34  were classified into different phenotypical groups Table 1:COL1A2 consequent to skipping of exon 16) displayed rather severe phenotypes: with more than 30 times total fracture times (2.9 times yearly of fracture frequency), short stature (Z score = −6.32), presence of dentinogenesis imperfecta and disability of walking.Those exhibited severe phenotype (type III OI), the minigene analysis showed no aberrant splicing (confirmed as no aberration for intronic variants or missense mutation for exonic variants) or exon skipping effect. For instance, PUMC-371  often expressed relatively milder phenotypes: only 11.76% (2 in 17) of them were OI type III, and 88.24% (15 in 17) were OI type I or type IV. Regarding the exonic variants, a large proportion led to a severe type III phenotype, being 30.77% (4 in 13).The splicing effects of 29 suspected atypical splicing variants associate with OI were examined in current study. Among 29 variants, 17 were identified with aberrant splicing, and 12 were not observed any abnormal splicing effect. The splicing effects can be classified as (i) exon skipping or (ii) alternative splice site induced intron retention or partial exon deletion. We further conducted skin fibroblast RT-PCR sequencing and confirmed the findings in the minigene assay, suggesting it is a reliable approach to assess the splicing consequences.Pre-mRNA splicing occurs when exons and introns are precisely recognized. Two theories were proposed about the splicing initiation: the intron definition and exon definition . In intrCOL1A2), such variant changed the consensus sequence AAGgt to AAAgt (COL1A1) belongs to this instance, and the disappearance of the canonical donor site induced exon skipping or the activation of a cryptic donor site.This can be resulted from the alteration of an adjacent nucleotide, for example in patient PUMC-371 . The deletion changed 5′ intronic consensus sequence gtaag to gtag   Figure 6COL1A2) . Wilkins A/G/C/T . Therefo A/G/C/T . Sanz etCOL1A1 and COL1A2, have two main types of collagen defects: quantitative defect and structure defect  Table 1.e defect . The stre defect . The col OI type ; (II) The levels .Among the aberrant splicing in this research, we noticed that all OI patients with more than one mutant transcripts  have mild phenotypes, being either type I or type IV OI Table 1.Similarly, variants locate at the polypyrimidine tract (PPT) region  have mild phenotypes as well. What need to be noted here is PUMC-15, 105, 369, and 189, among whom all the variants resulted in an insertion of part of PPT sequence, generated the premature termination codon and therefore resulted in the degradation of the defective transcript . Their pThe most dominant splicing effect is exon skipping. However, we did not observe a strong correlation between exon skipping and phenotype Table 1.Although a large proportion of structural defects of collagen was due to the classical splicing mutations , atypicaHttp://Oi.Gene.Le.Ac.Uk/). The Datasets Generated for This Study Can Be Found in the Osteogenesis Imperfecta Variant Database  of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China (015-2015). Informed consent was obtained from all adult participants/legal guardians of children under age 18.LL and YC performed the minigene assay, sequencing analysis, and wrote the manuscript. FZ, BM, and YY carried out plasmid construction. SL and TY conducted data collection as well as data analysis. XR and YW helped with recruiting patients and YG helped to discuss the data and helped writing the final manuscript. XZ conceived the study and supervised this research. All authors performed critical reading and approved the final version of manuscript. This study was supported by grants from National Key Research and Development Program of China , CAMS Innovation Fund for Medical Sciences  and National Natural Science Foundation of China (81472053).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IL-10RB mutations. In the three patients, sequencing of genomic DNA identified the same large deletion of exon 3 which precluded protein expression. This mutation was homozygous in two patients born from consanguineous families and heterozygous in the third patient born from unrelated parents. Microsatellite analysis of the IL10RB genomic region revealed a common haplotype in the three Portuguese families pointing to a founder deletion inherited from a common ancestor 400 years ago. In the third patient, surface expression of IL-10R was normal but signaling in response to IL-10 was impaired. Complementary DNA sequencing and next-generation sequencing of IL10RB locus with custom-made probes revealed a ≈ 6 Kb duplication encompassing the exon 6 which leads to a frameshift mutation and a loss of the TYK2-interacting Box 2 motif. Altogether, we describe two novel copy number variations in IL10RB, one with founder effect and one preserving cell surface expression but abolishing signaling.Mutations in interleukin-10 receptor (IL-10R) genes are one cause of very early-onset inflammatory bowel disease with perianal lesions, which can be cured by hematopoietic stem cell transplantation. Using a functional test, which assesses responsiveness of peripheral monocytes to IL-10, we identified three unrelated Portuguese patients carrying two novel  IL10RA, IL10RB, IL10B genes encoding the two chains of IL-10R or IL-10 respectively have been reported in approximately 100 children since 2009 [IL10RB mutations. One mutation consisted in a large deletion of exon 3, which abolished protein expression. It was shared by the three patients and a founder effect could be demonstrated. The other mutation identified in one patient was a heterozygous ≈ 6 Kb duplication comprising the exon 6 that preserved IL-10Rβ surface expression but abolished TYK2 phosphorylation and downstream STAT3 and STAT1 signaling.Mendelian bi-allelic loss-of-function mutations in the nce 2009 . Affectence 2009 . Interlence 2009 ,14. UsinPatients were included in each center after obtaining informed written consent for functional and genetic studies from both parents. The study was approved by the local ethics committee .5 PBMC were stained at 4°C for 30 minutes with PE-labelled anti-IL-10Rβ antibody, or IgG1 isotype (R&D systems), and CD45-APC, -CD3-PeCy7, -CD19-horizon V450  and -CD14-FITC  antibodies. To analyze STAT3 phosphorylation, 1x106 PBMC were stimulated with 25 ng/mL IL-6 or IL-10 (R&D systems) and surface-stained with the same antibody cocktail. After fixation and permeation, cells were labelled with anti-phosphorylated STAT3 antibody according to manufacturer’s instructions (BD Bioscience). Cells were analyzed on CANTO II (BD Biosciences) using the FlowJO software . To study TYK 2 phosphorylation, EBV cell lines were stimulated with 25 ng/mL IL-10 or IL-6 or with 100 pM IFNα for 15 minutes. Protein extracts (40 μg) were separated by SDS-PAGE gel, transferred to nitrocellulose membranes, and incubated with the indicated antibodies as in [Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll Hypaque, and Epstein-Barr virus (EBV)-cell lines were obtained according to standard procedures. Production of IL-8 by PBMC stimulated by lipopolysaccharide  in the presence of IL-10  was analyzed by enzyme-linked immunosorbent assay  as described [IL10RA (NM_001558) and IL10RB (NM_000628) was amplified from genomic DNA by polymerase chain reaction (PCR) as described [IL10RB, genomic DNA was amplified using two primers located in introns 2 and 3, respectively . For analyzing exon 6 duplication, IL10RB cDNA was amplified using two primers located in exons 4 and 7 . PCR products were purified with the QIAquick kit (QIAGEN) and Sanger sequenced on a Genetic Analyzer 3500XL . Segregation analysis of microsatellite markers that contain short tracks of dinucleotide repeats and flanking the IL10RB gene were performed using fluorescent primers. The polymorphic markers chosen from the Genethon Linkage Map [D21S1252/AFM261ZG1; D21S1895/AFMB280XD9; D21S1254/AFM276ZA5; D21S1898/AFMB308XE5; D21S262/AFM198TC5; D21S1888/AFMA218WB1; D21S1908/AFMC016YG9; D21S263/AFM211ZG9; D21S1916/AFMA052TC5; and D21S269/AFM263XF5. Primers sequences are available at the Working Draft of the Human Genome available at UCSC, Human Assembly (GRCh37/hg19). Amplified fragments were electrophoresed on a ABI PRISM 3500XL DNA Analyzer and analyzed using the GeneMapper Software 5 . Heterozygote frequency and the size range of alleles for each microsatellite marker were available at the Centre d’Etude du Polymorphisme Humain (http://www.cephb.fr/cephdb/browser.php).Genomic DNA and RNA were extracted from PBMC with QIAmp DNA Blood Mini Kit and RNA Extraction Mini kit respectively . cDNA was obtained with QuantiTect Reverse Transcription Kit (QIAGEN). Each exon of escribed . For anakage Map  were: D2IL10RB locus of the family 3 as described [IL10RB gene on chromosome 21  according to the February 2009 human reference sequence (GRCh37hg19). During capture, barcoded library molecules complementary to the biotinylated beads were retained on streptavidin-coated magnetic beads and amplified by PCR to generate a final pool of postcapture libraries covering the targeted region. A pool of libraries  covering a ≈150 kb region including the entire IL10RB gene was sequenced on an Illumina HiSeq2500 . Sequences were aligned with the GRCh37hg19 human genome with Burrows-Wheeler Aligner version 0.6.2.13 [http://www.repeatmasker.org/) in the UCSC genome browser (https://genome.ucsc.edu/) [To further analyze the exon 6 duplication, we performed high-throughput sequencing of the entire escribed . Briefly0.6.2.13 . Copy nu GRCh37hg. During Patients 1, 2 and 3  were born to three distinct Portuguese families, P1 and P2 to two consanguineous families while P3 was born from unrelated parents. The three patients presented with diarrhea, rectal bleeding, and perianal lesions since the age of 4 to 9 months. P1 also had severe chronic folliculitis. At diagnosis, endoscopy showed left colonic inflammation (P2) or pancolitis (P1 and P3). P1 was temporarily improved by ileostomy . This clIL10RA and two beta chains encoded by IL10RB [IL10RA but pointed out an identical large deletion in IL10RB encompassing exon 3 in P1 and P2. Exon 3 deletion (delE3) was homozygous in P1 and P2 but heterozygous in P3. Sanger sequencing showed the loss of 2979 base pairs (bp) and the insertion of 2 bp   . Accordiclature) . The parAluJb/AluSx3 element  in intron 3   [www.gnomad.broadinstitute.org) [In order to assess the role of genomic architecture in the mechanism of the large deletion of exon 3, we searched for known repetitive elements intersecting with the breakpoints. Breakpoints were localized respectively in a Tigger1 element  in intron 2  and in an 650,413) . Both aratabase) , nor in ute.org) .IL10RB revealed a common haplotype from loci D21S262 to D21S1254 in all individuals carrying the delE3  . Yet, their exact positions could not be determined precisely using either long-range PCR or PCR with primers framing the two breakpoints. Of note, duplE6 is described neither in DGV nor in gnomAD databases [In P3, sequencing all exons of  on PBMC . These r on PBMC  and with on PBMC . In orde(duplE6) . This duatabases ,23.The consequence of duplE6 is a frameshift at the end of the first exon 6, which precludes translation of exon 7 and results in an aberrant sequence of 50 amino acids . Accorditrans-phosphorylation of JAK1 and TYK2, recruitment and phosphorylation of the transcription factor STAT3 and to a lesser extent of STAT1 [In the IL-10R complex, α and β chains are associated with two tyrosine kinases, JAK1 and TYK2 respectively. Binding of IL-10 to the receptor complex results in of STAT1 . We analIL10RB copy number variations (CNV). One is a deletion and the first described founder IL10RB mutation; the second is a duplication preserving cell surface expression but abolishing downstream signaling.We report here three patients originating from Portugal with autosomal recessive complete IL-10Rβ deficiency. The combination of functional tests and genetic studies allowed us to describe two novel IL10RB mutations and CNV have been rarely identified. Only two patients carried CNV mutations. One was described by Pigneur et al (P5) and displayed a deletion of exon 2 [IL10RB reach 19% (5/26) and 3.8% (1/26) respectively, with an overall rate of CNV of 23%. Thus, CNV rate in IL10RB  is higher than in the human genome .IL10RB locus seems to be enriched in Alu-elements, the two delE3 breakpoints were non-homologous, reducing the probability that delE3 resulted from a recurrent rearrangement [D21S262 to D21S1254, a length compatible with an appearance approximately 400 years ago. To the best of our knowledge, delE3 is the first description of a mutation in IL10RB or IL10RA shared by a particular population.Interestingly, the same deletion of exon 3 was identified in three unrelated families, all originating from the same geographic origin. Even though angement ,28. FurtIL10RB locus captured with custom-made primers. TNGS, thanks to much higher coverage of each captured region of interest, emerges now as a powerful tool to identify CNV [IL10RB [Our observations in P3 illustrate difficulties in characterizing large CNV, and notably large duplications. Their identification requires combining several methods . Thus, itify CNV ,29. Of nIL10RA in two patients [It is likely that P3 cells fail to respond to IL-10 due to the inability of the duplE6 mutant receptor chain to bind TYK2. In recent structural studies, Lupardus and coll. identified residues in other cytokine receptors that are critical for binding TYK2 and map in the so-called Box2 motif . Inspectpatients ,32. DuplIL10RB mutation. While its exact frequency in the Portuguese population remains to be assessed, it may increase the risk of IL-10Rβ deficiency either due to homozygous delE3 or to the combination of delE3 with a compound IL10RB mutation as observed in P3, who was born in a non-consanguineous family of Portuguese origin. Importantly, early identification of the molecular defect has been instrumental to indicate HSCT in all patients, which is currently the only definitive treatment. P3 unfortunately died of severe graft-versus host disease 12 months after HSCT while P1 and P2 are free of symptoms after 4.5 years and 3 years respectively.In conclusion, our results provide the first description of a founder S1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PPTX)Click here for additional data file.


Osteoclasts play a critical role not only in bone homeostasis but also in inflammatory osteolysis, such as that occurring in inflammatory arthritis and systemic inflammation. In both inflammation conditions, inflammatory cytokines like Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α induce RANKL expression in osteoblasts, but the roles of these cytokines in osteoclast activation remain unclear. S100A12, an S100 family member, is a low-molecular-weight calcium-binding protein. Although it has a pro-inflammatory role, its effects on osteoclast differentiation have been unclear. Here we examined the direct effects of S100A12 on human osteoclasts in vitro. S100A12 facilitated osteoclast formation in the presence of RANKL, as judged by the cells’ morphology and elevated expression of osteoclast-related molecules, including NFATc1, ACP5, CALCR, and ITGβ3. In addition, S100A12 administration markedly enhanced the osteoclasts’ bone resorption ability, consistent with their increased expression levels of CTSK and CA2. Blocking RAGE and TLR4 cancelled the effects of S100A12. Our results indicate that S100A12 is a potential therapeutic target for inflammatory osteolysis. Osteoclasts originate from hematopoietic precursors of monocyte/macrophage lineage cells, which differentiate into multinucleated osteoclasts under the influence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclasts play a critical role not only in bone homeostasis but also in inflammatory osteolysis , 2. For The “alarmins” are endogenous substances that are released by dying cells or activated cells. They bind to certain pattern recognition receptors (PPRs) and induce tissue damage and/or inflammatory processes . RepreseRouleau et al. revealed that the S100A12 expression is increased in the synovial fluids of patients with RA, gout, and psoriatic arthritis, but not osteoarthritis, a non-inflammatory disorder [Liao et al. detected elevated levels of S100A12 protein in both the synovial fluid and serum of patients with erosive forms of RA compared with those with non-erosive forms [Among the S100 proteins, S100A12 is constitutively expressed in neutrophils and macrophages. S100A12 is released from activated neutrophils, has proinflammatory effects on immune cells, and promotes inflammatory arthritis . For exadisorder . MoreoveAlarmins like HMGB1 and S100A8 have been shown to stimulate osteoclast formation and an activity mediating osteoclastic bone destruction in experimental arthritis . HoweverRecombinant human M-CSF and recombinant human RANKL were purchased from R&D Systems . Recombinant human S100A8, S100A9, and S100A12 proteins were purchased from MBL . The toll-like receptor 4 (TLR4) inhibitory peptide VIPER was from Novus Biologicals , and the anti-human receptor for advanced glycation end products (RAGE) antibody was from R&D Systems.+ cells were isolated from PBMCs using positive selection with a MACS magnetic CD14 antibody . The isolated CD14+ cells  were incubated in α-minimal essential medium  containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin (supplemented α-MEM), and 50 ng/ml M-CSF, with or without 100 ng/ml RANKL. S100A12 , S100A8 , and S100A9  were added to cultures. Half of the medium was replaced every 3 days.The procedures were approved by the Ethics Committee of Kobe University Hospital (No. 160126), and oral informed consent was obtained from the healthy volunteers. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors, by centrifugation over Ficoll-Paque Plus  at 1,500 rpm for 30 minutes. After centrifugation, PBMCs at the interface between the plasma and the Ficoll-Paque Plus were collected and washed with ice-cold phosphate-buffered saline, followed by centrifugation at 2,000 rpm for 5 minutes. CD14+ monocytes (5×105 cells/12-well plate) were cultured in supplemented α-MEM containing M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 7–16 days.To induce osteoclast differentiation, CD14To elucidate the roles of TLR4 and RAGE in osteoclast differentiation and bone resorption, VIPER (30 μM) and/or an anti-human RAGE antibody (10 μg/ml) were used. In the bone resorption assay, VIPER (30 μM) and/or the anti-human RAGE antibody (10 μg/ml), and S100A12  were all added at the beginning of the culture. To examine the effects of these inhibitors on the mRNA expression of osteoclast-related genes, CD14+ cells were cultured in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 7 days, and then stimulated with S100A12  with or without VIPER (30 μM) and/or the anti-human RAGE antibody (10 μg/ml).TRAP staining was carried out using a kit  according to the manufacturer’s instructions. TRAP-positive multinucleated cells containing more than 3 nuclei were identified as osteoclasts and counted under a microscope .+ monocytes (2×105/well) were cultured on a bone resorption assay plate 48  in supplemented α-MEM with M-CSF (50 ng/ml), alone or in combination with RANKL (100 ng/ml) and with or without S100A12 . On day 16, the cells were removed by 5% NaOCl, and then each well was photographed by a microscope camera (Keyence), and the areas of the resorption pits were measured with digital image analysis software .CD14Total RNA was isolated using an RNeasy Mini kit , and 1 μg of the total RNA was reverse-transcribed with a QuantiTect reverse transcription kit (Qiagen). Quantitative real-time PCR was performed using a QuantiTect SYBR Green PCR kit (Qiagen) with a PikoReal 96 Real-Time PCR system  according to the manufacturer’s protocol. All primer pairs were purchased from Qiagen. Messenger RNA (mRNA) levels were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).t-test was used to evaluate the TRAP-positive cell counts, bone resorption area measurements, and mRNA expression levels of mature osteoclast markers. To evaluate the effects of inhibiting TLR4 and RAGE, one-way analysis of variance and Turkey’s multiple comparison test were used. P values less than 0.05 were considered to be statistically significant.Results are expressed as the mean and SD. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Prism Software). Student’s 2-tailed + monocytes were cultured in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml) to promote their differentiation into osteoclasts, and various concentrations of S100A12 were added to examine whether S100A12 enhances osteoclast differentiation. + monocytes differentiated into osteoclasts in the presence of M-CSF and RANKL. Adding S100A12 facilitated the human osteoclast differentiation in a dose-dependent manner (p< 0.05), as shown in + monocytes in the presence of only M-CSF did not induce osteoclast differentiation (data not shown). Thus, S100A12 enhances RANKL-induced osteoclastogenesis.Human blood CD14We next performed a bone resorption assay to determine whether S100A12 treatment enhances the bone resorption capacity of differentiated osteoclasts. Osteoclasts were generated on a bone resorption assay plate with or without S100A12  for 16 days. The added S100A12 appeared to increase the bone resorption levels. Representative patterns of the resorption pit formation are shown in Next, the messenger RNA (mRNA) expression levels of several osteoclast-related markers were determined by qRT-PCR. As shown in S100A12 has been shown to bind multiple receptors, including TLR4 and RAGE. We next examined whether TLR4 or RAGE was critical for the S100A12-induced osteoclast differentiation and bone-resorption capacity. To test this, we performed a bone-resorption assay with S100A12 in the presence of the TLR4 inhibitor VIPER and/or a specific RAGE-blocking antibody. Osteoclasts were induced for 16 days, and VIPER and/or the anti-RAGE antibody was added at the beginning of the culture. As shown in + monocytes were differentiated into osteoclasts with M-CSF and RANKL for 7 days, and then stimulated with S100A12 in the presence of VIPER and/or the specific RAGE-blocking antibody for 24 hours. As shown in p< 0.05, p< 0.01, We next examined the roles of TLR4 and RAGE in the S100A12-induced osteoclast-specific mRNA expressions. To this end, human CD14Finally, the effects of S100A12 on the differentiation and function of osteoclasts were compared with those of S100A8 and S100A9, which have been shown to induce osteoclast differentiation . We foun+ monocytes. Consistent with these findings, S100A12 enhanced the expression of mature osteoclast-related molecules, including NFATc1, ACP5, CALCR, and ITGβ3, and up-regulated CTSK and CA2, both of which are well known to be critical factors in bone resorption [In this study, we demonstrated for the first time that S100A12 protein directly enhances osteoclast formation and bone resorption through RAGE and TLR4 in the osteoclast differentiation from human blood CD14Inflammatory osteolysis has been ascribed to inflammatory cytokines such as TNF-α, IL-1, and IL-6. These cytokines up-regulate the RANKL expression by osteoblasts and thus facilitate osteoclastogenesis . HoweverAlarmins use various receptors. HMGB1 regulates RANKL-induced osteoclastogenesis through RAGE , while SElevated levels of other S100 proteins, including S100A8 and S100A9, are observed in RA serum and synovial fluid . Here weIn conclusion, we demonstrated that S100A12 is involved in RANKL-induced osteoclast differentiation and bone resorption. S100A12 and RAGE represent potential therapeutic targets for inflammatory bone resorption.


We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium . The SOX Understanding how transcription factors (TFs) orchestrate gene expression to instruct a phenotypic output is fundamental to biology and future therapeutics. Dynamic control of gene transcription is particularly important during development as cell lineages are established. In mammals, many members of the SOX SRY-related High-Mobility Group (HMG) box family act as central regulators of gene expression to govern cell fate in a variety of key processes , such asde novo mutations causing HLTRS have been identified within the HMG domain and the TAD and have been associated to a broad spectrum of the syndrome severity (Ragged mice) in SOX18, which lead to truncated proteins. The truncated SOX acts as a dominant-negative protein that suppresses the endogenous activity of SOX7 and SOX17  enrichment analysis of the subset of genes assigned to the SOX18 homodimer reveals a specific endothelial signature and include genes essential to vascular development. Finally, we validated that pharmacological disruption of SOX18 dimer interferes with the expression level of a subset of genes, linking physical interaction and transcriptional output.During a screen of small-molecules that could disrupt lymphangiogenesis in zebrafish, we showed that the SOX18 interaction network could be targeted pharmacologically . This woSOX18 gene was synthesized  and transferred to pCellFree vectors using Gateway cloning. Translation competent Leishmania tarentolae extract (LTE) was prepared as previously described , mCherry and cMyc (myc) tags, and cloned into the following cell free expression Gateway destination vectors: N-terminal GFP tagged (pCellFree_G03), N-terminal Cherry-cMyc (pCellFree_G07) and C-terminal Cherry-cMyc tagged (pCellFree_G08) . Human Rescribed ,39. GFP-® SV gel and PCR clean up system. These fragments were then cloned into the Gateway destination vectors (pCellFree_G03 or pCellfree_G08) by LR recombination  as described previously.The DNA sequences encoding the desired domains were obtained by PCR amplification of the SOX18 WT construct with the combination of primers listed in The SOX18DIM/SOX7 swap construct was made by exchanging the 50 amino acids following the HMG box of SOX18 WT:Y R P R R K K Q A R K A R R L E P G L L L P G L A P P Q P P P E P F P A A S G S A R A F R E L P P Lwith the 50 amino acids following the HMG box of SOX 7 WTY R P R R K K Q A K R L C K R V D P G F L L S S L S R D Q N A L P E K R S G S R G A L G E K E D R GThe swap construct was obtained as a gBlock (IDT), and was exchanged with the ccdB gene in the donor plasmid (pDONOR223) by BP recombination , then with the ccdB gene in the expression plasmid (pCellFree_G03 and pCellFree_G08) by LR recombination  as described previously.The SOX7DIM/SOX18 swap construct was created by exchanging the 50 amino acids following the HMG box of SOX7 WT:Y R P R R K K Q A K R L C K R V D P G F L L S S L S R D Q N A L P E K R S G S R G A L G E K E D R Gwith the 50 amino acids following the HMG box of SOX 18WTY R P R R K K Q A R K A R R L E P G L L L P G L A P P Q P P P E P F P A A S G S A R A F R E L P P LThe swap construct was obtained as a gBlock (IDT), and was exchanged with the ccdB gene in the donor plasmid (pDONOR223) by BP recombination , then with the ccdB gene in the expression plasmid (pCellFree_G03 and pCellFree_G08) by LR recombination  as described previously.Mus musculus, Danio rerio, Xenopus tropicalis, Gallus gallus, Anolis carolinensis, Orcinus orca, Monodelphis domestica, Latimerica chalumnae and Callorhinchus milii) were obtained using the 50 amino acid human SOX18 homodimerization domain as a query in Protein Blast (NCBI). Multiple sequence alignment of the SOX18 homodimer domain of 10 different species (including human), as well as the corresponding 50 amino acid region of the human SOXF family  was performed using Clustal Omega.Putative SOX18 homodimeriation domains from 9 different species . The resulting bell-shaped curve is an indication of a positive interaction, while a flat line reflects a lack of interaction between the proteins. Measurements of each protein pair were performed in triplicate. A binding index was calculated as: I is the highest signal level (top of the hook effect curve) and Ineg is the lowest (background) signal level. The signals were normalized to the Iref signal obtained for GFP-SOX18/SOX18-Cherry-myc.The assay was performed as previously described ,40, usinN = 6, n = 3).All experiments were performed using independent and technical triplicates . Plates were analysed on a Zeiss LSM710 microscope with a Confocor3 module, at room temperature. Two lasers (488 and 561 nm) were co-focussed in the well solution using a 40 × 1.2 NA water immersion objective ; fluorescence was collected and split into GFP- and Cherry-channel by a 560 nm dichroic mirror. The GFP emission was further filtered by a 505–540 nm band pass filter and the Cherry emission was filtered by a 580 nm long-pass filter .mVENUS-based FosLZ/JunLZ plasmids  were used as a starting point to construct the SOX18 homodimer BiFC reporters (NmVENUS155-SOX18S and CmVENUS155-SOX18S) and the SOX18 homodimer mutant BiFC reporters used as a negative control (NmVENUS155-SOX18SΔ84-205 and CmVENUS155-SOX18SΔ84-205). pCS2+-NmVENUS155-SOX18S and pCS2+-CmVENUS155-SOX18S were generated using In-Fusion cloning  by replacing FosLZ and JunLZ with SOX18S. Circular polymerase extension cloning (CPEC) was used to remove the HMG, NLS and homodimerization domains of SOX18S (SOX18SΔ84–205), which was then inserted into the pCS2+-mVENUS constructs via In-Fusion cloning to generate pCS2+-NmVENUS155-SOX18SΔ84-205 and pCS2+-CmVENUS155-SOX18SΔ84-205.in vitro mRNA synthesis . Synthesized BiFC reporter mRNA was purified  and 1 nl of 100 ng/μl mRNA was co-injected with phenol red into the yolk sac of single-cell zebrafish embyros. Embryos were maintained in E3 media  at 28°C until they reached 4–5 hpf.Restriction enzyme digestion was performed to linearize 5 μg of each mVENUS-based BiFC reporter construct. Following linearization, BiFC reporter DNA was purified , 1 μg of which was used as a template for 4–5 hpf zebrafish embryos were screened for fluorescence using a fluorescent stereo microscope . Embryos identified as fluorescent had the chorion manually removed prior to being embedded in a 2% methylcellulose-containing 35 mm glass-bottom dish. Zebrafish embryos were imaged live using confocal laser scanning microscopy , whereby a 514 nm laser was used to visualise mVENUS-based BiFC reporters. Fluorescent and brightfield images were taken as Z-stacks at 10 × magnification with a 0.45 NA dry objective and at 40 × magnification with a 1.3 NA oil objective. Post-acquisition image processing was performed using FIJI (FIJI Is Just ImageJ) to generate maximum intensity projections and fluorescence/brightfield composites. Time-lapse images were taken over a 10 h period.Sox18 was PCR amplified and cloned into the pOPIN-GST vector, to generate N-terminally tagged HIS-GST-SOX18. A sequence-verified construct was co-transfected with flashBACULTRA  baculovirus DNA onto Spodoptera frugiperda Sf9 cells to obtain recombinantly expressed HIS-GST-SOX18. High Five cells (BTI-TN-5B1-4) in Sf-900™ II serum-free medium were infected at cell density of 1.5 × 106 cells/ml with a multiplicity of infection (MOI) of 5 PFU/cell, and incubated at 21°C for 144 h before harvest. The cell pellet from 100 mL of expression culture was resuspended in 30 mL of phosphate lysis buffer  and sonicated on ice for 20 s. The cell lysate was centrifuged at 17 000 × g for 40 min at 4°C. Supernatant was incubated with Benzonase Nuclease (Merck Millipore) for 1 h at room temperature for DNA degradation, before being mixed with 500 μl GST resin  and incubated on a rotating wheel at room temperature for 1 h. The sample was centrifuged at 500 × g for 1 min to remove unbound protein in the supernatant. The resin was further washed with 50 resin volumes (RV) wash buffer , with unbound protein removed by centrifugation as above. Bound protein was eluted from the resin with 3 × 1 RV elution buffer , collecting the supernatant by centrifugation as above.A cDNA clone of mouse Sox18: 3967084) into a pDONR™221 pENTRY vector, sequenced and recombined into a pETG20A or a pHisMBP expression plasmid using Gateway®LR Technology (Escherichia coli BL21(DE3) cells  and purified as described above.The HMG domain of mouse SOX18 was BP cloned from cDNA templates (IMAGE cDNA clone IDs: chnology . ConstruA double-strand (ds) 37 bp-long DNA probe centred on a synthetic IR5 element was designed. GC-rich flanking and spacer sequences were used to avoid confounding off-site protein–DNA binding. Three spacer lengths were designed: 1 (IR1), 5 (IR5) and 10 (IR10) bp. The DNA probes were obtained from IDT .Sequences for the probes are:2, 100 mM KCl, 10% glycerol, 2 mM β mercaptoethanol, 0.1 mg/ml bovine serum albumin (BSA), 0.1% (v/v) NP-40 and 5% DMSO) in a reaction volume of 10 μl, for 1 h at 4°C in dark. Samples were loaded into a pre-run 12% (w/v) 1× Tris-glycine polyacrylamide gel, electrophoresed in 1× TG  buffer at 150 V for 50 min at 4°C and visualized by phosphorimaging .EMSAs were performed using a DNA elements with 5′ cy5 (Cyanine 5) label (Sigma Proligo) and Sox18_79, a protein construct encoding 79 amino acids of the HMG-domain of mouse Sox18. Experiments were carried out by incubating 15 nM mSox18 HMG with 1 nM DNA in binding buffer ; labelled DNA in presence of protein ; labelled DNA and protein in presence of 400 times excess of unlabelled DNA . Plates were sealed, briskly agitated in the dark at room temperature for 5 min then centrifuged at 1800 rpm for 10–20 s to flatten the sample meniscuses. Plates were allowed to equilibrate for another 15 min at room temperature, before reading fluorescence polarization on a Tecan M1000Pro . All experiments were performed using independent and technical triplicates .Protein-DNA binding was measured by fluorescence polarization, using fluorescein 5′-phosphate-tagged ds DNA probes. Three spacer lengths were tested: 1, 5 and 10 bp. The DNA-binding assay was performed in 20 μl, in black 384-well plates, using mouse full-length SOX18, or a SOX-HMG fragment incubated in 30 mM HEPES buffer pH 7.5, supplemented with 100 mM KCl, 40 mM NaCl, 10 mM ammonium acetate, 10 mM guanidinium HCl, 2 mM MgClAt given constant temperature and viscosity, the fluorescence polarization index (mP) is proportional to the molecular size of binding complex. Binding data were fitted to the Hill equation using GraphPad Prism version 7.03 for Windows, GraphPad Software .Sm4, dissolved in DMSO, at different final concentration. Control incubations used 0.7% (v/v) DMSO final concentration for Sm4. Incubations were in buffer B for 1 h. Percentage of interaction was calculated as: Disruption of protein-protein interactions was assayed to obtain IC50 values by expressing the desired protein pairs in LTE and incubating with IR5 or with the small molecule SpaMo analysis was performed on ChIP-seq peaks using a UniPROBE SOX18 motif  according to the manufacturers protocol, including on column DNA digestion. cDNA was synthetised from 1μg of purified RNA using the High Capacity cDNA Reverse Transcription kit . Amplification and quantitation of target cDNA was performed in technical triplicates of at least three biological replicates using the SYBR green  method. Reactions were run in 10 μl in 384-well plates using a ViiA 7 Real-Time PCR system. The housekeeper gene in vitro assays. Transcription factors are notoriously difficult proteins to work with, and SOXF proteins are no exception. The small DNA-binding domain can be expressed and purified in recombinant form, but the full-length proteins are difficult to obtain. The N-terminal and C-terminal domains of SOX18 are likely intrinsically disordered, reducing further the probability of high-resolution structural studies using crystallography.To assess whether members of the SOXF group have the potential to self-interact, we first measured physical interactions using different Leishmania tarentolae  to assess the effect of the fluorophore on protein-protein interactions (PPIs) .First, we performed a proximity assay  to measure interaction between protein pairs. The assays were performed directly from the cell-free co-expressions, without enrichment or purification steps that could perturb weak complexes. In AS, the interaction between the two proteins brings donor and acceptor beads into close proximity, generating a luminescent signal Figure . The ampTo further characterise SOX18 dimer complexes and their ability to recruit protein partners, we took advantage of single molecule spectroscopic assays. This approach measures the photon emission of individual molecules of GFP or mCherry in a defined confocal volume Figure . After cC = mCherry/(GFP + mCherry) = 0.5 , whereas binding to RXRA occurs in a 1:1 ratio (C = 0.5). These data provide evidence that the SOX18 dimers recruit MEF2C or RBPJ whereas monomeric SOX18 is able to recruit RXRA monomers.The identification of SOX18 homodimers prompted us to determine the stoichiometric ratios for different assembly complexes formed with protein partners such as MEF2C, RBPJ and RXRA Figure . In thisin vivo during development, we investigated the dimer formation using a zebrafish-based model system. To follow the formation of the SOX18 dimer in developing zebrafish larvae, we engineered a fluorescent reporter based on a split-fluorescent protein (split-FP) biosensor and took advantage of this construct in transient transgenic reporter experiments. Bimolecular fluorescence complementation (BiFC) assays are powerful tools for the visualisation of protein-protein interactions in both cell and zebrafish model systems  domain, we generated a series of truncated constructs and tested their binding ability in AS. The truncations were designed based on the known domains of SOX18 full-length, as shown in Figure To validate the importance of this region in the dimerization process Figure , we swapThe homodimerization ability of the two swap mutants were tested in AS Figure  and singMultiple sequence alignment of SOX18 DIM domain across 10 different species shows that the residues are mostly conserved throughout evolution Figure , especiaP-value = 0.005)  , to sear) Figure , B. Sinc) Figure .25,26. Electrophoretic mobility shift assay (EMSA) experiments demonstrated that two SOX18-HMG domains, as well as two SOX9-HMG domains could simultaneously bind to this IR5 motif  assay using FAM-labelled oligonucleotides harbouring IR motifs with different spacer lengths . In this assay, as proteins bind to DNA, the increase in molecular weight, as the protein-DNA complex forms, is reflected by an increase in the FP index (mP). This approach revealed that SOX18 full-length protein produces a maximum binding activity (higher mP index) in presence of an IR5 binding site Figure . There iIn order to tease apart a cooperative binding mechanism from a co-binding event that does not involve a PPI, we performed AS experiments using SOX18 and the SOX18DIM/SOX7-swap mutant in presence of an oligonucleotide harbouring the IR5 palindromic sequence. The lack of dimerization capability of the mutant protein only allows monomeric binding. Incubation of the IR5 probe in presence of SOX18 reaches a plateau phase almost instantly with only a mild increase of the maximum AS signal observed. In contrast, the SOX18DIM/SOX7-swap mutant responded in a dose-dependent manner to an increase of the IR5 probe concentration Figure . The maiSm4 interferes with SOX18-dependent PPIs, including its homodimerization  until all dimers are displaced by binding to individual DNA probes (>5 μM) Figure . This efFLT1, Endomucin, SEMA3D, MEF2A, MAP4K4 and NRP1, as well as other genes known to be involved in angiogenesis such as IL33 and KLF4   Figure . SOX18-dt Figure . These rHere, we describe the molecular basis for the dimerization of the SOX18 transcription factor, a key player during endothelial cell fate determination. We quantitatively describe this homotypic interaction, uniquely observed within the SOXF group and demonstrate the existence and functional relevance of SOX18 homodimer, showing the presence of an homodimer signature in the genome and controlling gene expression using pharmacological interferences with a small molecule inhibitor.In humans, 20 Sry-related high-mobility-group box (SOX) genes have been identified, characterised, and categorised into 8 groups  (A-H). AOnly a handful of SOX protein have been shown to dimerize  evenSOX2 is another protein able to form a dimer in a DNA-dependent fashion. It has been shown that both monomeric and dimeric forms are present in human neutrophils . The dimrd α-helix of the HMG-box. This localization is in good agreement with our previous observation that binding of an antibody raised against the 3rd α-helix of the HMG-box prevents homodimerization14. As with the SOXD group, we speculate that the self-assembly process of SOX18 might be DNA-independent, since dimerization occurs both in the in presence or absence of an IR5 oligonucleotide.In contrast to the DNA-dependent dimerization processes of the SOXE, members of the D-Group (SOX5/SOX6/SOX13) are known for dimerizing via a leucine zipper motif in a DNA-independent manner . A coileCol2a1 transcription. In a similar fashion, we show that the SOX18 dimer has the capability to recruit the notch effector RBPJ or the transcription factor MEF2C (this study, Figure As for SOX2, SOX9, and SOX10, a subset of SOX-responsive genes are specifically regulated by SOX18 dimer activity. In the case of SOXE proteins, dimerization partially drives transcriptional output specificity. For instance, SOX10 homodimer binding sites are found in enhancers of several SOX10 target genes, including connexin-32, protein zero and myelin basic protein. Occupation of both SOX binding sites is required to drive promoter activities . SOX10 dIn conclusion, structural and functional variations within different members of the SOX family make the identification and characterisation of the dimerization process a tedious exercise. Different modalities of self-assembly, involving the DNA, the HMG-box and specific motifs in C-terminal and N-terminal positions outside the DNA-binding domain, contribute to the diversity of self-assembly mechanisms. Our work shows that the mechanism of SOX18 dimer formation is a unique feature within the F-group, and involves a distinct binding motif, which permits the transcriptional signature of SOX18 to be distinguished from confounding, closely related, and redundant, SOX7 and SOX17 activities.Supplementary DataClick here for additional data file.


H3F3A) or H3.1 (HIST1H3B). H3K27M mutant glioma responds more poorly to treatment and is associated with worse clinical outcome than wild-type tumors, so mutation detection is now diagnostic for a new clinical entity, diffuse midline glioma H3K27M mutant, as defined in the most recent WHO classification system. We previously reported patterns of histone H3 trimethylation (H3K27me3) and acetylation (H3K27Ac) associated with H3K27M mutation that impact transcription regulation and contribute to tumorigenesis. Given the clinical implications of the H3K27M mutation and these associated H3 post-translational modifications (PTMs), we set to determine whether they can be characterized via immunohistochemistry (IHC) in a cohort of pediatric glioma (n = 69) and normal brain tissue (n = 4) specimens. We observed 100% concordance between tissue IHC and molecular sequencing for detecting H3K27M mutation. In turn, H3K37M and H3K27me3 results, but not H3K27Ac staining patterns, were predictive of clinical outcomes. Our results demonstrate H3K27M and H3K27me3 staining of pediatric glioma tissue may be useful for diagnosis, stratification to epigenetic targeted therapies, and longitudinal monitoring of treatment response.Pediatric diffuse midline glioma is a highly morbid glial neoplasm that may arise in the thalamus or brainstem . Because tumor anatomic location precludes surgical resection, diagnosis and treatment is based on MR imaging and analysis of biopsy specimens. Up to 80% of pediatric diffuse midline gliomas harbor a histone H3 mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3 variant H3.3 ( Pediatric high-grade glioma (HGG) persists as the leading cause of cancer death in children. A subset of pediatric HGG, known as diffuse midline glioma, presents a particularly significant diagnostic and therapeutic challenge due to its midline location, infiltrative nature, and aggressive tumor biology Figure . Somaticn = 69), and to further analyze these results with respect to tumor clinicopathologic features and clinical outcomes in order to determine the clinical utility of this approach.Importantly, patients with H3K27M mutant glioma have poorer overall response to therapy and clinical outcomes relative to H3 wild-type. As a result of these significant implications on tumor biology and patient clinical outcomes, molecular detection of the H3K27M mutation in thalamic or brainstem glioma is now diagnostic for a new clinical entity known as diffuse midline glioma K27M-mutant, which receives a WHO grade IV designation regardless of tissue histological features . While Dn = 18, 26%), thalamus , cerebral hemisphere  and cerebellar hemisphere . Four normal brainstem tissue specimens were also analyzed as controls. Subject age ranged from nine months to 20 years  = 7.2 months). Female and male genders were similarly represented in the cohort . The majority of specimens (56%) were high-grade, with 20% designated Grade III and 36% Grade IV by histopathological features.Demographic and descriptive statistical results for the cohort of cases analyzed are summarized in Table n = 69) and normal brain tissue (n = 4) was stained for H3K27M. In addition, 61 glioma cases in our cohort were stained for H3K27me3, and 33 for H3K27Ac. Immunohistochemical staining results for H3K27M and H3K27me3 across our cohort were consistent with prior investigations and reports in the literature , with midline tumors more likely to harbor the mutation compared to those arising in the cerebral or cerebellar hemisphere (p = 0.012). Fifty percent of our H3K27M mutant cases occurred in children between 5 and 10 years of age. There was no relationship between H3K27M and tumor histopathological grade (p = 0.594) or gender (p = 0.23). H3K27me3 signals significantly differed by tumor grade (p < 0.02), with greater H3K27me3 loss in high-grade tumors (Grade III and IV) tumors relative to low-grade lesions . Midline tumors also demonstrated significantly lower H3K27me3 signals compared to non-midline tumors (p = 0.036). No relationship was observed between H3K27me3 signal intensity and gender (p = 0.59) or age (p = 0.62). As expected, H3K27M mutation correlated with reduced H3K27me3 signal . In contrast, there was no association of H3K27Ac positivity with H3K27M status . There was no relationship between H3K27me3 and H3K27Ac detection either by direct comparison (p = 0.86) or when parsed by degree of H3K27Ac signal intensity (p = 1.00). Similarly, there was no relationship between H3K27me3 and H3K27Ac positivity when evaluated by tumor anatomic location .All tumor tissue (n = 4), and one normal brainstem specimen as a negative control, via PCR-amplification and Sanger sequencing of the H3F3A gene , with a trend towards increased risk of mortality in patients 5–10 years of age (p = 0.06) tumors. Overall, we also observed increased risk of mortality with H3K27me3 loss .Association between IHC staining patterns and clinical disease progression was also evaluated in cases for which this data was available (H3F3A and HIST1H3B) occur in up to 80% of pediatric diffuse midline gliomas, including thalamic and diffuse intrinsic pontine gliomas (DIPGs). This mutation causes Lys27Met (K27M) substitution at a critical regulatory location on the Histone H3 N-terminal tail, affecting chromatin structure and regulation of gene transcription [Somatic missense mutations in Histone H3.3 and H3.1 genes  via exclusion of PRC2 subunits EZH2 and SUZ12 from chromatin binding. This exclusion permits H3K27 acetylation (H3K27Ac), promoting active transcription at specific loci throughout the genome that contributes to tumorigenesis and is the basis for investigation of novel epigenetic targeted therapies for H3K27M mutant tumors . We alsoIn our cohort of 69 pediatric gliomas, 55% were H3K27M positive by IHC staining . This finding is in line with our published cell line data and other studies that demonstrate loss of H3K27 trimethylation in the setting of H3K27M mutation , 22, 24.We demonstrate in this study the clinical feasibility and utility of IHC staining for H3K27M mutation and two associated H3 post-translational modifications, H3K27me3 and H3K27Ac, in pediatric glioma. Recent advances in neurosurgical techniques now enable safe stereotactic biopsy of brainstem and thalamic gliomas . As a reIn our cohort, H3K27M mutation was the strongest predictor of clinical outcome in our cohort, independent of tumor grade or location, while H3K27me3 negativity, but not H3K27ac positivity, was predictive of clinical outcomes. While H3K27M status will not change in response to epigenetic therapies, the proportion of H3K27me3 and H3K27Ac may. Our results suggest that monitoring tumor H3K27me3 levels vial simple IHC could be useful for stratification to epigenetic therapies, and evaluating therapeutic response. As H3K27me3 loss was not consistently accompanied by a reciprocal gain in H3K27Ac staining, our results suggest H3K27Ac detection via IHC may not be as useful as predicted by our preclinical studies.Overall, given the relative lack of diffuse midline glioma tumor tissue for advanced molecular analysis compared to other brain tumors that are anatomically accessible for surgical removal, our study suggests that tissue IHC staining for H3K27M and H3K27me3 may serve as a cost-effective and efficient way to evaluate the efficacy of promising new therapies on the tumor epigenetic landscape, with the potential to portend clinical outcomes for these patients. In addition, we and others have shown detection of tumor proteins in CSF , and aren = 59) or post mortem (n = 14), and preserved via formalin fixation and paraffin embedding  were also analyzed. Informed consent for specimen collection and analysis was obtained under protocols approved by Ann & Robert H. Lurie Children's Hospital of Chicago and Northwestern University Institutional Review Boards . All patient identifiers were removed at the time of specimen analysis, and a numerical identifier was assigned to each specimen before processing.Tissue specimens from children with glioma treated at our institution between 2004–2014 were collected during the course of treatment , and incubated in the Biocare Medical Decloaking Chamber for five minutes at 110° C, followed by incubation in PBS for five minutes. 200 μL peroxidase I (Dako K4011) were then added to the sections, incubate for ten minutes at room temperature, and rinse sections with PBS. Subsequently, two to five drops of background sniper  were added to the sections, incubate for fifteen minutes at room temperature, and rinse sections with PBS.Primary antibodies were prepared to a final volume of 200 μL using antibody diluent (Dako S0809) as follows: rabbit polyclonal anti-Histone H3K27M (Millipore ABE419) 1:1000, rabbit monoclonal anti-Histone H3K27me3  1:200, and rabbit monoclonal and anti-Histone H3K27Ac  1:200. Tissue sections were incubated with primary antibody at 4° C overnight followed by wash in TBST/Tween (Dako S3306) for three minutes. Sections were then incubated with polymer-HRP anti-rabbit secondary antibody (Dako K4011) for 30 minutes, followed by PBS wash. Immunohistochemical reactions were visualized by incubating sections for three minutes in DAB chromogen (dilute one drop DAB into 1mL DAB buffer from Dako Kit K4011). The sections were then counter stained with hematoxylin for one minute at room temperature, washed with tap water and dehydrated with graded alcohol and xylene, and finally coverslip using a xylene-based mounting medium. Hematoxylin and Eosin (H&E) staining was performed on each specimen by the Pathology Department at the Ann & Robert H. Lurie Children's Hospital of Chicago in order to validate tissue diagnosis and assign WHO-grade.et al. [Results of tissue IHC staining was evaluated independently by three pathologists, including one board certified neuropathologist, who were blinded to all specimen identifiers including histological diagnosis and anatomic tumor location at the time of tissue evaluation. H3K27M mutation and H3K27me3 positivity was defined as nuclear staining in >80% of tumor cells visualized in the absence of staining in tumor vascular epithelial cells , as previously described by Venneti et al. . In caseA retrospective chart review of pediatric glioma patients from whom tissue specimens were collected and analyzed was performed to determine: patient gender; age at diagnosis; tumor anatomic location; tumor histologic diagnosis; tumor WHO-grade; date of surgery; extent of tumor resection; date of recurrent and/or progressive disease; date and nature of adjuvant therapy; overall survival; progression free survival; and mortality. Tumor recurrence was defined as MR- confirmed radiographic evidence of new disease burden after confirmation of gross total resection (GTR) of their tumor on initial post-operative MR imaging. Disease progression was defined as radiographic evidence of increased disease burden after tumor biopsy or subtotal resection (STR), or evidence of recurrent and increasing disease burden after GTR.Fresh frozen tumor tissue was used for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen) per manufacturer's protocol. Template DNA isolated from tumor tissue was amplified via PCR using H3F3A primers (0.8 mM) flanking a 300 base pair exonal region encoding Lys27 and Gly34 in Histone H3.3. Conventional PCR was performed in a thermocycler (Bio-Rad) under the following conditions: two minutes at 95° C, 40 cycles of , and five minutes at 72° C. PCR products separated in 2% agarose gel and full-length H3F3A DNA purified using the QIAquick Gel Extraction Kit (Qiagen). Extracted DNA was submitted to Sanger sequencing of H3F3A for K27M mutation using the ABI 3730 High-Throughput DNA Sequencer (Applied Biosystems). Sequenced data were visualized with FinchTV (Geospiza) and MegAlign (DNASTAR).t-tests and chi-square tests for continuous or dichotomous variables, respectively, to test the null hypothesis that patient outcomes, demographics or mutation status were the same across subgroups. When appropriate, the Fischer's exact test was implemented for categorical variables requiring small sample adjustment. Differences in survival and recurrence were analyzed using Kaplan–Meier method and the log-rank test was used to test the null hypothesis that the subgroups have identical recurrence or survival patterns. Patient data on follow-up time (in months) were compared for patients based on H3K27M mutation status (wild-type versus mutant), presence or absence of K27me3, or H3K27 acetylation expression  based on immunohistochemistry for the survival analysis. Statistical tests were considered significant for p-values < 0.05. Data were analyzed using Intercooled Stata, Version 14.0 .Study measures were estimated using Frozen tissue was thawed and dissociated with gentleMACS Dissociator . Protein was extracted in Tissue Extraction Reagent  and concentration determined with Pierce BCA Protein Assay Kit . 60 μg of protein was separated by electrophoresis in a 4–15% precast protein gel  and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution  overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000  for 1 hour. Pierce ECL Plus  was used to detect protein bands. Blots were then stripped  and re-probed with anti-total H3 primary antibody at 1:1000 dilution  as a loading control. HRP-conjugated anti-Mouse IgG antibody  was used to detect total H3 signal. Protein from a patient-derived DIPG cell line SF8628 was extracted with RIPA buffer  and processed in parallel with tissue specimens as a positive control. Densitometry analysis was performed with image J.


Kd ∼ 1.8 μM). Mutation of charged residues in the predicted HS-binding motif of E2 to alanine resulted in reduction of HS binding. Molecular dynamics (MD) simulations on E2, after docking HS, predicted allosteric domain movements. Fluorescence spectroscopy, far-UV circular dichroism spectroscopy, fluorescence resonance energy transfer experiments on HS-bound E2 corroborate our findings from MD simulations. We propose a mechanism where receptor-binding results in allosteric domain movements in E2, explaining E2–E1 dissociation.Receptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly understood in alphaviruses. Chikungunya virus (CHIKV), an alphavirus, re-emerged as a global pathogen in recent past. CHIKV surface envelope proteins, E2 and E1, function as receptor binding and fusion protein, respectively. Site of heparan sulfate (HS) receptor binding on E2–E1 heterodimer and its effect on E2–E1 heterodimer conformation is not known. Using molecular docking, we mapped HS binding to a positively charged pocket on E2 that is structurally conserved in alphaviruses. Based on our results from docking and sequence analysis, we identified a novel HS-binding sequence motif in E2. Purified E2 binds to heparin and HS specifically through charge interactions. Binding affinity of E2 to HS is comparable with other known HS–protein interactions ( Chikungunya virus (CHIKV) is an aedes mosquito-transmitted re-emerging alphavirus that causes chikungunya fever, a debilitating disease . Since iCHIKV is an enveloped virus (has a protective lipid bilayer on surface). Similar to other enveloped viruses, CHIKV enters cells in a sequential, two-step process: (i) virus binding to cell surface receptor, followed by endocytosis and (ii) fusion of viral envelope with endocytic vesicle membrane that leads to opening of a fusion pore. Nucleocapsid escape into cytoplasm follows membrane fusion ,5,6. TheIn CHIKV envelope proteins, E2 and E1 are anchored into viral membrane through short transmembrane segments. A third protein, E3, is on viral surface through its interaction with E2 . Role ofEctodomains of E2 and E1 have predominantly β-sheet secondary structure. E2 and E1 have three domain architecture: from viral membrane proximal to distal – domains III, I and II in E1; C, A and B in E2 refer A 8]. In. In8]. IOn CHIKV surface , E2–E1 heterodimers follow the icosahedral symmetry of the viral capsid, because of E2 C-terminal tail interaction with the capsid protein. As a result, three E2–E1 heterodimers assemble to form a spike-like structure, with E1 at the periphery and E2 at the center B 8]. At. At8]. ACHIKV shows broad cell and tissue tropism. CHIKV can infect keratinocytes, fibroblasts, muscle stem cells, monocytes and macrophages . Though In this study, using molecular docking and biochemical approaches, we characterized HS-receptor binding site on CHIKV E2, and studied conformational changes in E2 upon HS binding. We found that HS binds to a structurally conserved, positively charged pocket on E2. Interestingly, HS-binding sequence in E2 does not match to other consensus heparin/HS-binding motifs. By sequence analysis, we identified a new HS-binding sequence motif , that is present in all alphaviruses. Using purified E2 protein, we show that E2–HS interaction is specific. Mutation of charged residues (Arg104 and Lys107) in predicted CHIKV E2 HS-binding pocket to alanine resulted in significant loss of HS binding. Molecular dynamics (MD) simulation on HS-docked E2 structure revealed dramatic changes in tertiary structure of E2 conformation upon HS binding. Results from intrinsic tryptophan fluorescence, far-UV circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) studies on E2 in absence or presence of HS support our MD simulation predictions. Based on our results, we propose a model to explain mechanism of E2–E1 dissociation after receptor binding: binding of receptor on domain A results in allosteric movement of domain C of E2, pushing E1 away from trimeric spike central axis.We used PyMol plugin version of Autodock Vina  to perforep ± 0.40Eattr + 600Eelec + 1EDARS, in the equation E = w1Erep + w2Eattr + w3Eelec + w4EDARS, where Erep and Eattr denote the repulsive and attractive contributions to van der Waals interaction energy, and Eelec is an electrostatic energy term, as implemented in ClusPro protein-protein and protein–heparin docking algorithm. EDARS is a pairwise structure-based potential constructed by the decoys as the reference state (DARS) approach. It primarily represents desolvation contributions to free energy change due to removal of water molecules from the interaction interface.For protein–protein docking using ClusPro server , we usedWe searched for HS-binding motif in E2 sequences from different alphaviruses using Multiple Em for Motif Elicitation server . First, Escherichia coli. Overexpressed E2 and E3E2 proteins were in insoluble fraction of cell lysate. We solubilized the proteins from inclusion bodies using 20 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl and denaturant 8 M Urea. Phenyl methyl sulfonyl fluoride  (100 μM) was added to lysate as protease inhibitor. Proteins were purified from urea-solubilized fractions using Ni-NTA His-tag affinity chromatography. Purity of proteins was assessed by SDS-PAGE. Purified proteins were refolded by exchanging into TN buffer containing 1 mM EDTA (TNE), and concentrated, using ultrafiltration method. Protein concentration estimation was done using absorbance at 280 nm and molar extinction coefficients.PCR amplicons of full-length E2 and E1-coding regions from CHIKV LR2006 isolate are a kind donation from Prof. Scott C. Weaver, UTMB, Galveston, Texas. We prepared E3-coding DNA fragment, using overlapping short oligonucleotides in an assembly PCR method, following a published protocol . We clonMutations in predicted HS-binding motif on E2  were generated by site-directed mutagenesis approach using primer-extension method. We also created serine to cysteine mutations, S154C and S296C in β-ribbon connector and domain C of E2, respectively, for coupling a FRET pair of fluorophores. All mutant constructs were sequence verified before expression and purification. Expression and purification of mutant proteins was done exactly the same way as that used for the wild-type protein. Mutant proteins were characterized for structure and function (HS binding) in the same way as wild-type protein.To characterize E2 binding with heparin, we used heparin sepharose  affinity chromatography as a binding assay. We applied 10 μg of recombinant E2 protein onto a heparin column in TNE buffer (conc. of NaCl is 150 mM). After an intermittent wash step with TNE buffer, we eluted the bound protein with buffer containing increasing concentration of NaCl . Aliquots of equal volume from each fraction  were analysed on SDS-PAGE followed by Coomassie Brilliant Blue R-250 (CBB) staining. BSA was used as a control to assess non-specific binding of proteins to heparin. The amount of protein eluted with 300 mM or higher NaCl concentration is taken as a measure of specific binding of protein to heparin through electrostatic interactions. To prove the specificity of binding, we performed a competition assay by pre-incubating E2 with HS  before loading on to heparin column.We also used E3E2 or E3E2 Arg/Lys to Ala mutants in HS-binding assay to measure the extent of binding. To calculate the fraction of protein bound to heparin column specifically, we performed the binding assays as described above, except that the elution was done in a single fraction with 1 M NaCl. Extent of protein-heparin binding is represented as percent of protein loaded (fraction bound), by quantifying the band intensities using imageJ software. Mean ± SD values of three independent experiments is presented in a bar graph and compared for statistical significance of differences in one-way ANOVA analysis using GraphPad Prism 7.0a software .Purified E3E2 protein was fluorescently  labelled using N-hydroxysuccinimide labelling protocol. The protein concentration  was kept constant, while the concentration of the non-labelled HS varied from 0.03 to 15 μM. After 30-min incubation at RT, the complex samples were loaded into Microscale thermophoresis (MST) standard coated capillaries and analysis was performed using the Monolith™ NT.115 instrument with 40% MST power. Raw data were analyzed with the software MO. Affinity Analysis , and the final curve fitting was done with the data points  using GraphPad prism software.We carried out MD simulations, energy minimization and trajectory analyses on the HS-docked E2 complex structure with the GROMACS 5.1.4 package  using thPyMol was used for structure visualization, making figures and distance measurements between domain center of mass.Far-UV CD and intrinsic tryptophan fluorescence spectra were recorded with protein alone and with protein-HS mixture (at 1:50 molar ratio). Samples were incubated at room temperature for 30 min before recording the spectra. Protein was used at 0.2 mg/ml concentration in TNE buffer.http://cbdm-01.zdv.uni-mainz.de/∼andrade/k2d3/.The far-UV CD spectra were recorded using a 0.1 cm path length quartz cuvette. Mean residue ellipticity (θmrw) values against wavelength spectra presented in the figures are average of three spectral accumulations each, and blank corrected. The percentage of secondary structure content of the protein was predicted from the far-UV CD spectra, using K2D3 software available at Fluorescence emission spectra were recorded using a 1 cm cuvette and excitation wavelength was set to 295 nm. Average of three spectral accumulations is presented in the figures, after blank correction.Cysteines were introduced, replacing serine residues at 154 and 296 position, in E3E2 to enable labelling with a FRET fluorophore pair. The mutant protein was reacted with Ellman’s reagent and number of free cysteines available were estimated from Ellman’s reagent absorbance at 412 nm. After ensuring that only introduced cysteines are available for labelling, E3E2 S154C + S296C protein was labelled with the FRET pair, Alexa Fluor™ 488 and 594 C5 Maleimide  through cysteine thiol-reactive maleimide chemistry, following protocol prescribed by the manufacturer. Protein and fluorophore maleimide were used at a molar ratio of 1:20 in the labelling reaction. Reaction mixture, in TNE buffer, was incubated at RT for 2 h in dark. The labelled protein was separated from free dyes using Sephadex G-25 in PD-10 desalting columns .max abs. for Alexa Fluor™ 488) and emission was recorded with wavelength range 500–700 nm. Spectra were collected either with the labelled protein alone or with the labelled protein incubated with HS (with 50 molar excess to protein) for 30 min at RT. Excitation and emission slit widths were set to 5 nm.Fluorescence emission spectra were recorded with excitation wavelength set to 493 nm (λTo predict HS-binding site(s) on E2-E1 trimeric spike surface, we performed molecular docking of HS disaccharide structure on to E2-E1 trimeric spike structure. Best docking pose, as assessed by least Gibbs free energy score  is used to analyze for HS-binding site on E2-E1 trimer. 104CPK107G), did not match to any of the known HBDs. Then, we mapped mutations in E2  that were reported in published literature [Further, we compared HS-binding motif on E2 that we predicted from docking studies, with consensus HS-binding motifs (HBDs) reported for other HS/heparin-binding proteins. There are two consensus HBD-sequence motifs: (i) XBBBXXBX and (ii) XBBXBX, where X is any residue and B is a basic residue (arginine/lysine) . Other Hterature ,16 to eiTo experimentally validate our docking results of HS-binding site on E2, and to study conformational changes in E2 upon HS binding, we expressed and purified E2 or E3E2 proteins . Binding of E3E2 R104A / K107A / R104A+K107A to heparin was significantly reduced compared with wild-type protein (From the predicted HBD on E2 (docking studies), AR protein F. HoweveFurther, we observed that extent of loss of HS binding to E3E2 in double mutant (R104A+K107A) is not an additive effect of each single mutant (R104A or K107A). Several earlier studies characterizing HS and other glycosaminoglycan–protein interactions have reported similar results with HS-binding site mutations. For example, a recent study by Richard et al.  has repoKd) value of 1.8 ± 0.6 μM shows that the binding affinity is weak. Our observations are in line with Kd values reported for other proteins binding to HS [n=2)  G. This fTaken together, our docking predictions and comparison of heparin binding to purified E2, E3E2 or E3E2 R104A / K107A / R104A+K107A mutant proteins strongly suggest that HBD between domain A and arch 1 of β-ribbon connector is the primary-binding site for HS/heparin receptor. On virion surface trimeric spike this site (of the four HBDs on E2) would be the most accessible site for HS binding.Receptor binding on E2 is the first triggering event in alphavirus entry. However, how receptor binding leads to breaking of E2–E1 interactions and dissociation is not known. Does HS binding on E2 lead to conformational changes? To test this, we performed MD simulations on HS-docked E2 structure. Clustering analysis of simulation trajectory, with 4 Å cut-off, revealed two major clusters in a six clusters group: cluster1 with 692 and cluster 2 with 284 structures A. We comThree out of five tryptophans present in E2 are located in domain C. We reasoned that, if domain C moves upon HS binding (as predicted in MD simulation), tryptophan residues (at least for those in domain C) environment is likely to change. Tryptophan fluorescence emission spectra in presence and absence of HS are shown as an overlay in To further validate, we generated a cysteine double mutant of E3E2 to introduce FRET acceptor-donor pair onto domain C and β-ribbon connector and confirmed that the mutant protein is similar to wild-type with respect to secondary structure content and tertiary structure, as deduced from intrinsic tryptophan fluorescence and far-UV CD spectroscopy . We probed available free cysteines with Ellman’s assay, to make sure that only introduced cysteines are available for fluorophore labelling–Ellman’s reagent absorbance at 412 nm for wild-type is same as buffer blank, whereas for double cysteine mutant it increased (Table S2). Double cysteine mutants, labelled with FRET fluorophore pair bound to HS, to similar extent as wild-type protein . Using the labelled E3E2 protein, we studied domain movements upon HS binding by FRET. We carefully chose places for cysteine introduction, to avoid any possible effect on HS binding to the cysteine double mutant. We measured the distance between sidechains of Ser154 and 296  to be 2in silico and biochemical approaches, we characterized heparan sulfate and heparin interaction with CHIKV E2 protein. We studied changes in E2 structure upon HS binding, that can explain E2–E1 dissociation mechanism—an essential step before membrane fusion. Here, we discuss our results in the light of literature available on alphavirus E2–E1 dissociation mechanism during entry.CHIKV has spread globally since its re-emergence in recent past. Similar to other alphaviruses, CHIKV enters cells through endocytic pathway. Viral envelope protein E2 is the receptor-binding protein. Despite several years of study, knowledge on E2–receptor interaction is very limited. Also, mechanistic details of E2-E1 action after receptor binding are not understood. In this study, using CHIKV ,23,24 anOther viral envelope proteins such as HIV gP120  and RespKd = 1.8 ± 0.6 μM). Mutation of either Arg104 or Lys107 or both from predicted HS-binding motif sequence lead to significant loss of HS binding. Taken together, our docking studies and biochemical studies affirm that the charged pocket at the boundary of domain A and β-ribbon connector on E2 is the binding pocket for HS receptor.We validated our docking predictions using purified E2 or E3E2 proteins. E2 and E3E2 binds to HS and heparin specifically, though through weak electrostatic interactions (Structures of trimeric spike from CHIKV ,10, SINVBased on our observations, and interpreting our results in the light of structure information available on different alphavirus E2-E1 proteins, we propose a mechanistic model for receptor binding as a triggering event in alphavirus entry . We propDespite several studies on receptors of alphaviruses, mechanism of receptor binding as trigger in cell entry remained enigmatic. Our study provides first clues to understand receptor binding as a regulation mechanism in alphavirus cell entry.