SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of  Cancer patient mortality is overwhelmingly due to distant organ metastases. Epithelial-mesenchymal transition is a process thought to facilitate local invasion and dissemination of cancer cells, thereby promoting metastasis. The conversion of epithelial cells into mesenchymal, fibroblast-like cells requires profound gene expression changes. A few transcription factors like SNAIL1 can initiate these changes, but are unlikely to be solely responsible for all of them. In our study we asked, whether destabilization of epithelial gene expression programs could involve FOXA transcription factors. FOXA factors represent a special subgroup of regulatory proteins, so-called pioneer factors, with unique roles in the activation of transcriptional enhancers which are key regulatory DNA elements that orchestrate spatio-temporal gene expression. In a model of colorectal cancer we found that SNAIL1 represses FOXA factors, and demonstrate that FOXA factors are associated with enhancer elements at epithelial signature genes. Indeed, FOXA factors are sufficient to initiate enhancer activation and necessary to maintain their activity. Our findings indicate that SNAIL1 induces pervasive repression of epithelial genes through a hierarchical scheme of alterations in transcription factor expression which may be applicable to other instances of cell fate changes and transcriptional reprogramming. The formation of distant organ metastases and acquired therapy resistance represent major challenges for the successful treatment of cancer. The process of epithelial-mesenchymal transition (EMT) is widely believed to promote initial steps in the invasion-metastasis cascade . EMT wascis-acting DNA elements with pivotal functions in gene regulation [Transcriptional enhancers are gulation . Aside fgulation –10. The gulation : Inactivgulation . AccordiCDH1 (coding for E-CADHERIN) in human breast cancer cells [FOXA1, FOXA2, and FOXA3 are closely related DNA-binding proteins which are known to function as pioneer factors ,12. Theyer cells .CDH1, EPHB3 and CDX2 which are integral to the differentiation and tissue integrity of the intestinal epithelium [During EMT, a pervasive repression of epithelial genes takes place. A possible mechanistic explanation for this could be that EMT-inducing transcriptional regulators target transcription factors at strategically important positions in gene-regulatory hierarchies to inactivate enhancer elements at a broad range of epithelial genes. Here we tested this hypothesis and show that SNAIL1 downregulates all three FOXA family members in a colorectal cancer (CRC) model of EMT. The loss of FOXA factors in turn impairs the structure and function of enhancers at the tumor suppressor genes ithelium –26. It aFOXA1, FOXA2, and FOXA3 clustered together with epithelial genes that are involved in the formation of adherens junctions (CDH1) and tight junctions   and EMT inducers  formed a separate cluster of co-expressed genes  and Western Blotting. In this model, the expression of FOXA1 turned out to be mutually exclusive with that of SNAIL1, while elevated SNAIL1 expression was not paralleled by lower levels of FOXA2 and FOXA3  inducible expression of epitope-tagged murine Snail1-HA in the CRC cell lines LS174T and HT29 . Upon Do29 cells  panel C.29 cells . In bothFOXA1 gene. Among 56 cell clones analyzed we obtained 22 in which genome editing had not been effective. These cells morphologically resembled the parental LS174T cell line  or their complete absence (KOA2A3neg). The molecular basis for the concomitant reduction or loss of expression of FOXA2 and FOXA3 upon knockout of FOXA1 in these cell clones is not clear. Irrespective of this, the acquisition of a more mesenchymal morphology by FOXA1KOA2A3lo/neg cells was accompanied by the upregulation of SNAIL2/SLUG, and LEF1, two transcription factors with EMT-inducing capacities [KO clones. Likewise, the increase in LEF1 levels was seen in all FOXA1KO clones, albeit FOXA1KOA2A3neg clones showed considerably higher LEF1 expression compared to FOXA1KO and FOXA1KOA2A3lo clones. SNAIL2/SLUG induction was restricted to the three FOXA1KOA2A3lo/neg clones and did not occur in the FOXA1KO clone 4F3. When inspecting intracellular distribution in FOXA1 wild-type (FOXA1WT) and FOXA1KOA2A3lo/neg cells by immunofluorescence staining, SNAIL2/SLUG unfortunately remained below the detection limit, but LEF1 showed the expected nuclear localization. As anticipated, CADHERIN11 appeared membranous and marked cell boundaries  [FOXC1, S100A4/FSP1, VCAN) [KOA2A3lo and FOXA1KOA2A3neg clones were not identical but nevertheless closely related. Therefore, for subsequent comparisons the transcriptome data of FOXA1KOA2A3lo/neg clones were merged. Gene set enrichment analysis based on the KEGG and REACTOME databases pinpointed the features “Pathways in cancer”, “Hemostasis”, “Focal adhesion”, “WNT signaling pathway”, and “Adherens junction” as the five most significantly upregulated in FOXA1KOA2A3lo/neg clones  ,32, EMT,1, VCAN) –35 , and integrated this information with the results of the FOXA1 and FOXA2 ChIP-seq analysis. As a result, we examined in detail the impact of FOXA factors on enhancer regions at the CDH1 locus at +7.8 kb and +11.5 kb that were activated during mesenchymal-epithelial transition in NMuMG cells [CDH1 locus revealed DNA sequence conservation with a predicted FOXA binding motif at +7.8 kb  and both genes are silent in this cell line. Thereby, at the CDX2 and EPHB3 loci the impact of inducible FOXA1-HA and FOXA3-HA expression in a naïve chromatin setting could be studied. In addition, the CDH1 gene which displayed occupancy of the +7.8 kb enhancer by FOXA3 and which is expressed in HCT116 cells , respectively , representing the SNP rs1078621 (MAF/MinorAlleleCount: A = 0.4541/2274 (1000 Genomes)). To mutate the FOX binding sites, the Stratagene QuikChange site-directed mutagenesis protocol was used with the primers listed in FOXA1 luciferase reporter construct was generated as previously described [For the construction of FOXA1-HA, FOXA3-HA and dominant negative (dn) FOXA2-HA expression plasmids, the respective coding regions were PCR-amplified from cDNA from LS174T cells (FOXA1), from the CCSB human ORFeome V5.1 collection (FOXA3), and from cDNA from SW403 cells (dnFOXA2). The resultant cDNA fragments were cloned into a pCS2+ vector carrying the coding region for a hemagglutinin (HA)-epitope tag. The amplified coding region of FOXA1 contains a base pair exchange representing a natural variant , which leads to an amino acid change (A82T). This sequence variation however does not influence functional properties . The seqribed in . To geneescribed . All pla2. Stable cell lines with Dox-inducible protein expression were generated by retroviral transduction using the pRetroX-Tight-Pur vector system. As recipient cells either LS174T and HT29 cells stably transfected with the pN1pβ-actin-rtTA2S-M2-IRES-EGFP plasmid [LS174T, SW480, HCT116, HT29 and SW403 colorectal cancer cells were cultivated in DMEM supplemented with 10% (v/v) FCS, 10 mM HEPES, 1/100 (v/v) MEM non-essential amino acids solution and 1/100 (v/v) penicillin/streptomycin at 37°C and 5% CO plasmid  or HCT11 plasmid . To indu5 cells per well were seeded in 24-well plates. 4–6 h after seeding cells were transfected with 500 ng firefly reporter and 10 ng Renilla luciferase expression plasmids using the FuGENE6  transfection reagent. For Snail1 cotransfection experiments 5 ng Snail1-HA expression plasmid were used. After 48 h cells were harvested and lysed with lysis buffer (0.1% (v/v) NP-40; 250 mM KCl; 50 mM Tris-phosphate pH 7.8; 10% (v/v) glycerol) for at least 30 min at 4°C with gentle agitation. Lysates were cleared from cellular debris by centrifugation . Firefly and Renilla luciferase activities were measured in duplicates using a luminometer. For calculation of the relative luciferase activity (RLA) the Firefly luciferase activity was normalized to the Renilla luciferase activity to compensate for differences in transfection efficiency.For luciferase reporter assays, 1 x 105 cells were seeded in 24-well plates on round cover slips (12 mm diameter) coated with 0.2% gelatine. The following day, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Thereafter, cells were washed three times with PBS, followed by permeabilization at 4°C with 0.2% Triton X-100 for 30 min. After three further washing steps with PBS cells were incubated in 5% sheep serum at 4°C for 1 h. Subsequently, cells were washed once and incubated with the primary antibodies listed in ™, Canvas GFX, Inc., Fort Lauderdale, USA) whereby all panels in For immunofluorescence stainings, 4 x 10-ΔCt method with the signals from the FAIRE samples relative to the signals from noncrosslinked control samples. Primers are listed in Formaldehyde-assisted isolation of regulatory elements (FAIRE) was performed as described  with sli3) for 10 min at 67°C with agitation  were performed. As reference samples, aliquots with 20 μg chromatin were brought to a volume of 300 μl with elution buffer and processed in the same way. The eluates were mixed with 10 μg RNaseA and 300 mM NaCl and incubated at 67°C for 5 h to reverse the DNA-protein crosslinks, followed by digestion with proteinase K at 45°C for 1 h. DNA was purified using the QIAquick PCR Purification Kit according to the manufacturer’s protocol. For qPCR analysis, 1 μl precipitated ChIP DNA and 2% input material was used as template. Primers are listed in For chromatin preparation, cells were treated with 1% formaldehyde for 10 min at RT, followed by addition of 125 mM glycine for 5 min to quench crosslinking. After two washing steps with ice-cold PBS, cells were scraped off from the plates, spun down  and washed one more time with PBS. For cell lysis, cells were resuspended in NP-40 buffer (0.5% (v/v) NP-40; 85 mM KCl; 5 mM HEPES pH 7.9; 1 x Complete protease inhibitors  and incubated for 10 min on ice, followed by mechanical cell disruption by applying 50 strokes with a dounce homogenizer with tight fitting pestle. Cell nuclei were pelleted by centrifugation , resuspended in nuclei lysis buffer  SDS; 1 x Complete protease inhibitors) and incubated for 10 min on ice. To shear the chromatin to approximately 100–750 bp fragments, lysates were sonicated for 20–30 cycles (30 sec on/ 30 sec off) with high amplitude setting in a Bioruptor Plus device . Lysates were cleared from cellular debris by centrifugation  and the chromatin concentration was measured using a NanoDrop 2000 device . For immunoprecipitation, aliquots with 100–200 μg chromatin were filled up to a volume of 1 ml with sonication buffer  Triton X-100; 0.1% (w/v) sodium-deoxycholate; 0.1% (w/v) SDS; 0.5 mM PMSF; 1 x Complete protease inhibitors) and mixed with antibody  according to the supplier’s protocol. For the EMSA binding reaction, 10 fmol biotinylated probe, 1 μl in vitro translated protein, 1 μg dI:dC, 60 μg BSA, 5 mM DTT and 1x Complete protease inhibitor were mixed in EMSA buffer  and incubated on ice for 30 min. Samples were loaded onto a 6% polyacrylamide gel and separated by electrophoresis in TBE buffer  at 120 V for 3 h 15 min. Transfer of DNA::protein complexes onto a HybondN+ membrane was performed in TBE buffer at 340 mA for 1 h, followed by UV crosslink for 15 min. For the detection of DNA::protein complexes the Chemiluminescent Nucleic Acid Detection Module kit  was used according the manufacturer’s instructions followed by exposure of light-sensitive films.Probes for EMSA were generated by PCR using biotinylated primers. Primers are listed in For detection of FOXA1, FOXA1/A2, FOXA3 and Snail1 protein levels in CRC cells, nuclear extracts were used. Expression of all other proteins was analyzed using whole cell lysates. Protein isolation was performed as described . The antGAPDH expression and calculated using the 2-ΔCt method. Primers are listed in For RNA isolation the PeqGOLD total RNA kit  was used. The cDNA synthesis was done with the SuperScript II Reverse Transcriptase (RT) kit  according the manufacturer’s protocol with the following modifications: 500–2000 ng total RNA, 5 μM oligo-dT primer and 50 units of SuperScript II RT were used. qRT-PCR was performed with the CFX96 multicolor realtime PCR detection systems . For the PCR reaction, KAPA SYBR green reaction mix  was mixed with 0.2 μM primer and a cDNA amount equivalent to 20 ng total RNA. For gene expression analyses, data were normalized to EPHB3 enhancer and for the FOXA1 knockout was done with the CRISPR design tool developed by the Zhang lab (http://crispr.mit.edu) [EPHB3 enhancer are the following: FOX binding sites 5’-CTGGCTGGCTGTGTTTGTCCAGG-3’; TCF binding site 5’-GCACCAAGCTCCTTTGTTCTTGG-3’ and RBPJ binding site 5’- TCTCCAGGAAGCATTCCCGTGGG-3’. For the generation of a FOXA1 knockout the gRNA with the sequence 5’- CATGTTGCCGCTCGTAGTCATGG-3’ was used. Cloning of the gRNAs into the gRNA cloning vector  was performed as described in the gRNA synthesis protocol (https://www.addgene.org/static/cms/filer_public/13/af/13af84f5-50ee-472e-96f1-35c5b0533645/hcrispr-grna-synthesis.pdf) [EPHB3 locus was PCR amplified from genomic DNA from LS174T cells and transferred into a pUC18 vector. Mutagenesis of the transcription factor binding sites was done according to the QuikChange site-directed mutagenesis protocol with the primers listed in 6 cells were transfected with 0.5 μg Cas9-GFP, 0.5 μg gRNA, 0.5 μg dsRed expression vectors and, in the case of targeting transcription factor binding sites at the EPHB3 enhancer, 2.5 μg donor DNA plasmid by nucleofection using the Cell Line Nucleofector kit L . 72 h post nucleofection, GFP+/RFP+ cells were single cell sorted. After approximately 3 weeks cells were used for analyses.Design of the gRNAs targeting different transcription factor binding sites in the mit.edu) . The seqsis.pdf) . For CasEPHB3 enhancer, genomic DNA from LS174T single cell clones and LS174T wild-type cells was isolated using the DNeasy Blood & Tissue kit  according to the manufacturer’s instructions. The genomic DNA was used to amplify regions of the EPHB3 locus spanning the CRISPR/Cas9 cutting site by semiquantitative polymerase chain reaction using the primers listed in To identify genome-edited cell clones carrying mutations in the transcription factor binding sites in the WT clone 1C6, and the FOXA1KOA2A3lo/neg clones 2F8 and 2F12. Biotinylated cRNA was prepared with the Ambion MessageAmp kit for Illumina arrays according to the manufacturer’s protocol. Quality of cRNA was controlled using RNA Nano Chip Assay on an Agilent 2100 and hybridized to Illumina HumanHT12-v4 BeadChips  according to the manufacturer’s protocol. Raw microarray data were chip-wise processed using the Bioconductor R package beadarray [Total RNA was extracted in biological triplicates from untreated parental LS174T cells as well as the FOXA1eadarray  and subseadarray . P-valueeadarray , which teadarray . Only gehttp://epigenomegateway.wustl.edu/).We reanalyzed six ChIP-Seq data sets from the GEO database for FOXA1 binding in T47D, HepG2 and A549 cells  and for FOXA2 binding in Caco2, HepG2 and A549 cells . Individual fastq files were trimmed using trimmomatic and aligned to the Human Genome version 19 (hg19) by bwa-mem ,85. The VIM (VIMENTIN), FN1 (FIBRONECTIN) and CDH2 (N-CADHERIN), the EMT inducers SNAI1 (SNAIL1), SNAI2 (SNAIL2), ZEB1, ZEB2, TWIST1 and TWIST2, the epithelial marker gene CDH1 (E-CADHERIN), the EPHB receptors EPHB2 and EPHB3, the intestine-specific differentiation markers CDX1 and CDX2, and the tight junction proteins OCLN (OCCLUDIN), TJP1, TJP2, TJP3, and CLDN (CLAUDIN) 1, 2, 3, 4, 5, 7, 8, 12, 15 that are expressed in the human intestine [Pairwise correlation analyses and calculation of relative gene expression levels based on microarray data from CRC tumors and cell lines were performed as described ,41. The ntestine .t-test. Unless otherwise stated, the comparison was between Dox-treated versus untreated cells and between parental versus genome-edited cells. Significant changes are shown by the respective p-values represented with *: p < 0.05; **: p < 0.01; ***: p < 0.001. Non-significant changes are denoted with ns.Statistical analysis was performed using the unpaired, two-tailed Student’s S1 FigFN1, SNAI1, SNAI2, TWIST, VIM, ZEB1) and epithelial cells  was analyzed. Color scale represents relative expression levels as depicted by the bars on the left side of the panels. Unsupervised hierarchical clustering of relative gene expression levels from 290 primary CRCs (GSE14333) (A) and 151 CRC cell lines (GSE59857) (B). Expression of FOXA family members in relation to that of genes characteristic for mesenchymal cells ((TIF)Click here for additional data file.S2 Figin vitro translated FOXA proteins. Immunodetection of FOXA1 (A), FOXA2 (B) and FOXA3 (C) revealed a marginal crossreactivity of the FOXA1 antibody with FOXA2 and a considerable crossreactivity of the FOXA2 antibody with FOXA1. The FOXA3 antibody showed no signs of crossreactivity with FOXA1 or FOXA2 proteins.Western Blot analyses to test the specificity of the FOXA antibodies. Immunodetection of the HA-tag served to verify equal protein amounts of the (TIF)Click here for additional data file.S3 FigSnail1, FOXA1, FOXA2, and FOXA3 relative expression (rel. expr.) levels in HT29 cells stably transduced with Dox-inducible retroviral control or Snail1-HA expression vectors. Shown is the mean and SEM; n = 3. (B) Western Blot to analyze Snail1-HA, FOXA1, FOXA1/2, and FOXA3 protein levels upon Dox-induced Snail1-HA expression in HT29 cells. MW = molecular weight in kDa. To monitor equal protein loading RNA polymerase II (RNAPII) was detected. (C) ChIP analysis to test for Snail1-HA occupancy at the FOXA1 promoter in HT29 cells. Data were calculated as percent input. Shown are the mean and SEM; n = 4.(A) qRT-PCR analyses to assess (TIF)Click here for additional data file.S4 FigFOXA1 promoter. Mutations of the respective E-boxes are indicated by red crosses. E-box I apparently has a dual function. It is involved in activation of the FOXA1 promoter in the absence of Snail1-HA. Additionally, E-box I in part mediates the repressive effect of Snail1-HA. Shown is the mean and SEM; n≥3. RLA: relative luciferase activity. Statistical significance was calculated between samples without and with Snail1 expression. Luciferase reporter assay in LS174T (A) and HT29 (B) cells with constructs harboring the (TIF)Click here for additional data file.S5 FigFOXA1, FOXA2, FOXA3, CDH1, CLDN3, SNAI2, LEF1 and CDH11 in parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus was assessed by qRT-PCR analyses. rel. expr.: relative expression. Data are shown as mean and SEM; n = 3.Expression of (TIF)Click here for additional data file.S6 FigExpression of LEF1, CADHERIN11, E-CADHERIN and CLAUDIN3 in parental LS174T cells and cell clones subjected to CRISPR/Cas9-mediated genome editing of the FOXA1 locus was assessed by immunofluorescence studies. Areas within yellow frames were enlarged fivefold and are presented on the right. Scale bars: 50 μm and 10 μm (fivefold enlargements).(TIF)Click here for additional data file.S7 Fig(A) Total numbers and genic distribution of FOXA1/FOXA2 ChIP-seq peaks in different cell lines. The p-values refer to the results of bootstrapping analyses  to test whether the number of FOXA1/FOXA2 ChIP-seq peaks at epithelial and mesenchymal genes is significantly different from random groups of genes. (B) FOXA1 ChIP-seq data from HepG2 cells were analyzed by a bootstrapping approach to estimate whether the number of binding regions at epithelial genes is significantly high or low. Out of all 22,000 annotated genes random groups of N = 45 or N = 54 genes representing the sample size of epithelial and mesenchymal gene groups, respectively, were selected, and the numbers of associated peaks were counted. The resulting distribution of associated peak numbers from 10,000 trials is shown. The red lines indicate the number of associated peaks for the epithelial and mesenchymal gene groups. The p-values shown were calculated from fitting a skewed normal distribution to the histogram.(TIF)Click here for additional data file.S8 FigCDH2, FN1) and epithelial  signature genes. Localization and width of FOXA1/FOXA2 ChIP-seq peaks are represented by the differently sized bars at the bottom of the scheme. Vertical white lines were added to facilitate aligning ChIP-seq peaks and chromatin states. (B) Box-whisker plots depicting the pileup values from the MACS output for the transcription factors FOXA1 and FOXA2 for the two cell lines A549 and HepG2. The boxplots indicate median values of the population and the boxes indicate the interquartile range (IQR) from the first to the third quantile of the pileup distribution. The notches indicate the +/-1.58 IQR/sqrt(n), where n denotes the number of respective peaks. The whiskers indicate the 1.5 IQR. sqrt: square root.(A) Genome browser view of the 15-state chromatin model in relation to gene structure and FOXA1/FOXA2 binding regions for mesenchymal ((TIF)Click here for additional data file.S9 FigCDH1 +7.8 kb (A), the CDX2 +10.0 kb (B) and the EPHB3 −2.3 kb (C) ECRs. The consensus of the FOXA motifs is highlighted by a grey box. Sequence identity is indicated by asterisks. The indicated base positions are relative to the transcriptional start site of the human DNA sequence based on the Ensembl genome browser.Alignment of the DNA sequences of the FOXA motifs in the (TIF)Click here for additional data file.S10 FigCDH1 (A), CDX2 (B), and EPHB3 (C) in the indicated CRC cell lines. Data are shown as mean and SEM; n = 3.(A-C) qRT-PCR to analyze expression of (TIF)Click here for additional data file.S11 FigCDH1, the CDX2, and the EPHB3 enhancers in LS174T cells stably transduced with Dox-inducible retroviral control or Snail1-HA expression vectors. Data are given as percent input; n = 3. As control (ctrl) the EPHB3 locus at -10.0 kb was analyzed. (B) qRT-PCR to analyze expression of Snail1-HA, CDH1, CDX2, and EPHB3 in LS174T cells stably transduced with Dox-inducible retroviral control or Snail1-HA expression vectors. Data are shown as mean and SEM; n = 2. (C) Western Blot to analyze CDX2, E-CADHERIN (E-CAD), and EPHB3 protein levels in nuclear extracts  and whole cell extracts , respectively, upon Dox-induced Snail1-HA expression in LS174T cells. To monitor equal protein loading RNA polymerase II (RNAPII) and α-TUBULIN (α-TUB) was detected. MW = molecular weight in kDa.(A) ChIP analyses to test for binding of FOXA1 to the (TIF)Click here for additional data file.S12 FigEPHB3 −2.3 kb enhancer. Asterisks: non-specific bands. mut: mutated. (B) ChIP analyses using anti-HA antibodies showing recruitment of dnFOXA2-HA to the CDH1 +7.8 kb, the CDX2 +10.0 kb and the EPHB3 −2.3 kb enhancers upon Dox treatment in LS174T cells stably transduced with Dox-inducible dnFOXA2-HA expression vectors. As control (ctrl) the EPHB3 locus at -10.0 kb was analyzed. Data are given as percent input. Shown is the mean and SEM; n≥4.(A) EMSA to demonstrate binding of dnFOXA2-HA to the FOX binding motifs at the (TIF)Click here for additional data file.S13 FigEPHB3 enhancer exemplified by targeting the FOX binding motifs. The structure of the EPHB3 enhancer with the FOX binding sites is schematically shown at the bottom. The blue line denotes the position of the guide RNA. The structure of the donor plasmid is shown in the upper part. The left and right homology arms  are schematically indicated by grey trapezes. (B) CRISPR/Cas9-mediated mutation of the FOX, TCF and RBPJ binding sites within the EPHB3 enhancer. A part of the DNA sequence of the EPHB3 enhancer with the FOX binding sites (upper), the TCF binding site (middle) and RBPJ binding site (lower) is given. The sequence of the transcription factor binding sites is written in bold. The sequence targeted by the guide RNA is highlighted by light blue shading. The PAM is indicated by the red line. The cutting site of the Cas9 nuclease is marked by the arrowhead. The mutations introduced in the transcription factor binding sites by integration of the donor plasmid are shown in lowercase. The recognition sequence of the restrictions enzymes (written in green) used to test for successful integration of the donor plasmid is indicated in the DNA sequence by the green line. (C) Scheme of the EPHB3 enhancer and its known transcription factor binding sites. The positions of the EMSA probes P1 and P2 are shown in red and blue, respectively.  EMSA showing that the mutations introduced into the genomic DNA of LS174T cells interfere with binding of RBPJ (D), the TCF factor TCF7L2 (E) and the FOXA protein FOXA1 (E). Arrows mark the protein::DNA complexes.  Western Blot analyses to control the expression of the in vitro translated proteins used for EMSA shown in . MW = molecular weight in kDa.(A) Scheme of the strategy to mutate transcription factor binding sites at the (TIF)Click here for additional data file.S14 Fig(A) Surveyor assay to test for genome alterations in LS174T cell clones subjected to CRISPR/Cas9 genome editing to mutate the FOX binding sites (left panel), the TCF binding site (middle panel), and the RBPJ binding site (right panel). Changes in the genomic DNA of cell clones were detected by cleavage of a mixture of PCR products from wild-type and cell clone DNA. The specific cleavage pattern is indicated by the arrowhead. To test for homozygous DNA alterations, PCR products of LS174T cell clones were subjected to Surveyor nuclease digest without admixture of wild-type PCR products. Asterisks: non-specific PCR products. (B) Restriction enzyme digest to test for successful integration of the donor plasmid and thus mutation of the FOX binding sites (left panel), the TCF binding site (middle panel) and the RBPJ binding site (right panel) in LS174T cell clones subjected to CRISPR/Cas9 genome editing. Changes in the cleavage pattern indicative of successful binding site mutations are shown by the arrowheads. Asterisks: non-specific PCR products. (C) Electropherograms of the sequencing analyses of PCR products from LS174T cell clones subjected to CRISPR/Cas9 genome editing showing mutations of the FOX binding sites (upper panel), the TCF binding site (lower left panel) and the RBPJ binding site (lower right panel) in the respective cell clones. The DNA sequences with the transcription factor motifs (written in bold) are given above the electropherograms. The bases that are changed compared to the wild-type sequence are written lowercase. The sequences of the mutated transcription factor binding sites are underlined in red in the electropherogram.(TIF)Click here for additional data file.S15 FigFOXA1, FOXA2, and FOXA3 relative expression (rel. expr.) levels in LS174T cell clones subjected to CRISPR/Cas9 genome editing. Data are shown as mean and SEM; n = 3. Statistical significance was calculated between the respective cell clone and the LS174T wild-type cell pool. (B) Western Blot to analyze abundance of the FOXA proteins FOXA1, FOXA1/2, and FOXA3 (left panel), NICD , and β-CATENIN (β-CAT) and TCF7L2  (right panel) in LS174T cell clones subjected to CRISPR/Cas9 genome editing. MW = molecular weight in kDa. α-TUBULIN (α-TUB), GSK3β and RNA polymerase II (RNAPII) immunodetections served as loading controls. (C) qRT-PCR analyses to test for expression levels of the FOXA1/2 target gene MUC2, the Notch target gene NRARP and the Wnt/β-Catenin target gene AXIN2 in LS174T cell clones subjected to CRISPR/Cas9 genome editing. Shown are the mean and SEM; n = 3. Statistical significance was calculated between the respective cell clone and the LS174T wild-type cell pool.(A) qRT-PCR analyses to assess (TIF)Click here for additional data file.S16 FigFOXA1, FOXA3, CDH1, CDX2, and EPHB3 relative expression (rel. expr.) levels in HCT116 cells stably transduced with Dox-inducible retroviral control, FOXA1-HA or FOXA3-HA expression vectors. Data are shown as mean and SEM; n = 3. (B) Western Blot to analyze FOXA1-HA, FOXA3-HA, E-CADHERIN, CDX2 and EPHB3 protein expression in HCT116 cells stably transduced with Dox-inducible retroviral control, FOXA1-HA or FOXA3-HA expression vectors. MW = molecular weight in kDa. GSK3β and α-TUBULIN (α-TUB) immunodetections served as loading controls.(A) qRT-PCR analyses to assess (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Vitamin D deficiency is associated with an increased risk of Alzheimer’s disease and increased beta-amyloid (Aβ) in animals. Hence we sought to investigate the relationship between plasma 25-hydroxyvitamin D (25(OH)D) and cerebral Aβ in older adults with subjective memory complaints.18F]-florbetapir positron emission tomography. Plasma 25(OH)D was measured at study baseline using a commercially available electro-chemiluminescence competitive binding assay. Standard uptake value ratios (SUVRs) were generated using the cerebellum as a reference. Brain regions assessed included the cortex, anterior cingulate, anterior putamen, caudate, hippocampus, medial orbitofrontal cortex, occipital cortex, parietal cortex, pons, posterior cingulate, posterior putamen, precuneus, semioval centre and temporal cortex. Associations were explored using fully adjusted multiple linear regression models.This is a secondary analysis of the Multidomain Alzheimer Preventive Trial. Participants were 178 dementia-free individuals aged 70 years or older with data on plasma 25(OH)D and cerebral Aβ load assessed by [p > 0.05) between baseline 25(OH)D levels and Aβ in any of the brain regions studied.Participants had a mean (SD) age of 76.2 years (4.4) and 59.6% were female. The mean (SD) plasma 25(OH)D level was 22.4 ng/ml (10.8) and the mean (SD) cortical SUVR was 1.2 (0.2). We did not find any cross-sectional associations (These preliminary results suggest that circulating 25(OH)D is not associated with cerebral Aβ in older adults. Further longitudinal studies with the measurement of mid-life vitamin D status are required to explore the relationship between vitamin D and Aβ accrual over time, thereby circumventing the shortfalls of a cross-sectional study. Vitamin D is a fat-soluble steroid hormone that seems crucial for brain health in humans –5. Low vDespite the controversy surrounding the role of vitamin D in cognitive function and AD, animal studies suggest that vitamin D hypovitaminosis results in increased brain beta-amyloid (Aβ)  and vita18F]-florbetapir positron emission tomography (PET) study carried out as an ancillary project to the Multidomain Alzheimer Preventive Trial (MAPT) [n = 1680). Both the MAPT and the ancillary [18F]-florbetapir PET study were approved by the ethics committee in Toulouse (CPP SOOM II) and written consent was obtained from all participants.Data were obtained from a -florbetapir as described previously [18F]-Florbetapir. Radiochemical purity of [18F]-Florbetapir was superior to 99.5%. Regional standard uptake value ratios (SUVRs) were generated from semi-automated quantitative analysis with the whole cerebellum used as the reference region. Cortical-to-cerebellar SUVRs  were obtained using the mean signal of the predefined cortical regions: frontal, temporal, parietal, precuneus, anterior cingulate and posterior cingulate as described previously -florbetapir imaging data and plasma 25(OH)D measurements, providing a more accurate value for vitamin D levels, as opposed to vitamin status assessed through dietary questionnaires. In addition, we considered several cofounders, including a possible interaction between vitamin D and ApoE ε4 status. The limitations of our study included the cross-sectional design, which precluded the examination of the relationship between plasma 25(OH)D and change in cerebral Aβ load over time. PET scans were also performed throughout the 3-year period of the MAPT; therefore the study design was not truly cross-sectional. The inclusion of the time interval between baseline 25(OH)D measurements and the PET scan as a confounder in the linear regression models probably served to mitigate this bias. Furthermore, we only had data availability for vitamin D status in later life which might perhaps not be the optimal time window for studying the associations between vitamin D and cerebral Aβ.The strengths of the current study are the relatively large sample size and the availability of PET [We have shown here that circulating 25(OH)D was not associated with cerebral Aβ in older adults at risk of dementia cross-sectionally. However, a longitudinal observational study including mid-life vitamin D measurements is warranted to examine the relationship between plasma 25(OH)D and cerebral Aβ accrual over time.


Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression.Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted  de novo super-enhancer-like element to drive high viral oncogene expression. This provides insight into the genesis of super-enhancer-like elements and is a novel mechanism by which HPV integration can promote oncogenesis.Oncogenic human papillomavirus (HPV) infection is responsible for ~5% human cancers. A key event in the development of many of these cancers is integration of the viral genome into host chromatin. Integration results in dysregulated expression of the viral oncogenes, which promotes unregulated cellular division and the accumulation of cellular mutations, ultimately leading to cancer. Genetic rearrangement and/or amplification of cellular sequences flanking sites of integration are frequent in HPV positive tumors, and this can influence transcription of the viral oncogenes from integrated loci. We show an example where the HPV16 genome integrated adjacent to a cellular, tissue-specific enhancer and subsequently the viral DNA and flanking cellular sequences were co-amplified into a tandemly interspersed viral-cellular sequence array. The genetic and epigenetic signature of this site promoted the formation of a  High oncogenic-risk human papillomaviruses (HPVs) are the etiological agent responsible for virtually all cervical cancers , and a gE2 gene, which eliminates its repressive function over the E6/E7 oncogenes -dCTP-labeled HPV16 DNA in hybridization buffer . Radiolabeled probe was generated from 50 ng of gel-purified linear HPV16 DNA with a Random Prime DNA labeling kit (Roche). Hybridized DNA was visualized and quantitated by phosphorimaging on a Typhoon Scanner (GE Bioscience).Total DNA was harvested with the DNeasy Blood and Tissue kit (Qiagen). 1 μg total DNA was digested with either a single-cut linearizing enzyme (BamHI) for the HPV16 genome or a non-cutter (HindIII) to linearize cellular DNA. After digestion, samples were separated on 0.8% Tris-acetate-EDTA (TAE) agarose gels. A known quantity of linear HPV16 DNA was run on the same gel as the samples. DNA was visualized with 0.5 mg/ml ethidium bromide and transferred onto nylon membranes with a TurboBlotter downward transfer system (Whatman). Membranes were UV cross-linked, dried, incubated with prehybridization blocking buffer , and then incubated overnight with [6 W12 cells using the DNeasy Blood and Tissue kit (Qiagen) and sequenced (2 x 150 bp paired-end reads) using the Illumina HiSeq 4000 platform . Alignment of WGS data was performed using Bowtie 2 (version 2.2.5) [Total cellular DNA was purified from 2 x 10n 2.2.5) , 47.To confirm virus-host insertional breakpoints, we designed a custom panel of Agilent SureSelect XT v.G.1 HPV16 genome sequences to detect viral genomic sequences and flanking host genomic DNA sequences in various samples . We perCells were cultured on coverslips and fixed at room temperature in 4% paraformaldehyde (PFA)–PBS for 20 minutes. Cells were permeabilized in 0.1% Triton X-100 and stained with primary and fluorescent secondary antibodies using standard procedures. Coverslips were mounted in ProLong Gold containing 4’,6-diamidino-2-phenylindole (DAPI) for analysis by confocal microscopy. Anti-BRD4 CW152 rabbit polyclonal antibody was used at 1:100 dilution [Cells grown on coverslips were fixed in cold methanol-acetic acid (3:1) for 1–3 minutes followed by 4% paraformaldehyde–PBS for 10 minutes. Cells were treated with RNase A for 1 hour at 37°C and dehydrated in a 70%, 90%, and 100% ethanol series for 3 minutes each. Green 5-Fluorescein dUTP labelled BAC clone RP11-1140H22  was purchased from Empire Genomics. DNA-FISH probe against full-length HPV16 DNA was fluorescently labeled using the Alexa Fluor-594 FISH Tag DNA Multicolor Kit (Life Technologies) following manufacturer’s protocol. To each coverslip, 40–50 ng labeled DNA FISH-probe in hybridization buffer (Empire Genomics) supplemented with 50 μg/ml human Cot-1 DNA (Invitrogen) was added. DNA denaturation was performed at 75°C for 5 minutes, followed by hybridization at 37°C overnight. Cells were washed for 5 minutes each with 1x phosphate-buffered detergent (Qbiogene) at room temperature, 1x wash buffer  at 65°C, and 1x phosphate-buffered detergent at room temperature. Coverslips were mounted in ProLong Gold (ThermoFisher) containing DAPI for analysis by confocal microscopy.Cells grown on coverslips were fixed in cold methanol-acetic acid (3:1) for 1–2 minutes followed by 4% paraformaldehyde–PBS for 10 minutes. Cells were processed for immunofluorescence staining followed by DNA FISH using methods described above and as described previously .7 cells were crossed-linked with 1% formaldehyde and sheared to 100–500 bp DNA fragments using a Bioruptor sonicator (Diagonode) on high power settings. An aliquot of the lysate (5%) was processed for input control. Chromatin samples (20 μg per ChIP) were incubated overnight at 4°C with antibodies against Brd4  and H3K27ac . No-antibody controls were included to measure non-specific binding. Chromatin-immunocomplexes were precipitated for 1 hour at 4°C with blocked Dynabeads Protein G (Invitrogen), subjected to multiple wash steps and the chromatin eluted. Recovered DNA was reverse cross-linked overnight at 65°C with 5 M NaCl, followed by RNase A and proteinase K treatment, and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). ChIP DNA was analyzed by real-time qPCR, using primers listed in Supplementary W12 cells were processed as previously described . BrieflyW12 cells were processed as described above for ChIP-qPCR. ChIP DNA samples were pooled and sequenced (2 x 150 bp paired-end reads) using the Illumina HiSeq 4000 platform . Alignment of ChIP-seq data was performed using Bowtie 2 (version 2.2.5) , 47.Total RNA was purified from W12 cells using the RNeasy Mini Kit (Qiagen) and RNA integrity determined using the Bioanalyzer 2100 (Agilent Technologies). Polyadenylated RNA was sequenced (2 x 150 bp paired-end reads) using the Illumina HiSeq 4000 platform . RNA-seq data was aligned to reference genomes using TopHat2 (version 2.1.0)  and exprHPV16 DNA inserted into the BamHI site of pUC18/19 was digested with BamHI and extracted from a 0.8% agarose gel to remove pUC18/19. Biotinylated HPV16 genome probe was synthesized with BioPrime DNA Labeling System  using 500 ng of digested HPV16 DNA incubated at 37°C for 4 hours. Biotinylated probe was purified using QIAquick PCR Purification Kit .6 cells per ml. Diluted cells were warmed to 37°C for 1–2 minutes, and an equal volume of 1.5% SeaPlaque agarose  in 1x PBS, warmed to 45°C, was added. Cell/agarose mixture was aliquoted into plug mold wells  and solidified for 20 minutes at 4°C. Solid plugs were ejected into lysis buffer . Melted plug was cooled for 5 minutes in a 42°C water bath. 3 units of β-agarase  were added to cooled DNA solution and incubated overnight in a 42°C water bath. DNA solution was carefully poured into a 2 ml Teflon reservoir. A silanized coverslip was inserted into the DNA solution and incubated for 5 minutes. The coverslip was removed and adhered to a glass slide with cyanoacrylate glue. A test coverslip was pulled, stained with YOYO-1 , and visualized with a microscope to ensure quality and density of DNA fibers. Coverslips were baked for 30 minutes at 60°C to crosslink DNA fibers to coverslip.DNA fibers were denatured for 20 minutes in 0.2 M NaOH. Slides were neutralized 5 times with 1x PBS (pH 7.4) for 1 minute. Slides were dehydrated for 3 minutes each in successive baths of 70%, 90%, and 100% EtOH and air dried for 8 minutes. 500 ng of HPV16 biotinylated probe was mixed with 5 μg of human Cot-1 DNA and hybridization buffer  up to a total volume of 20 μL. Probe mixture was added to coverslip, a clean coverslip was placed on top and sealed with rubber cement. Slides were denatured for 5 minutes at 75°C and then incubated for 16 hours at 37°C. The rubber cement and top coverslips were removed from the slides and the slides rinsed with 2x SSC. The slides were washed three times with 2x SSC/50% formamide for 5 minutes, and then washed three times with 2x SSC for 5 minutes. Slides were blocked for 1 hour in a humidified chamber with 5% BSA in 1x PBS and washed 3 times for 5 minutes with PBS-T (0.1% Triton X-100 in 1x PBS). To visualize biotinylated probe, slides were incubated in a humidified chamber at 37°C in the following layers. Layer 1: 1:100 streptavidin, Alexa Fluor-488 conjugated  in 5% BSA/1x PBS for 30 minutes. Layer 2: 1:200 biotinylated anti-streptavidin  in 5% BSA/1x PBS for 1 hour. Layer 3: 1:200 streptavidin, Alexa Fluor-488 conjugated in 5% BSA/1x PBS for 30 minutes. Layer 4: 1:200 biotinylated anti-streptavidin and 1:200 anti-ssDNA IgG2a  in 5% BSA/1x PBS for 1 hour. Layer 5: 1:200 streptavidin, Alexa Fluor-488 conjugated, and 1:100 goat anti-mouse IgG2a, Alexa Fluor-647 conjugated , in 5% BSA/1x PBS. Three 5 minute washes with PBS-T were performed in between each layer. Slides were rinsed with 1x PBS, mounted with ProLong Gold .Digoxigenin labeled probes were synthesized with BioPrime DNA Labeling System  using 500 ng BsaBI and XcmI digested HPV16 DNA. The biotinylated nucleotide mix from the kit was replaced with PCR DIG Probe Synthesis Mix  and the labeling reaction incubated at 37°C for 4 hours. The DIG probe was purified using QIAquick PCR Purification Kit . Biotinylated probes were prepared as described previously. Following combing, DNA fibers were denatured for 20 minutes in 0.2 M NaOH, and neutralized five times with 1x PBS (pH 7.4) for 1 minute. Slides were dehydrated for 3 minutes each in successive baths of 70%, 90%, and 100% EtOH and air dried for 8 minutes. Biotinylated/DIG probes in an equivalent ng/kb ratio, 200 ng of an 8 kb probe and 620 ng of a 25 kb probe, were mixed with 5 μg human Cot-1 DNA in hybridization buffer  to a total volume of 20 μL. Probe mixture was added to the coverslip, a clean coverslip was placed on top and sealed with rubber cement. Slides were denatured for 5 minutes at 75°C and incubated for 16 hours at 37°C. Slides were washed three times with 2x SSC/50% formamide for 5 minutes and three times with 2x SSC for 5 minutes. Slides were blocked for 1 hour in a humidified chamber with 5% BSA in 1x PBS and washed 3 times for 5 minutes with PBS-T (0.1% Triton X-100 in 1x PBS). To visualize the biotin and DIG-labeled probes, slides were incubated in a humidified chamber at 37°C with the following layers: Layer 1: 1:100 streptavidin, Alexa Fluor-555 conjugated , and 1:100 anti-digoxigenin, FITC conjugated , in 5% BSA/1x PBS for 1 hour. Layer 2: 1:200 biotinylated anti-streptavidin  and 1:200 anti-FITC  in 5% BSA/1x PBS for 45 minutes. Layer 3: 1:200 streptavidin, Alexa Fluor-555 conjugated in 5% BSA/1x PBS for 30 minutes. Layer 4: 1:200 biotinylated anti-streptavidin, 1:200 anti-rabbit, FITC conjugated , and 1:200 anti-ssDNA IgG2a  in 5% BSA/1x PBS for 45 minutes. Layer 5: 1:200 streptavidin, Alexa Fluor-555 conjugated, and 1:100 goat anti-mouse IgG2a, Alexa Fluor-647 conjugated , in 5% BSA/1x PBS. Three 5 minute washes with PBS-T were performed in between each layer. Slides were rinsed with 1x PBS, mounted with ProLong Gold . All slides were imaged using a Leica TCS-SP5 laser scanning confocal imaging system.Measurement of signal lengths from raw Leica files was performed using Bitplane Imaris x64. Images were analyzed in Surpass with an orthogonal camera. Measurement points of equal diameter were placed on the first and last points of signal for each HPV16 genome. Dots of signal were considered contiguous if they were within 0.6 μm of a previous dot. Genomes continuous in a fiber were measured with a single line of measurement points to determine the length of any interspersing DNA. Base pair length was determined using 1 μm = 2 kb conversion.S1 FigA, Representative image of a fiber containing the 20861 type II integration site. The DNA strand was visualized with a single stranded DNA antibody, shown in red. The HPV16 FISH signal is shown in green. B, Scatter plot showing number of HPV16 genomes per individual DNA fiber. Average signal count per fiber and SD is shown along with total number of fiber counts. Greatest number of genomes found in a single fiber is 16.4. HPV16 genome copy number was calculated by dividing site length (kb) by 7.9 kb (unit length of HPV16 genome). Data are from two biological replicates.(EPS)Click here for additional data file.S2 FigAlignment of WGS data from the 20861, 20862, 20831 and 20863 cell lines to the HPV16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional (red) breakpoints. The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in (EPS)Click here for additional data file.S3 FigBrd4 was detected in NIKS (HPV-negative), W12 sub-clones  and cervical carcinoma derived cell lines (C-33A (HPV-negative), C-411, CasKi, HeLa, ME-180 and SiHa) by indirect immunofluorescence with the CW152 Brd4 antibody (green). Data and images shown were obtained from z-stacks of the entire cell combined using maximum projection. DAPI was used as a nuclear stain.(PDF)Click here for additional data file.S4 FigA, Representative image of a fiber containing the 20831 Type III integration site. Image is representative of a conserved pattern. The DNA backbone was visualized with a single stranded DNA antibody, shown in red. The Type III site of 20831 cells contains two sizes of integrated HPV16, whole and partial, and three sizes of interspersing host DNA, large, small, and smallest. B, Scatter plot showing number of HPV16 genomes per individual DNA fiber in the Type III integration site. Average signal count per fiber and SD is shown along with total number of counts. Data are from two biological replicates. Greatest number of genomes found in a single fiber is 18. C, Scatter plot showing measured HPV16 signal lengths. Two populations of HPV16 signal size represent whole (N = 240) and partial (N = 72) genomes shown in A. Data are from two biological replicates. D, Scatter plot showing measured length of the interspersing space between HPV16 signals from every fiber. Three populations of interspersing host DNA lengths represent the large (N = 96), small (N = 85), and smallest (N = 69) lengths seen in A. Measurements were based on the conversion 1 μm = 2 kb. Average measured length in kb and SD is shown along with total number of fibers. Data are from two biological replicates. E, Representative image of a fiber containing the 20831 Type II integration site. The DNA backbone was visualized with a single stranded DNA antibody, shown in red. F, Scatter plot showing number of HPV16 genomes per individual DNA fiber of the Type II integration site. Average signal count per fiber and SD is shown along with total number of fibers. Greatest number of genomes found in a single fiber is 56.7. HPV16 genome number was calculated by dividing site length (kb) by 7.9 kb (unit length of HPV16 genome). Data are from two biological replicates.(EPS)Click here for additional data file.S5 FigA, Representative image of a fiber containing the 20862 Type III integration site. The DNA backbone was visualized with a single stranded DNA antibody, shown in red. B, Scatter plot showing number of HPV16 genomes per individual DNA fiber of the Type III integration site. Average signal count per fiber and SD is shown along with total number of counts. Greatest number of genomes found in a single fiber is 13. Data are from two biological replicates. C, Scatter plot showing measured HPV16 signal length for the Type III integration site. D, Scatter plot showing measured length of the interspersing space between HPV16 signals from every fiber. Two populations of differing interspersing host length units could represent separate patterns of Type III integration in 20862 cells. Measurements were based on the conversion 1 μm = 2 kb. Average measured length in kb and SD is shown along with total number of fibers. E, Representative image of a fiber containing the 20862 Type II integration site. The DNA backbone was visualized with a single stranded DNA antibody, shown in red. F, Scatter plot showing number of HPV16 genomes per individual DNA fiber of the Type II integration site. Average signal count per fiber and SD is shown along with total number of fibers. Greatest number of genomes found in a single fiber is 59.8. HPV16 genome number was calculated by dividing site length (kb) by 7.9 kb (unit length of HPV16 genome). Data are from two biological replicates.(EPS)Click here for additional data file.S6 FigA, Southern blot analysis of W12 cell lines. Genomic DNA was digested with either HindIII  or BamH1 . DNA fragments were separated by electrophoresis, transferred to membranes and probed with 32P-labeled HPV16 DNA. Arrows indicate Type II or Type II integration patterns. Southern blot is duplicated from B, Predicted BamHI and HindIII restriction fragments at the 20861, 20831 and 20862 Type III integration loci. C, Summary of properties of the 20861, 20831 and 20862 Type III integration loci. a Determined from Southern blot analysis (see A and B); b Fold difference in Type III fusion mRNA expression relative to 20861 cells; c With respect to transcriptionally active Type III integration site, determined by ChIP-seq and immunofluorescence. D, Total DNA was extracted from 20861 and 20863 cells and qPCR analysis performed using primers targeting the cellular flanking sequence at the 20861 integration site. Copy number was determined against a standard curve and normalized to ACTB. Average copy number was calculated from two independent experiments. Error bars represent SD. See Supplementary E-F, Total RNA was extracted from 20831, 20861 and 20862 cells and RT-qPCR performed using primers targeting E6*I splice transcript and viral-host fusion mRNAs. mRNA expression levels were calculated against a standard curve and normalized to PPIA. Error bars represent SD across three independent experiments. See Supplementary (EPS)Click here for additional data file.S7 FigA, Alignment of RNA-seq reads from W12 20831 and 20862 cells to the human (hg19) and HPV type 16 isolate 16W12E (AF125673.1) reference genomes showed expression of a viral-cellular fusion transcript spliced into a cryptic acceptor within an intergenic region at chr3: 189,662,800. Data are averaged from three biological replicates. B, Alignment of ChIP-seq reads to the human reference genome (hg19) in 20863 cells containing extrachromosomal HPV16, and 20861, 20831 and 20862 cells containing integrated HPV16 genomes. Brd4 and H3K27ac signatures encompassing the integration locus at chr3: 189,662,800 are similar across all four W12 sub-clones. Data are averaged from two biological replicates. C, Alignment of ChIP-seq reads to human and HPV type 16 isolate 16W12E (AF125673.1) reference genomes in 20831 and 20862 cells did not show enrichment of super-enhancer markers over the Type III integration site as is the case in 20861 cells Click here for additional data file.S1 TableBold font; the most strongly supported insertional breakpoints matched analysis of WGS data Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in  Since their boosting effect on biomass degradation was discovered in 2010 .TaLPMO9A [PaLPMO9B [In the Carbohydrate Active Enzyme database (CAZy), LPMOs are currently classified in Auxiliary Activity (AA) family 9, 10, 11 and 13 ,6,7. LPMPaLPMO9B , but thePaLPMO9B .Neurospora crassa LPMOs [Several hypotheses have been formulated on the underlying causes for these differences in regioselectivity. Based on the accumulating structural data, the accessibility of the solvent-facing axial position of the catalytic copper site is believed to determine the C1/C4-oxidation ratio ,18. Strusa LPMOs . The possa LPMOs ,12,20,21sa LPMOs . It is cExcept for a colorimetric assay to test the catalytic efficiency based on a side reaction of LPMOs , all preHjLPMO9A’s aromatic surface residues in the C1/C4 oxidation by site-directed mutagenesis.Here, we present an indicator diagram to quantify the impact of a rational engineering strategy on regioselectivity. The diagram was successfully used to confirm the role of Neurospora crassa LPMO9C (Uniprot ID Q7SHI8) and Phanerochaete chrysosporium LPMO9D (H1AE14) were synthesized using the GeneArt gene synthesis service and codon optimized for Pichia pastoris. P. pastoris strain CBS7435 and the pPpT4 plasmid were provided by the Institute of Molecular Biotechnology in Graz, Austria. See The genes for P. pastoris and integrated into the pPpT4 vector downstream of the methanol-inducible AOX1 promotor, preceded by their native secretion signal and with a His6 tag attached to their C-terminus. The cloning of Hypocrea jecorina LPMO9A gene (O14405) and PcLPMO9D gene was described earlier [NcLPMO9C gene was inserted into the pPpT4 backbone using the CLIVA method [PfuUltra High-Fidelity DNA Polymerase (Agilent). Escherichia coli strain BL21 (DE3) was used for plasmid construction and was transformed by electroporation.In all cases, the LPMO genes were codon optimized for  earlier . The NcLA method . All priSwaI-linearized and transformed into freshly prepared electro-competent P. pastoris CBS7435 cells [P. pastoris control strain was prepared by transforming an empty SwaI-linearized pPpT4 vector into the wildtype strain. Transformants were grown on YPD plates supplemented with 100mg/L zeocin.After confirming the sequence (LGC Genomics), plasmid DNA was 35 cells . A P. paHjLPMO9A sequence using a two-stage PCR reaction based on the Sanchis method using Q5 High-Fidelity DNA Polymerase (Bioke) [DpnI, the mutagenized plasmid was transformed in E. coli BL21 (DE3) cells by electroporation.Enzyme variants were obtained by site-directed mutagenesis of the  (Bioke) . TogetheSwaI-linearized and transformed into freshly prepared electro-competent P. pastoris CBS7435 cells. Transformants were grown on YPD plates supplemented with 100 mg/L zeocin.After confirming the sequence (LGC Genomics), plasmid DNA was E. coli LPMO clones were cultivated in Luria-Bertani broth supplemented with 25 mg/L zeocin.P. pastoris LPMO transformants were grown on BMGY medium  and induced with BMM2Y (BMGY medium without glycerol but with 2% (v/v) methanol) and BMM10Y (with 10% (v/v) methanol).P. pastoris strains were maintained on YPD plates , if necessary supplemented with 100 mg/L zeocin.LPMO transformants were cultivated and expressed in 96-deep-well plates (Enzyscreen) tilted under an angle of 25° following the microscale protocol described by Jacobs et al. , with smThe best-producing transformant was cultivated and expressed in a 250mL shake flask at 30°C and 200 rpm. Growth was started in 25 mL BMGY medium, induction followed 60 hours later by adding 25 mL BMM2Y medium. Methanol induction was maintained by adding two shots of 1 mL BMM10Y a day. After 5 days of induction cultures were harvested by centrifugation at 1500 x g for 20 minutes (4°C).The culture supernatant was concentrated and washed with 10 mM sodium acetate buffer (pH 5) by ultrafiltration using 10 kDa Ultracel ultrafiltration disks (Millipore) in an Amicon stirred ultrafiltration cell. A final volume of 2 mL concentrated enzyme was obtained.For protein purification, culture supernatant was first ultrafiltrated as described above, using PBS buffer (50 mM sodium phosphate and 300 mM sodium chloride at pH 7.4), to a final volume of about 5 mL. After adding imidazole to a concentration of 10 mM, the ultrafiltrated samples were applied to 1.5 mL equilibrated Ni-NTA agarose slurry (MC-lab) in 10 mL purification columns. The columns were incubated for 1 h at 4°C while gently rotating to allow binding to the resin. Next, the columns were washed with 3 x 8 mL of 20 mM imidazole in PBS-buffer and the protein was finally eluted in 10 mL of 250 mM imidazole solution. The buffer was exchanged for 10 mM sodium acetate (pH 5) and the sample was concentrated to 2 mL using Vivaspin 20 columns with 10 K PES membrane (Sartorius).HjLPMO9A, a bioreactor cultivation was performed in a 5L fermentor vessel (Infors HT Labfors) starting with 4L of BSM medium containing 6% glycerol at 30°C, 2 vvm of air and 1200rpm. After the batch phase, six methanol pulses between 0.5–2.0% were performed for induction  using 20 mM PBS buffer (500 mM NaCl) supplemented with 20 mM imidazole and further filtrated using 0.2 μm filters (Roth). The concentrated culture supernatant was purified by immobilized metal affinity chromatography (IMAC) using 3x 1 mL HisTrap columns (GE). After elution using 20 mM PBS buffer (500 mM NaCl) with 500 mM imidazole, the buffer was exchanged for 10 mM sodium acetate buffer (pH 5) using Amicon-Ultra 15 spin tubes with 10 kDa cut-off (Millipore). Although this protocol had a higher yield, it did not have additional benefits in removing the endoglucanase background and was therefore not routinely pursued.For large scale production of wildtype Culture supernatant and concentrated enzyme were analyzed using 12% SDS-PAGE gels to confirm the presence of the LPMO enzyme. Protein bands were visualized by staining with colloidal Coomassie G-250 stain (Bio-Rad) and the PageRuler prestained protein ladder (Thermo Fisher Scientific) was used for mass determination.A dilution series of bovine serum albumin (0.3–0.05 mg/L) was added to each gel to get an estimation of the protein concentration of the ultrafiltrated culture supernatant, using the digital imaging software ImageJ as described earlier ,31.-1.cm-1, 52870 M-1.cm-1 and 52870 M-1.cm-1 for HjLPMO9A wildtype, variant Y24A and variant Y211A, respectively.The concentration of purified enzyme was measured using a Nanodrop device with extinction coefficients of 54360 MHjLPMO9A and NcLPMO9C) or 1 mM MES buffer (PcLPMO9D) at a final pH of 4.5 and 6.1, respectively. The regioselectivity indicator diagram was developed by using a HjLPMO9A dilution series of 20–250 μg enzyme. All measurements and calculations for the development of the indicator diagram were done independently in triplicate. In the final indicator diagram the slope of the WT line is calculated as the average of the independently calculated lines.Reaction mixtures of 500 μL contained 0.5% Phosphoric Acid Swollen Cellulose (PASC) and 1 mM ascorbic acid in 1 mM sodium acetate  at 50°C (HjLPMO9A and PcLPMO9D) or 40°C (NcLPMO9C) while shaking at 1400 rpm using an Eppendorf Thermomixer.The activity tests with 25 μL samples were taken every 30 min and the reaction was stopped by heat-inactivation (10 min at 95°C), which was proven to inactivate the enzymes. Samples were diluted 10-fold before HPAEC-PAD analysis using the protocol described by Forsberg et al., using a CarboPac PA-10 column  instead of the CarboPac PA1 column used in the original protocol .P. pastoris, namely P. chrysosporium LPMO9D [N. crassa LPMO9C [T. reesei LPMO9A [A characterized member of each LPMO regioselectivity type was recombinantly expressed in m LPMO9D , N. crasa LPMO9C ,34 and Ti LPMO9A  as C1, CThe soluble reaction products released upon incubation of the three LPMOs with the polymeric substrate PASC were analyzed by HPAEC-PAD . HPAEC-PPcLPMO9D as strict C1-oxidizer and HjLPMO9A as C1/C4-oxidizer, but signals of C1-oxidized products are also observed for the strict C4-oxidizer NcLPMO9C. However, this C1-oxidation response is not significantly higher than the C1-oxidative background activity in the P. pastoris CBS7435 wildtype strain , located on the LC-loop, was demonstrated to be important for interactions near subsite -3/-4, but due to the lack of mutagenesis studies, residues important for C4-oxidation have not been identified yet [As no crystal structure is available, a homology model, based on 3ZUD as a template, was used to identify all surface exposed aromatic residues in HjLPMO9A . ThroughHjLPMO9A . In partectivity . In NcLPfied yet .For the two mutations with the highest effect on regioselectivity, three more enzyme concentrations were examined and included in the indicator diagram . This coHjLPMO9A enzyme and its variants, the total amount of oxidized species was compared based on their peak areas in HPAEC-PAD chromatograms. The results are shown in In order to get an idea of the overall activity of the To further validate our findings, the effect on regioselectivity was also determined with purified enzymes, in which the endoglucanase background was no longer present. In that case, the HPAEC-PAD chromatograms became more complex, and showed a clear shift towards products with higher DP see .The results obtained with variant Y24A are in agreement with the effect observed after deleting the characteristic helix in enzyme NCU07760, although the single mutation Y24G did by itself not alter regioselectivity . Most liSince their discovery in 2010, many studies have addressed the structure and mechanism of LPMOs, but very few publications describe enzyme engineering experiments. One of the reasons could be the lack of a convenient screening assay, meaning that such studies would have to rely on lengthy incubations and exhaustive analyses of HPAEC-PAD chromatograms. The indicator diagram established here could partially alleviate this burden, as the incubation time can be kept relatively short (4 hours) and the interpretation is based on just 2 signals in the chromatogram. In future experiments, the workload could be reduced even more by using only one enzyme concentration instead of the complete indicator line that has been examined here to validate the diagram. This experimental cut down would indeed be justified as the relationship between the A2- and K3-rates was found to be constant over a wide range of enzyme concentrations .P. pastoris and time-course monitoring of cellulose cleavage led to the establishment of an indicator diagram that enabled quantitative determination of their regioselectivity. As proof of concept, the effect of four point mutations was examined in detail by performing activity tests at different enzyme concentrations. By using this diagram, we were able to demonstrate that aromatic surface residues are important for regioselectivity in Hypocrea jecorina LPMO9A. This is in accordance with the hypothesis that by carefully orienting the oxidative force of the copper ion towards the C1 or C4 glycosidic position, these aromatic residues can determine the exact site of oxidation [Expressing a representative member of each LPMO class in xidation . FurtherS1 FigHypocrea jecorina LPMO9A native enzyme; lane 2: Phanerochaete chrysosporium LPMO9D native enzyme; lane 3: Neurospora crassa LPMO9C native enzyme; lane 4: PageRuler prestained protein ladder (Thermo Scientific); lane 5: histag purified HjLPMO9A; lane 6: histag purified HjLPMO9A variant Y24A; lane 7: HjLPMO9A variant Y211A.Lane 1: (DOCX)Click here for additional data file.S2 FigHjLPMO9A.Time courses of the three aldonic acid  and three 4-ketoaldose  peaks released upon incubation of PASC with a dilution series of (DOCX)Click here for additional data file.S3 Fig12 + Stdev22 + Stdev32)).The total amount of oxidized cello-oligosaccharides released by 70μg of LPMO after 2.5h incubation at 50°C is compared to gain insight in the overall activity changes of the mutations. The values are means of three replicates, error bars correspond to a cumulated total standard deviation Click here for additional data file.S4 FigHjLPMO9A and regioselectivity mutants Y24A (with higher C1-oxidative capacity) and Y211A (with higher C4-oxidative capacity). Two time points (after 1h and 4h incubation) are shown for each enzyme variant.Chromatograms of wildtype (DOCX)Click here for additional data file.S5 FigHjLPMO9A and regioselectivity mutants Y24A (with higher C1-oxidative capacity) and Y211A (with higher C4-oxidative capacity) after histag purification. A control sample only containing PASC, 1mM ascorbic acid and buffer (without enzyme) was run to verify the enzyme preparations lost their endoglucanase background activity. Two time points (after 1h and 9h incubation) are shown for each enzyme variant.HPAEC-PAD chromatograms of wildtype (DOCX)Click here for additional data file.S6 FigHjLPMO9A and mutants Y24A (with higher C1-oxidative capacity) and Y211A (with higher C4-oxidative capacity) after histag purification and confirms the effect on regioselectivity of the point mutations.This indicator diagram was obtained for wildtype (DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableNeurospora crassa LPMO9C and Phanerochaete chrysosporium LPMO9D.Sequence of (DOCX)Click here for additional data file.


TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a  Platelets are small, anuclear blood cells that are essential for blood clotting and the maintenance of vascular integrity , 2. TheyTHPO [thrombopoietin], GP1BA [glycoprotein Ibα], TUBB1 [tubulin-β1]) and survival (the apoptosis regulator BAK [encoding BAK]). However, the vast majority of identified SNVs were located near genes hitherto uncharacterized in the megakaryocyte lineage.The number, cellular volume, and function of platelets are tightly regulated within individuals. However, all 3 parameters vary widely at the population level, and represent independent risk factors for heart attack and stroke . PopulatTPM1) and tropomyosin 4 (TPM4), respectively. It has been reported that expression of transcripts from the TPM4 locus strongly increases in the later stages of megakaryopoiesis  and nemaline myopathies (TPM2 and TPM3). These disease phenotypes are recapitulated in mice carrying similar mutations  in the 2 pedigrees. Mutation of murine Tpm4 resulted in dose-dependent macrothrombocytopenia caused by a defect in the terminal stages of platelet production. This was recapitulated by shRNA knockdown in human megakaryocytes. In addition to a reduction in platelet number and an increase in mean platelet volume, a mild defect in platelet function was observed in both humans and mice. Our findings demonstrate that TPM4 encodes a tropomyosin with an important nonredundant role in platelet biogenesis, and define a novel, dominant, Mendelian platelet disorder.Here, we demonstrate that, in both humans and mice, TPM4 is an essential mediator of platelet biogenesis. Genome sequencing of 865 index cases with bleeding and platelet disorders of unknown molecular etiology revealed 2 pedigrees with a premature stop codon in TPM1, TPM3, and TPM4, and low levels of TPM2 (ref. https://doi.org/10.1172/JCI86154DS1). We performed reverse transcriptase–PCR (RT-PCR), which confirmed these findings, and demonstrated that TPM4 is the most highly expressed of the tropomyosins, followed by TPM1 . It was present on 1 allele in 2 unrelated BPD cases and was absent from the Exome Aggregation Consortium (ExAC) database  Bleeding and Platelet Disorders (BPD) collection. To date, over 1,000 participants have been enrolled and genome-sequenced as part of the BRIDGE-BPD study for genome sequencing along with approximately 5,000 participants with other rare diseases enrolled to the NIHR BioResource–Rare Diseases . Two higdatabase  as well database . The 2 cTPM4, resulting in a truncation at residue 69 of the major protein isoform TPM4.2, and residue 105 of the minor isoform TPM4.1 . The variant concerning the BPD patients encodes a premature stop codon in exon 3 of m TPM4.1 .9 per liter or the mean platelet volume above 13 fl or if there was presence of macrothrombocytes as observed by electron microscopy. The propositus of pedigree 1 carrying the premature stop codon was a woman of mixed European descent who was enrolled to the BRIDGE-BPD study at 56 years old with a history of mild bleeding  with mean platelet volumes well above 13 fl. All other blood counts were within normal range . Macrothrombocytes were observed by electron microscopy. She had suffered with menorrhagia from menarche that did not improve upon hormonal therapy and therefore required recurrent dilatation and curettages, and finally a hysterectomy at the age of 37 . He experienced no abnormal bleeding in response to a number of hemostatic challenges, including a laparotomy, orchiopexy, and 4 dental extractions. His sibling (2-III-7), who also carries the mutation, exhibited a low platelet count (146 × 109 per liter) and large mean platelet volume (14.6 fl), and a bleeding tendency similar to the propositus’ (see 9 per liter) and lowest mean platelet volume (10.8 fl) of all family members. Thus, the variant cosegregates with isolated macrothrombocytopenia.The propositus of pedigree 2 was a woman of mixed European descent enrolled in the BRIDGE-BPD study at 58 years of age. She had a platelet count of 230 × 10f 37 see . PedigreTPM4.2 mRNA levels were reduced to 59% and 78% of the control in the affected cases 1-II-5 and 1-III-7, respectively  mutagenesis screen in mice for mutations causing dominant thrombocytopenia . RT-PCR analysis of cDNA extracted from megakaryocytes revealed that the Plt53 mutation led to the inclusion of part of intron 7 in the Tpm4 mRNA. The predicted result is a protein bearing 8 intron-encoded amino acids followed by premature truncation due to the presence of an in-frame stop codon at nucleotide g.72,147,291  revealed the complete absence of TPM4 in latelets , top. Thlatelets . Using alatelets , middle.latelets , these dPlt53/+Tpm4) and homozygous (Plt53/Plt53Tpm4) animals appeared healthy, and histopathological examination revealed no obvious organ abnormalities or pathology. Relative to WT littermates, Plt53/+Tpm4 mice exhibited mild macrothrombocytopenia , increase in lymphocyte counts in the Plt53/Plt53Tpm4 cohort   . TogetheTransmission electron microscopy (TEM) analysis of platelets from the 2 affected cases from pedigree 1 revealed considerable heterogeneity in platelet size, some of them being very large. We also observed an increased presence of large vacuoles . BlindedPlt53/Plt53Tpm4 and Plt53/+Tpm4 platelets, with frequent vacuoles, indicating increased fragility of the mutant platelets (FLNA (the gene encoding filamin A) have recently been reported to result in fragile platelets and macrothrombocytopenia (ACTN1) are one of the most frequent causes of isolated thrombocytopenia with altered platelet morphology   .+/+Tpm4, Plt53/+Tpm4, and Plt53/Plt53Tpm4 platelets. No change in the kinetics of clearance or ultimate lifespan was observed, indicating that lack of functional TPM4 did not affect platelet turnover (IIbβ3 activation (JON/A antibody) and α-granule secretion (anti–P-selectin antibody) as markers. Plt53/+Tpm4 and Plt53/Plt53Tpm4 platelets were responsive to a range of agonists, indicating that the processes of integrin inside-out activation and degranulation are functional in the absence of TPM4  fluorescence signal . In WT megakaryocytes commencing proplatelet formation, TPM4 was organized in a fine punctuate network   , demonstPlt53/+Tpm4 and Plt53/Plt53Tpm4 bone marrow , and many exhibited an irregular morphology , the levels of phosphorylated (inactive) cofilin in Plt53/Plt53Tpm4 cells were significantly decreased  and cofilin, as these have been shown to directly compete with tropomyosins for actin binding . While Aecreased . This suecreased .+/+Tpm4 and Plt53/Plt53Tpm4 megakaryocytes. In line with that study, we observed that TMOD3 localized as puncta with proplatelet membranes , give rise to the “MYH9-related disorders,” all of which exhibit macrothrombocytopenia, and have been linked to the RHOA/ROCK pathway  , we inveembranes . While T of RHOA , 32. Inh pathway . We therobserved . Confoca network , very si network . This paembranes . In contterparts , right. TPM4, previously identified by genome-wide association studies as a candidate gene correlated to platelet count and volume, is a modulator of platelet production. TPM4 insufficiency causes macrothrombocytopenia in mice and humans in a dose-dependent manner.Cytoskeletal reorganizations are the driving force for the production of platelets by megakaryocytes . Here, wTPM4 are highly expressed in human and murine megakaryocytes, and the corresponding proteins are abundant in platelets  in cases with MYH9-related disorders cause autosomal dominant macrothrombocytopenia and bleeding associated with other complex pathobiologies , a component of the VWF receptor complex, is important for regulation of platelet size, as well as platelet adhesion and maintenance of membrane integrity under high shear , using an Advia 2120 hematological analyzer (Siemens). Megakaryocytes were counted manually in sections of sternum stained with H&E with a minimum of 10 visual fields (×200) analyzed. Images were acquired on a Nikon Eclipse E600 microscope equipped with AxioCam MRc5 (Zeiss) and AxioVision 4.8. Scale bars were inserted and the megakaryocyte area was determined using ImageJ (NIH). Acute thrombocytopenia was induced by injection of antiplatelet serum and assessed as previously described .Organ cellularity was determined by counting of cell suspensions from bone marrow, spleen, and thymus with calibrate beads (BD Biosciences). Cells were stained on ice for 30 minutes, washed once, and then incubated with streptavidin–PE–Texas red (Life Technologies). After a washing step, acquisition was performed on an LSR Fortessa 1 (BD Biosciences) and analyzed with FlowJo (Tree Star). Antibodies for flow cytometry either were labeled in house with FITC, PE, PECy7, APC, or Alexa Fluor 700 or were biotinylated. CD4 (GK1.5), CD8 (53.6.7), CD16/32 (24G2), CD19 (1D3), CD41 (MWReg30), Ter119 (Ly76), Gr1 (RB6-8C5), CD11b (M1/70), Sca1 (D7), Kit (ACK4), B220 (RA3-6B2), and CD34-FITC (RAM34) were purchased from BD Pharmingen, and CD150-PE (TC15-12F12.2) was purchased from BioLegend. Fluorogold (Sigma-Aldrich) was used to exclude dead cells.+/+Tpm4, Plt53/+Tpm4, and Plt53/Plt53Tpm4 embryos E13.5 were generated as described below. mRNA was isolated using the RNeasy Plus Kit (Qiagen), and cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocols. The primers used were 5′-AAAGAAGCCAAGCACATCAC-3′ (Tm4_F3) and 5′-AGCCCACATTCTCTTCTTTG-3′ (Tm4_R7). The expected product size for the WT was 360 bp, while an approximately 400-bp product was obtained in the presence of the Plt53Tpm4 allele. PCR products were sequenced and revealed the presence of an approximately 40-bp intronic inclusion in the mutant compared with the WT product.Fetal liver cell–derived megakaryocytes from GAPDH housekeeping gene for normalization. PCR primer pairs were designed to amplify only cDNA, had no reported off-target matches searching the human NCBI RefSeq database, and were within 80%–120% PCR efficiencies with single dissociation curves.Total RNA was extracted using an RNeasy Plus Kit (Qiagen) according to the manufacturer’s instructions. cDNA was prepared from 250–500 ng total RNA using SuperScript III First-Strand Synthesis system for RT-PCR (Invitrogen) with random hexamers. Two-step quantitative PCR (qPCR) reactions were performed in duplicates using SYBR Green chemistry  on an Mx3000P instrument (Agilent Technologies). Relative gene expression was calculated by the ΔΔCt method using TPM1-F, AGTCGAGCCCAAAAAGATGA; TPM1-R, TCGCTCTCAATGATGACCAG; TPM2-F, GGACAGAGGATGAGGTGGAA; TPM2-R, GCATCAGTGGCCTTCTTCTC; TPM3-F, CCTGCAAAAGCTGGAAGAAG; TPM3-R, TCTGCCTCTTCTGCAATGTG; TPM4-F, GGAGATGCAGCTCAAAGAGG; TPM4-R, CCTCCAGGATGACCAGCTTA; GAPDH-F, TATCGTGGAAGGACTCATGACC; GAPDH-R, TAGAGGCAGGGATGATGTTCTG.qPCR oligonucleotide primer pairs were as follows: Oligonucleotides were selected from the RNAi consortium database. Short hairpin or control nontarget sequences were annealed before cloning into the pLKO.1 lentiviral vector backbone (Addgene 10878) downstream of the human U6 promoter and checked for sequence integrity.Replication-deficient lentiviral vector particles (LVPs) were produced by transient cotransfection of HEK 293T/17 cells (ATCC CRL-11268) with pLKO.1–short hairpin constructs along with the psPAX2 and pMD2.G helper plasmids  using TurboFect transfection reagent (Thermo Fisher Scientific). Crude supernatants containing LVPs were concentrated by PEG-based precipitation  and functional titers determined by qPCR measurement of provirus copy number in genomic DNA of transduced HCT116 cells (ATCC CCL-247).The following oligonucleotides were used to construct short hairpin vectors: TPM4_shA forward, CCGGGGAAGAGGCTGACCGCAAATACTCGAGTATTTGCGGTCAGCCTCTTCCTTTTTG; TPM4_shA reverse, AATTCAAAAAGGAAGAGGCTGACCGCAAATACTCGAGTATTTGCGGTCAGCCTCTTCC; TPM4_shB forward, CCGGTCAGACACTAAACGAACTTAACTCGAGTTAAGTTCGTTTAGTGTCTGATTTTTG; TPM4_shB reverse, AATTCAAAAATCAGACACTAAACGAACTTAACTCGAGTTAAGTTCGTTTAGTGTCTGA; TPM4_shE forward, CCGGCCCAGTATCTAGTCGTGGATACTCGAGTATCCACGACTAGATACTGGGTTTTTG; TPM4_shE reverse, AATTCAAAAACCCAGTATCTAGTCGTGGATACTCGAGTATCCACGACTAGATACTGGG.To analyze the effect of TPM4 RNA and protein level on proplatelet formation, we performed logistic regression. A mixed-effects model was fitted to the log odds of a cell being proplatelet forming. A coefficient was included representing the effect of an increase in RNA or protein levels on the log odds and was adjusted for cord effects using a random effect parameter.g for 8 minutes, followed by centrifugation of the supernatant buffy coat at 125 g for 8 minutes. Platelets were washed by 2 sequential centrifugations at 860 g for 5 minutes in 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, and 12.5 mM sucrose, pH 6.0. The platelet pellet was resuspended in Tyrode-HEPES buffer  containing 5 mM glucose, 0.35% BSA, and 1 mM CaCl2.Murine blood was obtained by cardiac puncture into 0.1 volume of Aster-Jandl anticoagulant  or 150 g for 20 minutes (controls). Platelets were washed by centrifugation at 1,000 g for 10 minutes followed by resuspension in Tyrode-HEPES buffer containing 5 mM glucose before a final spin at 1,000 g for 10 minutes.Platelet-rich plasma was obtained from citrated human whole blood by centrifugation at either 50 4 per microliter and activated with the indicated agonists for 15 minutes at room temperature in the presence of JON/A–PE and anti–P-selectin–FITC antibodies (diluted according to the manufacturers’ instructions). Samples were diluted with PBS and analyzed on a FACSCalibur. To assess platelet receptor expression, anticoagulated whole blood was diluted 1:20 using Tyrode-HEPES buffer and stained with platelet-specific antibodies or negative controls for 20 minutes at room temperature. Samples were diluted with Tyrode-HEPES buffer and directly acquired by flow cytometry.Washed platelets were adjusted to a concentration of 5 × 10Citrated whole blood was incubated for 5 minutes with hirudin and aspirin, before being added to tubes containing HEPES-buffered saline, FITC–rabbit anti-fibrinogen antibody , and PE–mouse anti–P-selectin  or relevant negative controls . Platelets were agonized with a final concentration of 0.0005 μM ADP, 0.3 μg/ml CRP-XL, and 0.8 μM TRAP-6 or left untreated. Apyrase was added to platelets agonized with CRP-XL and TRAP-6. Flow cytometry was performed on an FC-500 (Beckman Coulter), and platelets were identified by light scatter. Results were recorded as the percentage of platelets positive for the relevant activation marker.6 platelets per microliter) were activated with 0.005 U/ml thrombin and incubated at room temperature for the indicated time periods. After fixation with 4% paraformaldehyde in PBS for 10 minutes, the coverslips were thoroughly rinsed with PBS, and platelets were visualized with a Deltavision Elite microscope equipped with CoolSnap2 CCD Detector (×100/1.4 oil immersion objective). Images were analyzed using ImageJ software.Coverslips were coated with 100 μg/ml human fibrinogen and blocked with PBS 1% BSA. After rinsing with Tyrode-HEPES buffer, washed mouse platelets (100 μl with 0.03 × 10Mice were injected i.v. with 0.15 μg/g body weight of X-488 (Emfret), a rat-derived IgG against the platelet glycoprotein Ibβ receptor, and platelet lifespan was measured as previously described .Mice were anesthetized using ketamine/xylazine. The tail was transected at 2 mm from the tip and immediately immersed in 37°C saline. The bleeding time was determined as the time from the tail transection to the cessation of blood flow or stopped after 10 minutes.t test or a Mann-Whitney test. When more than 2 experimental groups were compared, data were analyzed using 1-way ANOVA. When ANOVA indicated that a significant difference was present, individual differences were explored with the 2-tailed unpaired Student’s t test or Mann-Whitney test using Bonferroni correction for multiple comparisons . P values less than 0.05 were considered as statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001 or as otherwise stated. Data are presented as mean ± SD.Data distribution was analyzed using the Shapiro-Wilk test. Statistical significance between 2 experimental groups was analyzed using an unpaired 2-tailed Student’s The patients were enrolled to the BRIDGE-BPD study (UK REC10/H0304/66) and to the NIHR BioResource–Rare Diseases study (UK REC 13/EE/0325) after providing informed written consent. The control volunteers for functional work were enrolled to the Genes and Platelets study (UK REC10/H0304/65) after providing informed consent. For genetic analysis, control groups comprised other patients with bleeding or platelet disorders (BPD) of unknown genetic basis or with unrelated rare disorders enrolled to the NIHR BioResource–Rare Diseases study (UK REC 13/EE/0325).All animal experiments complied with the regulatory standards of, and were approved by, the Walter and Eliza Hall Institute and the University of New South Wales Animal Ethics Committees.Tpm4.2 knockout mouse. ECH conceived and supervised the generation and characterization of the Tpm4.2 knockout mouse. WHO directs the Bleeding and Platelet Disorders project of the NIHR BioResource–Rare Diseases. PWG contributed to the design and interpretation of experiments on the detection and function of tropomyosins. ET analyzed and interpreted human genetic, RNA, and proteomic data. MRT conceived the study in humans, designed experiments, interpreted data, and wrote the paper. BTK conceived the study, designed experiments, interpreted data, and wrote the paper.IP designed and performed experiments on mouse megakaryocytes and platelets, analyzed and interpreted data, and wrote the paper. JW designed and performed experiments on human megakaryocytes and platelets, analyzed and interpreted data, and wrote the paper. SC designed and performed experiments on mouse megakaryocytes and platelets and analyzed and interpreted data. VK designed and performed experiments on human megakaryocytes and platelets and analyzed data. NF designed and performed experiments on human megakaryocytes and platelets and analyzed data. JBB designed experiments for immunofluorescence stainings on human megakaryocytes and thrombus formation under flow and analyzed data. KA designed and performed experiments on mouse megakaryocytes and platelets and analyzed data. CL contributed to experiments on human megakaryocytes. RML contributed to animal experiments. GS interpreted results on the detection of tropomyosin isoforms. WSA and DJH contributed to the mouse ENU mutagenesis screen. WJA compared platelet parameters with distributions derived from INTERVAL data. KD performed functional analysis of human platelets. PN interpreted results from electron microscopy on human platelets. SKW and ADM recruited pedigree 1 and collected clinical phenotype data. SGO and PWC recruited pedigree 2 and collected clinical phenotype data. NIHR BioResource is a National Institute for Health Research research infrastructure that is responsible for the whole genome sequencing and phenotyping of 10,000 patients with rare diseases of unknown molecular etiology. FD, LMI, NSB, MH, and CAL generated and characterized the