However, it remains to be elucidated how m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation.N6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3′-RACE and poly(A) tail length assays.m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3′ untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3′ UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage.We characterized a dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.Our results address an essential role of dynamic mThe online version contains supplementary material available at 10.1186/s12967-022-03814-9. Mesenchymal stem cells (MSCs) residing in various tissues can still undergo self-renewal and differentiate into specific cell types to maintain tissue homeostasis and fulfill regenerative needs . Multipl6-methyladenosine (m6A) is the most prevalent posttranscriptional modification of messenger RNA (mRNA) and regulates almost every step of RNA metabolism in mammals [G > A]m6AC[U > A > C]), which is enriched in the CDS and 3′ UTR of RNA transcripts [6A-methylated transcripts are enriched in biological processes, such as neural stem cells, the cell cycle, and differentiation, which are essential to control the transcriptome composition of different stages [6A landscape in DPSC mineralization, and the m6A-mRNA profile was mainly related to transcriptional regulation and cell differentiation. Moreover, the increasing total m6A content and m6A distribution in the 3′ UTR might result from a pronounced elevation of METTL3 expression. METTL3 was reported to participate in tooth root development by modulating translational efficiency [RNA mnscripts , 26. Durt stages . We charficiency . The inhficiency . METTL3 ficiency , 29. METficiency . Consistficiency , 31. Ect6A modification, which in turn controls proper development and differentiation. METTL3-mediated m6A modification regulates the expression of some osteogenic markers and other related genes involved in bone metabolism [6A marks, which are essential in the cellular differentiation and cancer development [6A modification during DPSC differentiation. NOG is a key player in ectoderm development, and its disruption can lead to organogenesis defects such as craniofacial defects and hypoplastic teeth [6A peaks in the 3′ UTR of NOG mRNA increased during DPSC mineralization, which restricted its gene expression. METTL3 inhibited m6A-tagged NOG expression and promoted its degradation in differentiated DPSCs. Consistent with our data, m6A modification modulates RNA degradation and gene expression in neural stem cells, which is a critical epigenetic mechanism in the temporal control of neurogenesis [6A signaling clustered in the 3′ UTR is mainly responsible for cytoplasmic events related to RNA stability and translation [6A marks in the 3′ UTR of certain transcripts [6A modification dynamically modulates the stability of specific transcripts, which is required for the transcriptional prepatterning of DPSC mineralization.Various transcripts and signals are tagged in a timely manner by mtabolism . Parathyelopment , 33, 34.ic teeth , 36. Nogic teeth . BMSC osic teeth , 39. We ogenesis . m6A signslation –42, and nscripts . Taken t6A marks and poly(A) tail regulation. The transcriptional dynamics of certain genes are related to differences in poly(A) tail length via m6A modification and deadenylase complexes [6A signaling is capable of controlling RNA structural switching and RNA‒protein interactions [6A-HuR-dependent manner [6A reader YTH N6-methyladenosine RNA binding protein (YTHDF) 2 is reported to directly interact with the CCR4-NOT complex. YTHDF3 can recruit the poly(A) specific ribonuclease subunit (PAN) 2-PAN3 complex, contributing to its deadenylation and degradation [6A marks lead to shortened poly(A) tails in DPSC differentiation.Poly(A) tails are 150–250 adenosine nucleotides acquired by the end of the 3′ UTR in the nucleus that subsequently undergo deadenylation in the cytoplasm. The length of a poly(A) tail changes throughout the lifetime of mRNA and has essential effects on its stability, degradation and translation . In the omplexes . m6A sigractions . METTL3 t manner , 48. Theradation , 50. Nonradation , 52. In 6A RNA methylation is essential for heightened transcriptional coordination during DPSC differentiation. METTL3-mediated m6A marks tag the 3′ UTR of NOG and inhibit its stabilization via poly(A) tail regulation in a stage-specific manner. The present study identifies a critical role of METTL3-mediated m6A methylation in the temporal control of cell fate transition and sheds light on the epitranscriptomic machinery of m6A-dependent poly(A) tail regulation in transcriptional dynamics.The osteo/odontogenic differentiation of DPSCs and tertiary dentin formation are of particular interest in relation to dental repair. Identifying the key signaling in DPSC differentiation and mineralized matrix formation, and recapitulating these processes in clinical strategies could preserve pulp vitality. In the present study, we demonstrated that dynamic mAdditional file 1: Additional Table 1： The sequence of primers used in qPCR. Additional Figure 1: KEGG enrichment analysis of m6A-tagged transcripts and the relative expression of m6A-related genes during DPSC mineralization. Additional Figure 2: H&E staining of the composites of β-TCP/HA scaffolds and DPSCs. Additional Figure 3: Volcano plots of differentially expressed genes in OM-DPSCs after METTL3 inhibition. Additional Figure 4: Immunofluorescence staining of METTL3 and NOG in DPSCs. Additional Figure 5: Immunofluorescence staining of p-Smad3 and Smad in the composites of β-TCP/HA scaffolds with METTL3-interfering DPSCs.


SLCO1B1 (solute carrierorganicanion transporter family member 1B1) gene encoding the OATP1B1 (organicanion transporting polypeptide 1B1) protein are associated with alteredtransporter function that can predispose patients to adverse drugeffects with statin treatment. We explored the effect of six rare SLCO1B1 single nucleotide variants (SNVs) occurring in Finnishindividuals with a psychotic disorder on expression and functionalityof the OATP1B1 protein. The SUPER-Finland study has performed exomesequencing on 9381 individuals with at least one psychotic episodeduring their lifetime. SLCO1B1 SNVs were annotatedwith PHRED-scaled combined annotation-dependent (CADD) scores andthe Ensembl variant effect predictor. In vitro functionality studieswere conducted for the SNVs with a PHRED-scaled CADD score of >10and predicted to be missense. To estimate possible changes in transportactivity caused by the variants, transport of 2′,7′-dichlorofluorescein(DCF) in OATP1B1-expressing HEK293 cells was measured. According tothe findings, additional tests with rosuvastatin and estrone sulfatewere conducted. The amount of OATP1B1 in crude membrane fractionswas quantified using a liquid chromatography tandem mass spectrometry-basedquantitative targeted absolute proteomics analysis. Six rare missensevariants of SLCO1B1 were identified in the studypopulation, located in transmembrane helix 3: c.317T>C (p.106I>T),intracellular loop 2: c.629G>T (p.210G>V), c.633A>G (p.211I>M),c.639T>A(p.213N>L), transmembrane helix 6: 820A>G (p.274I>V), andthe C-terminalend: 2005A>C (p.669N>H). Of these variants, SLCO1B1 c.629G>T (p.210G>V) resulted in the loss of in vitro function,abolishingthe uptake of DCF, estrone sulfate, and rosuvastatin and reducingthe membrane protein expression to 31% of reference OATP1B1. Of thesix rare missense variants, SLCO1B1 c.629G>T (p.210G>V)causes a loss of function of OATP1B1 transport in vitro and severelydecreases membrane protein abundance. Carriers of SLCO1B1 c.629G>T might be susceptible to altered pharmacokinetics ofOATP1B1substrate drugs and might have increased likelihood of adverse drugeffects such as statin-associated musculoskeletal symptoms.Variants in the  SLCO1B1) gene encodes the organic anion transporting polypeptide 1B1 (OATP1B1),a transmembrane protein that is involved in the transport of drugssuch as statins and other compounds from the blood into the hepatocytes.2Statins are among the most common prescriptiondrugs in the worldand are used for the reduction of low-density lipoprotein cholesterolconcentration and prevention of cardiovascular disease. The solutecarrier organic anion transporter family member 1B1  and even rhabdomyolysis, impacting statin adherence and hinderingthe long-term effectiveness of statin therapy.7 Cardiovascular mortality is one of the leading causesof excess mortality in patients with schizophrenia.8 Antipsychotic medications, especially second-generationantipsychotic medications such as clozapine and olanzapine, can inducemetabolic abnormalities including dyslipidemias.9 Therefore, statins are commonly prescribed to patientswith psychotic disorders.Single nucleotide variants (SNVs) in 11The U.S. Food and Drug Administration(FDA) and European MedicinesAgency (EMA) acknowledge the significance of OATP1B1 variants in medicationsafety by providing guidelines for studying the drug transporter’seffect on pharmacokinetics in drug development.12 Rare variants of SLCO1B1 are mainly unreported,although their effect on drug safety can be as consequential as theeffect of common variants.14 Interpretation of geneticvariants from vast amounts of whole exome sequencing data remainsa challenge, and computational tools are needed to predict the functionalimpact of pharmacogenetic variants before the identified variantscan be explored at a biological level.17 Various algorithms thatare needed to predict the functional impacts of coding variants areavailable using standard criteria and scoring metrics or machine learningapproaches. Biological assessment of in silico evaluated variantsis necessary to generate clinically actionable recommendations.Pharmacogenetic research has suffered from small sample sizes,and finding a patient group of sufficient size is often challengingwhen exploring rare genetic variants.SLCO1B1 SNVs occurring among 9381 Finnish individuals with a psychotic disorderrecruited as part of the SUPER-Finland study. The variants were selectedfor in vitro expression and functionality experiments based on a damagingin silico prediction by the PHRED-scaled combined annotation-dependent(CADD) score algorithm19 and an Ensembl variant effectpredictor (VEP)20 annotation of missense.Cellular uptake studies in OATP1B1-overexpressing HEK293 cells wereconducted, and the amount of the OATP1B1 variant protein in crudemembrane fraction HEK293 cells was quantified using a liquid chromatographytandem mass spectrometry (LC–MS/MS)-based quantitative targetedabsolute proteomics (QTAP) analysis. Our results provide functionalannotations for SLCO1B1 c.317T>C (p.I106T), c.629G>T(p.G210V), c.633A>G (p.I211M), c.639T>A (p.N213K), c.820A>G(p.I274V),and c.2005A>C (p.N669H) variants.We studied the potential clinical relevance of rare 21 The SUPER-Finland study protocol will be describedin a separate cohort profile manuscript that is in preparation.The SUPER-Finland study recruited10,474 participants aged >18 with a severe mental disorder fromFinnishin- and outpatient psychiatric care, primary care, and housing unitsand additionally with newspaper advertising between the years 2016–2018.Subjects with a diagnosis of the schizophrenia spectrum disorder (ICD-10codes F20 and F22–29), bipolar disorder (F30 and F31), or majordepressive disorder with psychotic features (F32.3 and F33.3) wereincluded in the study. Exclusion criteria were inability to give informedconsent and age under 18 years. We put special focus on ensuring awide coverage of known Finnish internal population subisolates.DNA was extracted from participants’ blood samples collectedby venipuncture . A salivasample  was collected for DNA extraction whenvenipuncture was not possible. The samples were frozen (−20°C) within 60 min of sampling and sent to the THL Biobank  within 3 months for long-term storage(−185 °C). A PerkinElmer Janus chemagic 360i Pro Workstationwith the CMG-1074 kit was used to extract DNA from the EDTA bloodtubes. Extraction of DNA from saliva samples  was performed by Chemagen Chemagic MSM I robotwith the CMG-1035–1 kit. The DNA samples were genotyped andsequenced at the Broad Institute of MIT and Harvard, Boston Cambridge,Massachusetts, USA.SLCO1B1 SNVs with possible changesin OATP1B1 protein function, we used exome data from 9381 SUPER-Finlandstudy participants. Exome sequencing was carried out on the IlluminaHiSeq platform using 151 base pair paired-end reads at the Broad Instituteof Harvard and MIT. The samples were enriched with the Illumina Nexteracapture kit and sequenced until 80% of the target capture was coveredat 20×. The Picard sequence processing pipeline was used to processBAM files (http://broadinstitute.github.io/picard/), and the data were mapped to the human genome reference build 38(GRCh38) using BWA.22 This procedure followedstandard best practice alignment and read processing protocols asdescribed earlier.24 Variants were called using theGenome Analysis Toolkit (GATK26). Local realignmentaround indels and recalibration of base qualities in each sample BAMwere performed using GATK version 3.4. Each sample was called usingHaplotypeCaller to create gVCF files containing every position ofthe exome with likelihoods for variants alleles or the genomic referenceallele. All variants were annotated using the variant quality scorerecalibration (VQSR) tool in GATK version 3.6, resulting in a VCFwith germline SNVs and indels for all samples used in the analyses.The variant joint calling corresponded to the pipeline used to createthe GnomAD database.23Todiscover rare 27 matrix table file formatand annotated using CADD depletion19  scores and with the VEP20 (version95) tool through Hail . Thevariants were filtered using Hail to include only those having a callrate of > 0.99, a HWE p-value of > 1e-10, anallelecount of > 1, a genotype quality of > 20, and a depth of >10. The SLCO1B1 variants with a PHRED-scaled CADDscore of >10,predicted as missense variants by VEP and not included in the currentCPIC guidelines for SLCO1B1, were selected for furtherin vitro expression and functional analyses.The variants of chromosome 12 were convertedto the HailSLCO1B1 variants were createdas described by Kiander et al.14 The reference SLCO1B1 gene used was Genebank accession number AJ132573.1,and the mutagenesis primers are described in Supporting Information Table S1. GATC Biotech’s sequencing service confirmed the presence of the SNVs in the plasmids.Baculoviruses carrying the reference SLCO1B1 gene,the variant SLCO1B1 genes, and the previously clonedgene for enhanced yellow fluorescent protein (eYFP) as a negativecontrol were produced as described earlier by Tikkanen et al.28Plasmids carryingthe 2. The cells (0.5× 106) were seeded in each well of 48-well plates(Thermo Fisher Scientific Nunc coated with poly-d-lysinein-house) 24 h prior to transduction with the baculoviruses. To stimulatethe expression of proteins, sodium butyrate was added with the virusesat a final concentration of 5 mM (as per in-house optimization).HEK293 human kidneycells were cultured in Dulbecco’s modified Eagle medium (DMEM)and high-glucose GlutaMax culture medium supplemented with 10% fetalbovine serum (FBS) at 37 °C, 5% CO3 and 25 mM HEPES adjusted toa pH of 7.4 with NaOH) replaced the medium in the wells. After thebuffer was removed, cellular uptake began when 125 μL of thetest substrate solution was added into the wells. The uptake was stoppedduring the linear uptake phase by aspiration of the test solution(specific test times mentioned in the results). Three times wash with500 μL of ice-cold transport buffer followed this step, andthe cells were lysed with 125 μL of 0.1 M NaOH  or 150 μL of a 3:1 methanol/watermixture (rosuvastatin samples). Fluorescence measurement  using the multimode microplatereader Varioskan LUX  ofthe cell lysates was performed to quantify the DCF. The [3H]-estrone sulfate-containing cell lysate was neutralized with equivalentmoles of 1 M HCl before adding Optiphase HiSafe 3 scintillation liquidand measuring radioactivity of samples using a MicroBeta2 2450 MicroplateCounter . 10 μL ofthe cell lysate was mixed with 300 μL of the Coomassie Plusreagent and used for total protein amount quantification with absorbanceanalysis (595 nm) on Varioskan LUX. Rosuvastatin was quantified ona Sciex 5500Qtrap LC/MSMS system  interfacedwith an electrospray ionization (ESI) source as described previously.29The cellular uptake assay wasperformed 48 h post-transduction on a heated (37 °C) orbitalshaker plate. For a 3 min preincubation, transport buffer  were added to the HEK293 cells after thecells were cultured for 24 h in T175 flasks. After 48 h of culturing,the cells were pelleted by centrifugation  and broken down using a Dounce tissue homogenizer and resuspendedin Tris-sucrose (TS) buffer  while being kept on ice. The cell homogenate was centrifugedfor 30 min , separating the larger cellular organellesand the nucleus in the pellet. The resulting supernatant was subsequentlyseparated into new tubes and centrifuged  again, resulting in a pellet containing thecrude cell membrane. The protein sample was suspended in TS buffer,and the total protein concentration was quantified as previously described.Baculovirusescarrying eitherthe reference or variant 31 OATP1B1 and Na+/K+ ATPase signature peptideswere quantified from 50 μg of crude membrane fractions. Afterdenaturation and breakdown of the tertiary structure of the proteins,the crude membrane preparations were digested first with 1/100 LysCendopeptidase and after that with 1/100 TPCK-treated trypsin. A previouslyused isotope-labeled peptide mixture (3 fmol/μg protein) servedas an internal standard.14 Quantificationwas conducted on a 6495 QQQ MS with a 1290 HPLC system and an AdvanceBiopeptide Map Column, 2.7 μm, 2.1 × 250 mm  as described previously.31 The SNVs did not alter the amino acids in the analyzed peptide sequences.The peak area ratios of the analyte peptides and their respectiveinternal standards were compared using the Skyline application . The results of OATP1B1 expression arepresented as relative to the Na+/K+–ATPaseexpression level and normalized to the reference OATP1B1 protein.The absolute amount of OATP1B1 in proteomics samples is presentedin Supporting Information Table S2 andFigure S1.The LC–MS/MS-basedQTAP approach was used to quantify the absolute protein expressionof OATP1B1 in the crude membrane preparations. The method for theprotein sample preparation and quantitation using targeted LC–MSis described earlier.The uptake of DCFwas normalized to thetotal protein amount. The uptake into eYFP-expressing cells, representingpassive influx, was subtracted from the uptake into OATP1B1-expressingcells, yielding active OATP1B1-mediated transport. The transport activityin OATP1B1 variant cells was then normalized to the cells expressingwild-type OATP1B1. The statistical significance of the differencesin activity and expression levels was determined by one-way analysisof variance (ANOVA) with the Dunnett’s post hoc test for multiplecomparisons . Extra-sum-of-squares F-test was used to assess the statisticalsignificance of the observed changes in kinetic parameters of DCFtransport using the same software.The CoordinatingEthics Committee of the HospitalDistrict of Helsinki and Uusimaa (HUS) gave a favorable ethics statement(202/13/03/00/15) for the SUPER-Finland study. Prior to inclusion,written informed consent was obtained and archived from each participant.Individual-level data were pseudonymized.The computer code used inthe analysis of this study is available from the corresponding authoron reasonable request, and the SUPER-Finland study data are availablefrom THL Biobank when released from the original study.We evaluated the functionality of six SNVsof OATP1B1. The SNVswith their genomic positions, amino acid changes, and locations inOATP1B1 are shown in The variants were expressedin HEK293 cells, and theeffect ofthe SNVs on transport activity was evaluated with a cellular uptakestudy using DCF as a substrate. An LC–MS/MS-based QTAP approachwas used to quantify any changes in the protein abundance of OATP1B1in crude membrane fractions.SLCO1B1 variants are presented in In silico predicted consequencesof the six SUPER-Finland subjects are heterozygousfor the studiedvariants.The most frequent SNVs were c.639T>A, which was found in 16 subjects,and c.317T>C, which was found in four subjects. The other fourSNVswere found in only two subjects. Based on the in silico predictionsby CADD, SIFT, and PolyPhen, c.629G>T and c.2005A>C were mostlikelyto have a damaging effect, and c.317T>C was most likely to be tolerated,while the predictions on the three other variants were more conflicting.All the variants are predicted to be missense variants by VEP. Two-dimensionalprediction of the OATP1B1 transporter with the amino acid locationsof the identified variants is presented in Km) to the transporter or altered maximumvelocity (Vmax) (We discovered that c.629G>T (p.G210V) abolishes the transportactivityof OATP1B1 2 and 3. y (Vmax) 4.Km parameter, but reducedthe Vmax seemed to enhance transportactivity at a low concentration (1 μM) of DCF, which was onaverage 162% of reference OATP1B1 2. The cothe Vmax 4. The prSLCO1B1 variants. Among these, c.629G>T(p.210G>V) was found to be a loss-of-functionvariant in vitro. According to GnomAD,23 the variant has been detected only in Finnish and South Asian populations.We used computational algorithms to prioritize SLCO1B1 variants for in vitro validation. The PHRED-scaled CADD score forthis variant was 23.4, and it was predicted to be deleterious by SIFTand probably damaging by PolyPhen. A variant with a similar CADD scoreof 23.0 (c.2005A>C and p.N669H) and classified as deleterious andpossibly damaging by SIFT and PolyPhen, respectively, had normal functionaccording to our in vitro studies. This underlines the difficultyof computational functional annotation of missense variants. Due tothe enormous number of variants in whole exome sequencing studies,these studies need to rely on computational functional predictionsas not every potential loss-of-function variant throughout the genomecan be tested in the laboratory. The definition of a computationallypredicted loss-of-function variant is a matter of sensitivity andspecificity. Selecting a conservative definition would filter outpotential disease-causing mutations, while too liberal a thresholdcreates noise in association signals. As the CADD score is based onthe regional mutational constraint,19 it might notbe optimal in capturing pharmacogenetic variants as genes responsiblefor the metabolism and transport of xenobiotic compounds might notbe under as strong selection as genes related to, for example, braindevelopment.Wehave provided novel functional annotationsfor six SLCO1B1 c.629G>T results inthe replacement ofa glycine with a valine in position 210. Glycine has no side chainand has a very high tendency to build turns in the secondary structureof polypeptide chains.33 Valine, on theother hand, has a preference to form strands and obstructs the formationof turns and bends in the secondary structure. Thus, it is understandablethat this substitution could impair the function of OATP1B1. Indeed,our results show that SLCO1B1 c.629G>T reducestheuptake of three different substrates to less than 10% of the reference(SLCO1B1 c.317T>C (p.106I>T), c.633A>G (p.211I>M),and c.820A>G(p.274I>V) are all in codons that originally code for isoleucine,but the SNVs result in substitutions with amino acids with similarproperties. Nevertheless, all of these variants reduced the Vmax of DCF uptake significantly (SLCO1B1 c.639T>A(p.213N>K) changes an amidic asparagine into a basic lysine, whichin physiological pH has a positive charge. While the actual three-dimensionalstructure of the OATP proteins remain unknown, OATPs are predictedto form a positively charged pore.34 Sincemany OATP1B1 substrates are anionic compounds, an additional positivecharge near this putative pore in the intracellular loop where c.639T>A(p.N213K) is located might increase substrate affinity. Indeed, the Km value for DCF transport was significantlydecreased  and was shown in vitro to have substrate-dependent normalto enhanced OATP1B1 transport activity.35 Likewise,the effect of c.639T>A may be substrate-dependent, which needstobe clarified in future research. On the other hand, while SLCO1B1 c.2005A>C (p.669N>H) also gains a charge,it decreases Vmax of DCF transport butdoes not alter affinity(Km) or activity at a low DCF concentration could be characterized as a loss-of-function variantwhilethe other variants are most likely clinically benign.eference3A. SLCO1ficantly 4. SLCO1Becreased 4.Accordntration4. This c37 that an unfavorable genotypemight complicate. While, to date, according to The University of WashingtonDrug Interaction Database (DIDB),38 nocentral nervous system agents have been identified as OATP1B1 substrates,many other drugs can be prescribed to these patients to treat theirsomatic diseases. Among these are often statins to treat dyslipidemias,which are associated with antipsychotic medications.9 The association between statin use and muscle symptomshas been thoroughly investigated. Variants in SLCO1B1 are associated with myopathy in particular when there is a documentedincrease in creatinine kinase concentration in blood.39 A poor function phenotype of OATP1B1 can increase the probabilityfor adverse effects during statin treatment, thus reducing medicationadherence.Polypharmacyis common in patients treated with antipsychotic medication,leading to a high probability of adverse drug effects and drug–druginteractionsSLCO1B1*5 and SLCO1B1*15 haplotypesthat contain the function-impairing c.521T>C SNV are associatedwithreduced hepatic clearance of substrate drugs, increased systemic exposure,and increased risk of SAMS.7 The current gene-based prescribing guideline from the Clinical PharmacogeneticsImplementation Consortium (CPIC) states that individuals with decreasedand poor function phenotypes of OATP1B1 should limit statin dosesand those with poor function phenotypes should avoid simvastatin altogether.6 Based on our in vitro studies, c.629G>T (p.G210G>V)could be categorized as a poor function phenotype since the in vitroactivity and abundance is comparable to that of the SLCO1B1 c.521T>C genotype.14 However, homozygous carriersof a rare variant like c.629G>T (p.G210G>V) are very uncommon.Itis more likely to occur in a heterozygous state, as observed in ourstudy population  abolishesthe transport of all three of the tested OATP1B1 substrates, suggestingthat it might predispose patients to an increased risk of SAMS duringstatin treatment. This emphasizes the need to include diverse populationsin drug trials as adverse effects related to population-specific variantsmight otherwise be missed. While the kinetic parameters of DCF transportwere altered by several of the other tested variants, SLCO1B1 variants c.317T>C, c.633A>G, c.639T>A, c.820A>G, andc.2005A>C couldbe considered normal-function variants.We have identified anovel


Fibroblast growth factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress, FGF signalling must decline. Why these signalling dynamics are required has not been determined. Here, we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos, and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs genome-wide across neural genes. Importantly, ERK1/2 inhibition induces precocious neural gene transcription, and this involves dissociation of the polycomb repressive complex from key gene loci. This takes place independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. Transient ERK1/2 inhibition is sufficient for the dissociation of the repressive complex, and this is not reversed on resumption of ERK1/2 signalling. Moreover, genomic footprinting of sites identified by ATAC-seq together with ChIP-seq for polycomb protein Ring1B revealed that ERK1/2 inhibition promotes the occupancy of neural transcription factors (TFs) at non-polycomb as well as polycomb associated sites. Together, these findings indicate that ERK1/2 signalling decline promotes global changes in chromatin accessibility and TF binding at neural genes by directing polycomb and other regulators and appears to serve as a gating mechanism that provides directionality to the process of differentiation. FGF is a neural inducer in many vertebrate embryos, but does it regulates chromatin organization to coordinate the activation of neural genes? This study reveals how a decline in FGF effector kinase ERK1/2 signaling promotes neural differentiation by regulating chromatin accessibility and polycomb protein occupancy at neural genes in the mouse embryo and in human pluripotent cell-derived neural precursors. The identities of signals that induce particular cell fates are now well established, but how such signalling regulates chromatin to coordinate the transcription of differentiation genes and engage a differentiation programme are not well understood. Fibroblast growth factor (FGF) signalling has been implicated in the acquisition of neural cell fate in many vertebrate embryos /node streak border (which later form the tailbud) to maintain a bipotent cell population known as neuromesodermal progenitors (NMPs), which progressively gives rise to the spinal cord and paraxial mesoderm  11–19].–19.11–19tail end   activity in both chick and mouse embryos ,27. ThisPax6. This increased chromatin accessibility was also found in Raldh2 mutant embryos exposed to FGFR inhibitor in which retinoid deficiency resulted in the failure of Pax6 expression [A clue to the mechanism by which FGF regulates neural differentiation was revealed by analysis of chromatin organisation at key neural progenitor genes in the mouse embryo CLE . This shpression . This inPotential targets for FGF signalling include the polycomb repressive complexes 1 and 2 (PRC1 and 2), which cooperate in the repression of differentiation genes in many contexts  and polycomb occupancy (ChIP-seq for Ring1B) revealed that the decline in ERK1/2 activity promotes increased chromatin accessibility across thousands of neural genes, including a subset targeted by Ring1B in NMPs. Using genomic footprinting [Here, we investigate the role of ERK1/2 signalling in the engagement of a spinal cord neural differentiation programme in the caudal mouse embryo and in human spinal cord neural progenitors generated in vitro. We find inhibition of ERK1/2 activity rapidly increases chromatin accessibility across an exemplar neural progenitor gene, printing , we idenPax6 locus using FISH with fosmid probes hybridising either side of the locus [Pax6 transcribing neural tube cells and in CLE cells where Pax6 has yet to be expressed  to the small molecule MEK inhibitor (MEKi) PD184352 or vehicle-only DMSO control for one hour . The effhe locus  Fig 1D)Pax6 locuxpressed . In untrxpressed . Similarxpressed  and thescontrols . This dePax6 locus in vivo, we carried out chromatin immunoprecipitation (ChIP) for the PRC-mediated histone modification H3K27me3. Mouse embryo microdissection was performed to enrich for the caudal region at E8.5 for ChIP-qPCR (Pax6 transcription start site (TSS) was marked by high levels of H3K27me3 indicative of polycomb activity and consistent with repression of this gene in the CLE , which also transcribed the known PRC target HOXD11  cells versus NPs (D8). We assessed the occupancy of the core PRC1 protein Ring1B  [PAX6 and at the TSS of known PRC target HOXD11 in NMP-L (D3) cells. By contrast, reduced occupancy was detected at PAX6 and HOXD11 in NPs (D8) (while baseline detection in the gene desert region (Gw10) and the housekeeping gene GAPDH remained unchanged)   and for lopment)  and mouschanged) . Strikinntiation . This suPAX6 or reflects a general mechanism that mediates onset of neural gene transcription. To address this, we performed a global analysis of the chromatin accessibility landscape using ATAC-seq [PAX6 locus revealed increased accessibility at multiple sites, some as early as D5 and most prominent by D6  term analysis of the genes associated with regions of increased accessibility on D5, D6, and D8 revealed terms related to neural development and showed a progression with more genes associated with these terms over time . Focussily as D5 . OverallPax6 transcription [PAX6 transcript levels analysed by reverse transcription quantitative PCR (RT-qPCR). This revealed precocious PAX6 transcription beginning at D5 and peaking at D6 in the presence of MEKi  supported these conclusions, showing an increase over time and that this was advanced with MEKi treatment assessed on D5  found at open chromatin sites. For this, we carried out genomic footprinting, a computational approach that predicts TF occupancy from ATAC-seq data on the basis of the Tn5 transposase integration frequency detected at base-pair resolution, combined with motif enrichment . We assePAX6 locus  were decreased in NMP-L cells in comparison with hESCs, with a smaller set (679) constituting newly occupied sites in NMP-L cells. Such change in occupancy pattern further supports the conclusion that reduced Ring1B at the X6 locus  is not sX6 locus . This esPAX6) and D6 . This suggests that MEKi increases chromatin accessibility at canonical PRC and non-PRC-occupied sites.To assess the significance of Ring1B occupancy in NMP-L (D3) cells, we next compared all such sites (D3 consensus sites) to our previously (ATAC-seq defined) neural sites, which exhibit increased accessibility from D3 to D8. This revealed that a subset of neural sites  are occupied by Ring1B in the NMP-L cell population, while GO term analysis revealed enrichment for neural and anterior-posterior patterning associated with these Ring1B bound sites , linkingTo test whether Ring1B occupancy in NMP-L cells correlates with the binding of specific TFs, we further performed genomic footprinting on the Ring1B-bound subset and compared this with all other neural sites but found no differences in the factors enriched. However, comparison of predicted TF occupancy in MEKi and control conditions revealed that loss of ERK1/2 activity promoted the engagement of SOX family TFs (D5) and TFAP2 family TFs (D6) regardless of association with Ring1B. These findings indicate that while a decline in ERK1/2 signalling promotes increased accessibility at neural gene loci and promotes engagement of neural TFs, only a subset of the newly accessible loci was formerly occupied by Ring1B. Moreover, these data suggest that the activity of individual TF proteins such as SOX2 and SOX3 is subject to multiple regulatory mechanisms at different genomic sites. Overall, these data show that decline in ERK signalling broadly impacts accessibility at neural genes, can regulate PRC occupancy, and selectively promotes neural TF engagement signatures at neural gene loci.Pax6, within one hour  reduced dpERK1/2 levels   cells to MEKi for 12 hours and conducting the same panel of ChIP-qPCR experiments . At the eatment) . Importaeatment)  nor did nscripts . These dPAX6 locus using FISH. Significant decompaction of this region was found in MEKi-treated cells compared with both DMSO and untreated controls  indicated that the effects of ERK1/2 inhibition in differentiation conditions are not easily reversed. This contrasts with the findings in mES cells, where rescue of ERK1/2 mutant ESCs with ERK2 restored PRC mark H3K27me3 [A potentially direct mechanism by which ERK1/2 regulates PRC2 has been reported in mESCs, where ERK2 protein associates with PRC2/Jarid2 occupied chromatin . In thisin mESCs  might reH3K27me3 . Loss ofPAX6 locus was not accompanied by change in H3K27me3 levels, and PAX6 was not transcribed. These findings and those in differentiation conditions at D6 show that the mechanism(s) by which loss of ERK1/2 activity removes PRC are distinct from those that direct subsequent gene transcription. Such distinct regulation of chromatin accessibility is consistent with observations in Ring1B-deficient mES cells, where HoxB and D loci decompact while their gene promoters remain decorated with H3K27me3 [PAX6 (this mark is not significantly lost until D8)  Figs  and 3B. PAX6, but also other neural progenitor genes , which commence expression in the mouse and chick embryonic body axis, like Pax6, after FGF/ERK1/2 signalling decline [PAX6, which is known to regulate expression of other neural differentiation TFs, including the master neuronal differentiation gene Neurog2 [PAX6 transcription on D6, indicating that increased chromatin accessibility and neural TF binding are ERK1/2-regulated steps distinct from the machinery of transcription  is sharply down-regulated as FGF/ERK1/2 signalling declines in the elongating embryonic body axis [Jarid2 and also the histone deacetylase HDAC1 in the embryo: Moreover, here, HDAC1 expression depends on FGFR signalling [Further ways in which FGF/ERK1/2 signalling decline may direct chromatin reorganisation include alteration of BAF (or SWI/SNF) complex composition, leading to its recruitment and so eviction of PRC proteins from gene loci, as reported in mouse fibroblasts and ESCs . This isody axis . Interesgnalling . These oPAX6 in NMP-L cells and that its inhibition triggers precocious PAX6 transcription in differentiation conditions, suggest that promotion of PRC at neural genes by ERK1/2 signalling may function as a rite of passage for subsequent differentiation.The extent to which FGF/ERK1/2 signalling directs progression of differentiation by regulating PRC occupancy in contexts other than caudal epiblast/NMP-L cells requires further investigation. In mouse ESCs maintained in serum and LIF, PRC and H3K27me3 are detected at differentiation gene loci and this constitutes primed pluripotency, characterised by heterogenous cell states in a dynamic flux between pluripotency and differentiation ,106. ThiIn the future, it will be important to investigate whether ERK1/2 signalling dynamics operate to coordinate chromatin accessibility and expression of differentiation genes in other FGF-expressing progenitor cell populations in the developing embryo, including in limb, lung, and tooth buds –112, as 2 for 1 hour. For FISH, embryos were fixed immediately after 1 hour in hanging drop culture and processed as detailed below. For ChIP-qPCR, embryos were placed on ice in cold culture medium and the caudal region explants microdissected as shown in All animal husbandry and procedures were approved by the UK Government Home Office and were in accordance with European Community Guidelines (directive 86/609/EEC) under project licence number 6004454. CD1 mouse embryos collected at embryonic stage E8.5 were dissected in warm medium  and one half of the litter exposed to MEK inhibitor  and the other half exposed to vehicle control DMSO . Each embryo was placed in a drop of medium in the lid of a 35-mm plate, which was flipped over onto its base, resulting in a hanging drop . EmbryosJarid2 transcript variants were obtained from the database NCBI , and primers  designed to amplify the region homologous to the sequence used for the Jarid2 probe in the chicken [Jarid2 expression analysis in the mouse embryo.Mouse embryos for whole embryo in situ hybridisation were dissected at required stages (E8.5 to E13.5), fixed overnight in cold paraformaldehyde, and processed using standard protocols. Sequences for the  chicken . The seqWork with hESCs was undertaken in approval of the UK Stem Cell Bank steering committee (licence number SCSC14-29). Human ES cells  were authenticated by short tandem repeat (STR) DNA profiling. Cell banks were tested for sterility by direct inoculation of conditioned medium into tryptic soya broth and soya bean casein broth, and no contamination was observed; mycoplasma testing was carried out by DAPI staining of fixed cultures and with mycoalert mycoplasma detection kit (Lonza). Cell lines were tested to check for the uniform expression of pluripotency markers  and absence of differentiation markers  by immunofluorescence.−2) and provided for experiments by the Human Pluripotent Stem Cell Facility of the University of Dundee. For differentiation, cells were plated on Geltrex-coated dishes (20 μg cm−2) at a density of 4 × 103 cells cm−2 and allowed to attach overnight. To generate NMP-L cells, ES media was removed, and cells were cultured with neurobasal medium (Gibco) supplemented with N2, B27, GlutaMAX , 20 ng/ml bFGF (Peprotech), and 3 μM CHIR99021 (Tocris Bioscience) for 3 days -coated dishes (10 μg cms (after ). To gencribe in  to make Pax6 fosmid pair  and the human PAX6 BAC clone pair , then left to cool and washed in dH20. For probe hybridisation, 150 ng each of the labelled probes together with 15 μg mouse Cot1 DNA and 5 μg sonicated salmon sperm DNA were denatured and incubated overnight at 37°C. After a series of washes, the probes were detected using FITC-conjugated anti-Digoxigenin antibody  amplified with anti-sheep Alexa Fluor 488  and biotinylated anti-Avidin (1:100) together with Alexa streptavidin 594 ; nuclei were counterstained with DAPI, coverslips were mounted with Slowfade Gold (Molecular Probes) and imaged on a Deltavision (Applied Precision).The mouse For FISH on human ESC–derived cells, cells were directly grown and differentiated on glass coverslips coated with Geltrex and cells were untreated, exposed to DMSO or MEKi in DMSO as above. After a short fixation , the coverslips were washed in 0.05% Triton X100/PBS . For permeabilisation, the coverslips were incubated in 0.5% Triton X100/PBS for 10 minutes, then transferred to 20% glycerol/PBS for 1 hour at 4°C and then repeatedly dipped in liquid nitrogen until completely frozen, let to thaw, and soaked in 20% glycerol/PBS again (6 repeats). After the last snap freezing, the coverslips were washed in 0.05% Triton X100/PBS , rinsed and incubated in 0.1 N HCl , and washed in 0.05% Triton x100/PBS  again. To equilibrate, coverslips were washed in 2xSSC before prehybridisation (50% Formamide in 2xSSC) for 30 minutes at RT. For probe hybridisation, the same probe mix as for the FISH on mouse material was used (except probes specific for the human Pax6 locus). The probes were denatured on a heat block at 75°C for 3 minutes before hybridisation . The coverslips were then washed, and probes were detected using the same antibody reactions as for the FISH on mouse samples; nuclei were counterstained with DAPI, coverslips were mounted with Slowfade Gold and imaged using a widefield Deltavision Microscope. Using OMERO insight regions of interest (ROIs) were selected over at least 3 z-sections. The 3D interprobe distances in ROIs were measured using a custom script called OMERO mtools , by segmFor each IP and control IP, 25 μL of dynabeads coated with protein A (Invitrogen) were used. Beads were washed 3 times in blocking buffer (0.1% BSA in PBS) and resuspended in blocking buffer containing the antibody for immunoprecipitation or an equal amount of unspecific IgG  and quenched with glycine . Cells were then washed 3 times with PBS and harvested by scraping into protein low bind Eppendorf tubes and centrifuged . The pellets were snap frozen on dry ice and stored at −80°C. For the chromatin preparation, the cell pellet was resuspended in lysis buffer (50 mM Tris–HCl (pH 8.1); 10 mM EDTA (pH 8); 1% SDS and protease inhibitor), vortexed, and incubated (10 minutes on ice). Chromatin fragments of approximately 500 to 1,000 bp were generated by sonication with a probe sonicator . Sonicated lysate was diluted (1:5) in dilution buffer (20 mM Tris–HCl (pH 8.1); 150 mM NaCl; 2 mM EDTA (pH 8) 1% Triton X100 and protease inhibitor) and centrifuged . The supernatant was transferred into a fresh tube. DNA concentration was determined with NanoDrop 2000 Spectrophotometer.For ChIP on human ES cells, the cells were grown and differentiated in 10 cm culture dishes at 37°C and 5% COFor ChIP of chromatin associated proteins 100 μg of chromatin and ChIP of histone modifications 25 μg of chromatin were diluted to a final volume in 1 mL, added to the already prepared bead-antibody complexes, and incubated . An input for each ChIP (10% of ChIPed amount) was kept at −20°C. The next day, beads were washed 5 times in wash buffer (100 mM Tris (pH 8.8); 0.5 M LiCl; 1% NP40, 1% NaDoc and protease inhibitor) and then rinsed and washed in 1x TE (10 mM Tris and 1 mM EDTA (pH 8)). To elute, the beads were resuspended in elution buffer (50 mM Tris–HCl (pH 8); 10 mM EDTA (pH 8); 1% SDS) and incubated . The eluate was spun down quickly, and the supernatant was transferred into a fresh tube. Formaldehyde crosslinking was reversed by incubation at 65°C and 700 rpm for 6 hours. The input sample was diluted with elution buffer (1:1) and crosslinking reversed. All samples were then diluted with TE (1:1), and RNA and proteins were digested . DNA was recovered by Phenol/Chloroform extraction , Sigma-Aldrich) and washed with Chloroform:Isoamylalcohol  and then precipitated in 96% ice cold ethanol, with 3 M SodiumAcetate and linear polyacrylamide  overnight at 80°C. Precipitated DNA was washed with 70% ice cold ethanol, and pellet was air dried before resuspension in nuclease free water.A similar protocol was used to ChIP material from caudal mouse explants. Thirty fixed and snap frozen caudal explants were pooled and resuspended in lysis buffer, vortexed, and dissociated by pipetting before incubating on ice for 10 minutes and sonication. The lysate was diluted in dilution buffer, centrifuged , and the supernatant was transferred into a new tube (and the DNA concentration determined by NanoDrop 2000 Spectrophotometer 0.25 μg of chromatin were used to immunoprecipitate with 3 μg of the rabbit anti H3K27me3 antibody (Millipore) previously bound to dynabeads overnight at 4°C). After that, the same procedure of washing, eluting, uncrosslinking, and precipitation of DNA was used. For quantification of specifically immunoprecipitated DNA, quantitative real-time PCR was performed . Each sample was analysed in 3 biological replicates, and each biological replicate was analysed in technical triplicates using primer pairs in GAPDH using the Pfaffl method [GAPDH levels were checked by normalisation to HPRT1.For RNA extraction, human ES cells were grown and differentiated in 24-well plates. Cells were washed with PBS before being collected in 350 μL of lysis buffer from the Qiagen RNeasy Mini Kit, and RNA was prepared according to manufacturer’s protocol. An on-column DNA digest with RQ1 RNase-Free DNase (Promega) was performed . cDNA was generated from 500 μg purified RNA with the ImProm-II Reverse Transcription System (Promega) primed by random primers. The Aria Mx real-time PCR system with the Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies) was used to quantify the transcript levels. Each sample was analysed in biological triplicate, and each biological replicate was run in technical triplicates . The supernatant was transferred into a new tube and protein concentration determined by Bradford assay. Approximately 50 μg of protein were separated on a 4% to 12% precast Gel (Invitrogen) in 1x MES buffer (Invitrogen) next to a size marker before blotting on onto a PVDF membrane in transfer buffer . To minimise variances, every sample was loaded at least twice on the same gel and blotted onto a membrane at the same time. After blotting the membrane halved and one half was processed with a pan antibody and an antibody for the loading control α-Tubulin and the other half with a phosphorylation specific antibody and the loading control antibody. Membranes were first blocked in 3% BSA in TBST for at least 2 hours at RT, before incubation with primary antibody , kept on ice for 30 minutes while vortexing every 5 minutes, and centrifuged  and centrifuged . Nuclei extracts were resuspended in transposition buffer for 4 hours at 37°C. The reaction was purified using the Qiagen MinElute PCR Purification kit according to manufacturer’s instructions. To generate single-indexed libraries, the transposed DNA was PCR amplified with Nextera primers [ATAC-seq was performed on NMP-L cells, D5 and D6 cells in untreated, DMSO-treated, or MEKi-treated condition, and NP (D8) cells with the following modifications to the method described by Buenrostro and colleagues ,119. Cel primers . Library2+ and Mg2+ (Gibco) and proteins cross-linked with 2 mM disuccinimidyl glutarate  in Dulbecco’s PBS with Ca2+ and Mg2+ for 45 minutes at room temperature with gentle agitation. Cells were then washed three times with Dulbecco’s PBS (Gibco) and DNA cross-linked with 1% methanol-free formaldehyde (Sigma Aldrich) in Dulbecco’s PBS for 10 minutes at room temperature with gentle agitation. The formaldehyde was then quenched by adding glycine to a final concentration of 0.25 mM and incubating for 5 minutes at room temperature. The cells were then washed twice in Dulbecco’s PBS and harvested by scraping into Dulbecco’s PBS containing 0.02% (w/v) Triton X-100 on ice and collected by centrifugation at 500 x g for 5 minutes at 4°C. The supernatant was then removed using a pipette and the cell pellet frozen in liquid N2 and stored at −80°C.Samples were processed as previously described in . BrieflyFrozen pellets were thawed on ice, resuspended with a p200 in ice-cold shearing buffer ) with 0.3% SDS and protease inhibitors (Sigma) and transferred to a diagenode tube. Chromatin was sheered using the Diagenode Bioruptor Plus . Lysates were diluted to 0.15% SDS and cleared by centrifugation at 14,000 rpm for 10 minutes at 4 degrees. Around 1/20th of the lysate was snap frozen in liquid nitrogen and set aside for use as the input. The remaining lysate was incubated on a rotating wheel overnight at 4 degrees with 100 μl of protein G dynabeads (Invitrogen) that had been preloaded for 4 hours at room temperature with 2 μg ChIP-grade Ring1B antibody (MBL D139-3) diluted in sheering buffer with 0.15% SDS. Beads were immobilised using a magnet, and unbound supernatant discarded. Beads were sequentially washed under rotation twice with wash buffer 1 ), once with wash buffer 2 ), once with wash buffer 3 ) and twice with wash buffer 4 ) for 5 minutes each. Beads were captured using the magnet between each subsequent wash. Chromatin was eluted from the beads by incubating twice at 65 degrees for 10 minutes in 100 μl elution buffer  on a shaking heating block. Both eluted fractions were pooled together. Input samples were topped up to 200 μl with elution buffer. Both input and ChIP samples were decrosslinked overnight at 65 degrees with 6.4 μl of 5 M NaCl added to each sample. Samples were incubated for 2 hours at 37 degrees with 0.2 mg/ml PureLink RNAse A (Invitrogen), then 5 mM EDTA and incubated for a further 2 hours at 45 degrees with 0.2 μg/ml proteinase K (Thermo Scientific) before purifying the template DNA for library prep using the Qiagen PCR kit following manufacturer’s instructions. Samples were assessed on the Agilent TapeStation prior to library construction using the NEB Ultra II DNA. Biological triplicates were obtained for all samples from separate experiments. Libraries were generated using the Kapa HyperPrep Kit and sequenced as single-end 76 bp reads on the Illumina HiSeq 4000 (The Francis Crick Institute).http://broadinstitute.github.io/picard). Reads mapped to mitochondrial DNA were removed using the pairToBed command from BEDTools (version 2.26.0-foss-2016b) [Sequencing was performed by multiplexing 4 samples per lane on the Illumina HiSeq 2500 platform (The Francis Crick Institute). This typically generated approximately 86 million 51 bp or 101 bp paired-end reads per library. Raw reads from each sample were adapter-trimmed using cutadapt (version 1.9.1)  with pars-2016b) . Additios-2016b)  was useds-2016b) .To define peaks, regions of chromatin accessibility were identified genome-wide using MACS2 callpeak (version 2.1.1.20160309)  with thehttps://go.princeton.edu/cgi-bin/GOTermFinder. ATAC-seq metaprofiles were generated using deepTools (version 2.5.3) [Fragment-level BED files were derived from those created using the BEDTools bamToBed command with the option “-bedpe”. Differential chromatin accessibility sites between conditions were obtained using diffReps (version 1.55.4)  with then 2.5.3)  with thehttps://github.com/luslab/briscoe-nf-tobias) written in the Nextflow domain-specific language . Analys21.04.0; ).https://doi.org/10.5281/zenodo.3966161), written in the Nextflow domain-specific language . Gene annotation files in GTF format were originally downloaded from UCSC on 7 June 2017. For a condensed overview of the pipeline, the bioinformatics tools used in each step, and an extensive list of citations, please see the pipeline homepage. Differential ChIP-seq peaks (FDR 0.01) detected between day 0 and day 3 from three independent biological replicates using [The nf-core/chipseq pipeline   was use19.10.0; ). The co19.10.0;  downloades using  were subes using . Peak ines using  with thees using  relativeS1 FigA) Whole E8.5 embryo, the CLE is where NMPs are located, (A’) TS. Jarid2 expression continues (B) at E9.5, including in the NMP and for a time in NPs generated by this cell population, shown TS . This pattern of expression persists (C) at E10.5, shown in TS in (C’), and is then lost as the tailbud is depleted and axis elongation declines from (D) E11.5 and (E) 12.5, and ceases at (F) E13.5. Scale bars = 100 μm in A, 200 μm in B, C, D-F. CLE, caudal lateral epiblast; NMP, neuromesodermal progenitor; NP, neural progenitor; TS, transverse section.((PDF)Click here for additional data file.S2 FigComparison of peaks called from publicly available ChIP-seq data sets for H3K9me3, H3K27me3, H3K4me1, and H3K27ac from the ENCODE regulatory element database  with neu(PDF)Click here for additional data file.S3 FigA) ChIP-PCR for Jarid2 for untreated/WT, DMSO, and MEKi-exposed cells during differentiation from NMP-L (D3) to D6 (n = 3 independent experiments). These experiments provided low % input and just enriched for WT above IgG control, t test shows no significant difference between WT and DMSO or DMSO and MEKi conditions, although WT and MEKi are significantly different, error bars = SEM, * = p ≤ 0.05 (B) To assess levels of Jarid2 protein as hESCs differentiate into NMP-L cells and then NPs, we collected protein lysates and ran western blots using antibodies against Jarid2 and GAPDH. This is a sample blot showing one biological replicate and its 3 technical replicates. Bradford assays were used to determine protein concentration and 50 μg protein loaded, note both Jarid2 and GAPDH appear to reduce levels as hESCs differentiate. hESC, human ESC; IgG, immunoglobulin G; MEKi, MEK inhibitor; NMP-L, NMP-like; WT, wild type.(p ≤ 0.05 . These d(PDF)Click here for additional data file.S4 FigA) One biological replicate and its 3 technical replicates , and (B) quantification of these data. Data analysed using the Student t test, error bars ± SD, each dot represents a single data point, p = *p < 0.05, ***p < 0.001; NMP-L cells were differentiated towards neural progenitors in control (DMSO only) or MEKi /DMSO conditions for 3 days (assessed on D6) in 3 independent differentiations, replicates 1,2,3,;  exposure to MEKi lead to increased expression of PAX6 in all three replicates detected by RT-qPCR (p = 0.0024); western blot of 2 technical replicates from each of 3 biological replicates  confirm dephosphorylation of ERK1/2 with antibodies to p-ERK1/2 and compared with total ERK1/2 levels and show  Ring1B and GAPDH protein levels were unaffected by loss of ERK signalling; (G) ChIP-qPCR detecting ERK2 occupancy at PAX6 and control loci during differentiation (NMP-L (D3), D5, and D6: black, dark grey, and light grey, respectively) ; (H and H’) ChIP-qPCR investigating ERK2 occupancy at the PAX6 locus on D5 and D6 of the differentiation protocol comparing untreated, DMSO, and MEKi-exposed samples . All underlying numerical data in this figure can be found in To assess levels of Ring1B protein as hESCs differentiate into NMP-L cells and then NPs, we collected protein lysates from 3 independent differentiations and ran western blots, each with three technical replicates using antibodies against Ring1B and beta-Actin. Levels of Ring1B were normalised to b-Actin. ((PDF)Click here for additional data file.S5 FigA) Differentiation protocol used to generate NMP-L (D3) cells and treatment regime with vehicle control DMSO or MEKi for 3 hours; (B-B’) representative western blot of cell lysates probed with antibodies against total (panERK1/2) and dual-phosphorylated-ERK1/2 (dpERK1/2) and LiCOR quantification data ; (C-E) ChIP-qPCRs investigating Jarid2 and Ring1B occupancy and H3K27me3 levels at PAX6 and control regions in NMP-L (D3) cells treated with MEKi or DMSO for 3 hours , note low Jarid2 input, not enriched over IgG; (F-F”) transcription levels of PAX6, HOXD11, and JARID2 assessed by RT-qPCR in undifferentiated cell (hESCs), untreated, vehicle control (DMSO)-treated, or MEKi-treated NMP-L (D3) cells . All underlying numerical data in this figure can be found in ((PDF)Click here for additional data file.S6 FigA and B) Western blot analysis of NMP-L (D3) protein extracts using phospho-specific antibodies against ERK1/2 and PKB alongside pan antibodies. (C and D) Quantification of band intensity shows no reduction in ERK phosphorylation but increased levels of PKB phosphorylation . Underlying numerical data for western blots in this figure can be found in ((PDF)Click here for additional data file.S1 TableDuring in vitro neural differentiation from NMP-L (D3) to NP (D8) 7,877 regions open up associated with 4,001 genes. Regions of increased accessibility are defined by chromosomal position and their associated gene names have been identified by proximity. In the text, we refer to these as “neural sites.”(XLSX)Click here for additional data file.S2 TableDuring in vitro neural differentiation, ATAC-seq reveals that an increasing number of neural genes appear that are associated with open chromatin (as defined by proximity). By day 5 of differentiation, regions associated with 1,143 genes display increased accessibility by day 6 this increases to regions associated with 2,137 genes are open and by final day 8 regions associated with 4,001 genes are now more accessible.(XLSX)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(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S8 Data(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file.S10 Data(XLSX)Click here for additional data file.S11 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


In light of an issue identified post-publication, this article was republished on September 13, 2022, to replace S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Marichromatium gracile HOL-1 and another purple photosynthetic organism, strain H1R, that was originally isolated as an unidentified contaminant. Through genome sequencing, we have now identified organism H1R as a species of Afifella. A whole-genome-based phylogenetic analysis of both species is provided.We sequenced the genomes of both the purple sulfur gammaproteobacterium  Marichromatium gracile HOL-1, and although it was known to be a photosynthetic purple nonsulfur bacterium, its true nature has been elusive for decades. The species was simply referred to as “organism H1R” (red isolate from HOL-1), a purple photosynthetic bacterium of unknown taxonomy (1c5 and c3); however, the closest homologue of the c5 cytochrome appeared to be from Shewanella , in an effort to study novel cytochrome c proteins and photosynthetic mechanisms of purple sulfur bacteria. Strain H1R strain had been separated from it by one of us (T.E.M.) decades ago, by repetitive plating on Rhodospirillaceae medium (DSMZ medium 27) supplemented with 3% NaCl. This separation occurred before strain HOL-1 was deposited as a pure culture into the DSMZ culture collection (DSM 1712). Genomic DNA was isolated from glycerol freezer stocks of both species. DNA was purified using the GeneJET DNA purification kit (Thermo Scientific). Qubit and NanoDrop DNA analyses showed 260/280 nm absorbance ratios of 1.81 for strain HOL-1 and 1.60 for strain H1R. The sequencing libraries were prepared using the Illumina Nextera DNA Flex library prep kit. The genomes were sequenced with an Illumina MiniSeq using 500 μL of a 1.8 pM library. Paired-end (2 × 150 bp) sequencing was performed, and the number of obtained reads is given in de novo using Unicycler (version 0.4.8) through PATRIC (6 PATRIC 68. An Eva PATRIC 6. DefaultAfifella (formerly Rhodobium). The closest relative, with an ANI of 95.5%, is Afifella aestuarii JA968  and JAKEDY000000000 (Afifella sp. H1R). The versions described in this paper are versions JAKEDQ010000000 (HOL-1) and JAKEDY010000000 (H1R). The raw sequencing reads have been submitted to SRA, and the corresponding accession numbers are SRR17553093 (HOL-1) and SRR17553527 (H1R).This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers


Ex vivo treatment of primary patient-derived CRC tumor slice cultures with the LY6G6D/CD3 TcE led to IFNγ secretion in LY6G6D positive tumor samples. In vivo, LY6G6D/CD3 TcE monotherapy demonstrated tumor regressions in pre-clinical mouse models of engrafted human CRC tumor cells and PBMCs. Lastly, 2D and 3D cocultures of LY6G6D positive and negative cells were used to explore the bystander killing of LY6G6D negative cells after specific activation of T cells by LY6G6D positive cells. LY6G6D/CD3 TcE treatment was shown to lyse target negative cells in the vicinity of target positive cells through a combined effect of IFNγ, TNFα and Fas/FasL. In summary, LY6G6D was identified as a selectively expressed CRC antigen that can be utilized to potently re-direct and activate cytotoxic T-cells to lyse LY6G6D expressing CRC using a TcE. This effect can be spread to target negative neighboring tumor cells, potentially leading to improved therapeutic efficacy.Colorectal cancer (CRC) is one of the most common cancers worldwide and demands more effective treatments. We sought to identify tumor selective CRC antigens and their therapeutic potential for cytotoxic T-cell targeting by transcriptomic and immunohistochemical analysis. LY6G6D was identified as a tumor selectively expressed CRC antigen, mainly in the microsatellite stable (MSS) subtype. A specific anti LY6G6D/CD3 T cell engager (TcE) was generated and demonstrated potent tumor cell killing and T cell activation  Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and third most diagnosed cancer . CRC is The Lymphocyte Antigen 6 Family Member G6D (LY6G6D) belongs to a cluster of leukocyte antigens located in the major histocompatibility complex (MHC) class III region on chromosome 6 . DespiteBispecific T-cell engagers (TcE) have become a promising class of antibody-based immunotherapy. Their therapeutic efficacy is well established in hematologic malignancies  9), and, and9), in vitro assays where the expression of LY6G6D is homogeneous among cells. But it also demonstrates lysis of LY6G6D non-expressing cells in cocultures with LY6G6D expressing cells. Mechanistically this bystander killing of LY6G6D non-expressing cells is mediated by Fas/FasL, TNFα and IFNγ. Interestingly, these pathways seem to be dispensable for the direct killing of LY6G6D expressing cells upon engagement by the TcE. Since LY6G6D expression in tumors shows some level of heterogeneity, this finding implies that bystander killing of target negative cells in the tumor could increase the therapeutic effect of the TcE.In this report, we describe the identification of LY6G6D as a tumor selectively expressed antigen in CRC, and the generation of a LY6G6D/CD3 bi-specific antibody to potently redirect T cells into LY6G6D expressing CRC cells. The LY6G6D/CD3 TcE shows specific killing of target positive cells in Frozen human PBMCs were obtained from StemCell Technologies. Human tissues were obtained from the Department of Pathology at the Medical University of Vienna, Austria or Indivumed GmbH, Germany. All blood donors and patients provided written informed consent before sample collection.The human CRC cell lines used in this study were NCI-H508, LS1034, SK-CO1, obtained from ATCC; CL-14 from Leibniz Institute DSMZ: and HT55 from Public Health England and NCM460 from Incell. Human kidney cells 293 were obtained from ATCC and 293 cells expressing LY6G6D and Jurkat WT-NFATluc reporter cells were established in-house.2.All cell lines were cultured according to supplier’s instruction and maintained at 37°C, 5% CO+ in DMEM 10% HI-FBS, 2 mM L-Glutamine 1% NEAA and 1.0 mg/ml Geneticin (GIBCO) and Jurkat WT-NFATluc cells were cultured in RPMI 1640 (GIBCO), 10% HI-FBS, 2 mM L-Glutamine1% NEAA and 0.25 mg/ml Hygromycin (ThermoFisher Scientific).NCI-H508 and LS1034 cells were cultured in RPMI 1640 (GIBCO) for and HT55 Co and SK-CO1 cells in EMEM (Sigma) supplemented with 10% HI-FBS , 2 mM L-Glutamine 1% non-essential aminoacids .CL-14 cells were cultured in DMEM/F12 (GIBCO) containing 20% HI-FBS, 2 mM L-Glutamine 1% NEAA, NCM460 in M3-base medium (INCELL) containing 10% HI-FBS,recombinant 293 LY6G6D6) were transfected with 5 µg DNA of LY6G6D_NFLAG_pcDNA 3.1(+) A009 (LifeTechnologies) by using Cell Line Nucleofector™ Kit V (Amaxa/Lonza) according to manufacturer’s instructions. After 24 hours incubation at 37°C, 5% CO2, 95% humidity, 1.0 mg/ml Geneticin was added to the medium. Cells were analyzed in FACS for LY6G6D expression.293 cells (1x10Jurkat-E6.1 (from ATCC # TIB-152) were transfected with pGL4.30 NFATluciferase reporter plasmid (Promega cat. # E8481) according to manufacturer’s instructionsFFPE  tissue samples and TMA (Tissue Microarrays) were sliced into three micrometer sections and mounted on glass slides, stained with anti-LY6G6D clone 10C1 antibody or isotype control followed by OptiView DAB IHC Detection Kit (Roche Diagnostics). The stained tissue sections were evaluated by a pathologist including assessment of the percentage of stained tumor cells and the staining intensity. Sections containing at least 1% LY6G6D expressing tumor cells were scored as positive.To identify monoclonal antibodies that specifically bind to the human LY6G6D protein, mice were immunized with a human-IgG1-Fc-His6 fusion protein derived from the matured human LY6G6D sequence (amino acid 20-103). Serum titers were measured by ELISA using an analogous His6 fusion protein. Splenocytes from responsive mice were pooled and immortalized by PEG based fusion. Positive clones were selected by ELISA and monoclonal hybridoma cells were generated by limited dilution. Binding was confirmed by ELISA from purified IgG obtained from hybridoma supernatant.Next, two different screening processes were performed to select clones for IHC and TcE. To select clones for IHC and to ensure antibody specificity and selectivity in fixed tissues, clones were selected by staining of formalin-fixed paraffin-embedded cells mRNA positive/negative CRC cell lines. Clone 10C1 was selected eventually by this process. To select clones suitable as TcE and therefore to ensure recognition of LY6G6D in its native confirmation on live cells clones screened for binding to mRNA positive/negative CRC cell lines in FACS using unfixed cells. Clone 2C11A8 is derived from this later screen. Nucleotide sequence of 2C11A8 was determined after Vgene recovery.The LY6G6D specific T cell engager was based on LY6G6D specific clone 2C11A8 and CD3ϵ specific clone SP34 sequences including a C-terminal hexahistidine (His6 tag). The DNA sequence was synthesized (Geneart) and cloned in the pTT5 expression vector . Trans IT PRO transfection reagent (Mirus Bio) was used for transient transfection of CHO-3E7 cells  and the TcE was expressed for 10 days. Clarified culture supernatant was purified by IMAC affinity chromatography . Purified TcE was formulated in 50 mM sodium acetate, 100 mM NaCl, pH 5.0, and analyzed by analytical size-exclusion chromatography (Agilent PL1580-5301 column) coupled to multi-angle light scattering, SDS-PAGE, and intact mass spectrometry. The LY6G6D specific TcE showed the expected molecular weight with >99% purity and <3% aggregated species.Recombinant LY6G6D protein DNA was generated by gene synthesis (Geneart) and standard recombinant DNA technology. The protein was expressed in HEK293-6E cells by transient transfection using PEI MAX (Polysciences). Cells were harvested 96 hours post transfection, and supernatant was collected and clarified using G4 filter (Sartopore). The protein was purified from supernatant by IMAC chromatography  followed by size exclusion chromatography . Purified protein was formulated in 1 x PBS, 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, pH – 7.2 and quality and identity confirmed by analytical size-exclusion chromatography, SDS-PAGE, and intact mass spectrometry.Affinity analysis of the LY6G6D specific TcE was performed by surface plasmon resonance using BIAcore T200 device (Cytiva). In brief, the TcE was immobilized on CM5 Chip (Cytiva) and recombinant LY6G6D protein was injected at concentrations of 0-9000 nM in HBS-EP . Measurements were run at 25°C and data were analyzed according to the 1:1 Langmuir model.Cells were harvested and incubated with 0.5-0.0001 units of PI-PLC (ThermoFisher) for 30 min at 37°C with slight shaking. Flow cytometry analysis were performed to confirm release of LY6G6D after PI-PLC treatment.Target cells were seeded and incubated at 37°C. After 3 hours, purified T-cells from human peripheral blood mononuclear cells  were added in a target to T cell ratio 1:10 with different concentrations of LY6G6D/CD3 Target cell killing was assessed after 48 or 72 hours by quantification of LDH released following manufacturer’s instructions . Target cells with 3% Triton X-100 represents the maximum cell lysis (100%), and target cells with effectors cells the minimum lysis (0%). Percentage of specific cell lysis was calculated as [sample LDH release - spontaneous LDH release]/[maximum LDH release – spontaneous LDH release] x 100.To determine the total number of LY6G6D positive and negative cells in the coculture experiments after treatment, each population was labelled with different CellTrace™ dyes (Invitrogen) according to manufacturer’s instructions. Cell lysis after 72 hours was determined by quantifying absolute live cells by flow cytometry.To quantify the total number of cells after treatment, AccuCheck counting beads (Invitrogen) were added following manufacturer’s instruction before acquisition of samples on FACS Canto-II (BD Biosciences). Data was analyzed on FlowJo (TreeStar).Expression of activation markers on T cells were analyzed at end-point of the cytolysis assays. T-cells were stained with Zombie Green Viability Dye or Zombie NIR Viability Dye (BioLegend) and surface antibodies against human CD4, CD8, CD69 and CD25 and CD107a (BD Biosciences or BioLegend). For expression of GranzymeB and perforin, cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFisher) according to manufacturer’s instructions, and stained with anti- GranzymeB and anti-Perforin antibodies (eBioscience/ThermoFisher) for 30 min at 4°C.BioLegend)To assess LY6G6D expression on target cells, they were incubated with Zombie NIR Fixable Viability Dye and anti-human LY6G6D-PE . Before acquisition, AccuCheck counting beads (Invitrogen) were added to the cell suspension. Cells were acquired on FACS Canto-II or Fortessa flow cytometer (BD Biosciences) and data analyzed on FlowJo (Tree Star).®  according of manufacturer’s instructions.Quantification of LY6G6D cell surface molecules expressed on cell lines was performed by flow cytometry using the QIFIKIT® Luciferase Assay System; Promega) on EnSight™ (PerkinElmer) accordingly to manufacturer’s instructions.Target cells were seeded and 3 hours after LY6G6D/CD3 or untargeted control Fab-scFv,and reporter Jurkat NFATluc cells were added in a target to effector cell ratio of 1:10. After 24 hours of incubation at 37°C, activation of reporter cells was determined by luciferase expression (Steady-GloCytolysis assay was performed as described above. LY6G6D negative and positive cells labelled with cell tracer FarRed or cell tracer violet dye (BioLegend), respectively. T cells and Caspase 3/7 green dye (Sartorius) were added and cultured with or without TcE. Images of cells were acquired on an Opera Phenix Plus device (PerkinElmer) every 3 hours for 24 hours. Analysis of the images were performed using the Harmony Software, and display of images were done by the TIBCO Spotfire Software 4.9.® VF-300-0Z. Tumor slides were incubated with LY6G6D/CD3 TcE or TNP/CD3 control at 37°C, 5% CO2 for 48 hours. Then fixed in formalin and embedded in paraffin. LY6G6D and CD3 expression were analyzed by immunohistochemistry in baseline slices; Granzyme B was analyzed from treated tumor slices. IFN-γ, Granzyme B, IP-10, IL-2, TNFα and MCP-1 were quantified in the cell supernatant using U-PLEX Assays  following the manufacturer’s instructions. All slice culture experiments were performed in biological duplicates or triplicates. Cytokines were quantified from all biological replicates, the mean value of each sample was then normalized to the respective untreated (medium only) samples and depicted as fold change.Freshly excised tumor tissue was transferred to the lab within 30 min after surgery and 200 μm sections were prepared using a Compresstome6 LS1034 cells. When tumor size reached 100 mm3, mice were injected i.v. with 1 x 106 human PBMC. Treatment with TcE started 3 days after PBMC transfer, 0.5 mg/kg of LY6G6D/CD3 Fab-scFv negative control TNP-CD3 Fab-scFv were administered daily for 5 days per week. Animals were monitored daily for morbidity and mortality. Tumor volumes were measured twice per week with digital caliper, and the volume will be expressed in mm3 using the formula: “V = (L x W x W)/2, where L is tumor length and W is tumor width.All mice were housed and treated according to approved IACUC protocols. NOG mice were injected s.c. with 1 x 102.Isolated or cocultured NCM460D and LS1034 cells were seeded at 1000 cells/well in ultra-low attachment 96 well plates (costar) using RPMI (Gibco) supplemented with 10% FBS (HyClone), and incubated for three days at 37°C, 5% COAfter confirming spheroid formation by microscope 5000 PBMCs/well (StemCell Technologies) and treatments were added. Treatments consisted of LY6G6D/CD3 TcE at different concentrations. For blocking experiments, 10µg/ml Entanercept (EDQM), 5µg/ml CD178 monoclonal antibody (Invitrogen), or a combination or 1µg/ml human IFNγ antibody (bio-techne) with 1µg/ml human IFNγR antibody (bio-techne) were added. PBMCs and compounds were diluted with RPMI supplemented with 10% FBS. Spheroid growth was measured with an IncuCyte S3 (Sartorius) for the next 7 days and data analyzed bythe IncuCyte2021C software.Statistical analysis was performed using GraphPad Prism 9.0.0 software. A one-sided non-parametric Mann-Whitney- U-test was applied to compare treated animals with LY6G6D/CD3 TcE ITE and TNP/CD3 TcE control. ** p < 0.01.Potent re-direction of cytotoxic T-cells to tumors requires tumor selectively expressed antigens. Using public transcriptomic data, we identified LY6G6D as a CRC specific antigen within 11616 tumor samples from the Cancer Genome Atlas (TCGA), and 17588 normal samples from TCGA  and the Genotype-Tissue Expression project  . This data confirms the recent identification of LY6G6D as a CRC marker, mainly expressed in MSS tumors , by a similar in silico approach , as its staining was blocked with 50x recombinant LY6G6D protein, confirming its specificity for LY6G6D . The abundance of LY6G6D expression was analyzed in tumor samples of a CRC TMA (n=41) and non-malignant colon tissue using clone 10C1. We observed that similarly to other tumor specific antigens, LY6G6D expression was heterogeneous within the tumor area, and the percentage of positive cells ranged from 1 to 90% of the tumor content (via the (G4S)4 linker to a C-terminal CD3ϵ specific scFv derived from the murine clone SP34 , but not when the parental HEK293 (HEK293 LY6G6D-) cells were used  molecule. The GPI-anchored proteins (GPI-AP) can be released from the cell membrane by phospholipases and putative substrate-specific GPI-AP-releasing enzymes . We mimiWe interrogated whether the LY6G6D/CD3 TcE could activate tumor infiltrating lymphocytes (TILs) in precision cut tumor samples from CRC patients. For this, eight CRC tissue slices were selected: five with LY6G6D expression and three LY6G6D negative , and Granzyme B, IL-2, TNFα and MCP-1 (4/5 LY6G6D positive samples)  and tumor regression was observed in 2 out of 5 animals reconstituted with PBMCs from donor A . However, these antigens are scarce in solid tumors, and the field is searching for antigens selectively expressed in tumor cells, to guarantee a thesrapeutic window . TherefoIn this study, we describe the identification of LY6G6D as a high tumor specific antigen in CRC, specifically in MSS tumors, confirming the same observations made by other groups , 23. Thiin vitro and in vivo assays to validate its specificity and efficacy. In a homogeneous population of LY6G6D positive cells, the LY6G6D/CD3 TcE induced potent tumor cell lysis and T cell activation which correlated with target density in the cell membrane. In vivo studies showed strong tumor regressions in all animals treated with LY6G6D/CD3 TcE monotherapy. Tumors from treated animals presented a more inflamed phenotype, with higher infiltration of T cells (data not shown), as observed by others and our group previously  can be found below: in vivo studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to execution. During the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Short-time culture of human tumor samples was reviewed and approved by the Ethics Committee of the Medical University of Vienna (EK:2042/2019). Informed written consent was obtained from all participants in this study.The animal study was reviewed and approved by The protocol and any amendment(s) or procedures involving the care and use of animals for in vitro experiments and contributed to the preparation of the figures. NS: Designed the molecule and established the production process. IT: Supervised and coordinated organotypic in vitro experiments. K. Fuchs: Designed and supervised the antibody generation campaigns. BT: Executed and analyzed organotypic in vitro experiments. CW: Screened specific antibodies. KB: Screened specific antibodies. MF: Planned, executed, and interpreted tissue slice culture experiments. MB: Planned tissue slice culture experiments. CB: Excised tumor samples and supported pathological examinations of slice cultures. PC: Supported histological analysis of CRC samples in ex vivo studies. AV: Supervised and coordinated ex vivo studies. PA: Supported the design, supervised, and evaluated the manuscript. All authors contributed to the article and approved the submitted version.LC: Designed the manuscript, supervised the study, and wrote the manuscript. SH: supervised antibody campaigns and prevalence studies. KM: Executed and analyzed This study was supported by Boehringer Ingelheim and the Basisprogramm grants of the Austrian Research Promotion Agency .We thank Margit Zehetner, Andreas Schrenk, John Miglietta, Carolina Klicka, Sam Lukowski, Abdallah Souabni, Lisa-Sophie Heinz, Rene Hude and Katharina Kauer for their experimental support and help for this study; and our collaborator from Precision Antibody  for Ab generation of 10C1 and from Syngene  for generation of 2C11A8.LC, SH, KM, NS, IT, KF, BT, CW, KB, PC, AV, and PA are employees of Boehringer Ingelheim affiliates. MB received research funding and speakers fee by Boehringer Ingelheim and Bristol Myers Squibb.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Influenza A viruses (IAVs) in the swine reservoir constantly evolve, resulting in expanding genetic and antigenic diversity of strains that occasionally cause infections in humans and pose a threat of emerging as a strain capable of human-to-human transmission. For these reasons, there is an ongoing need for surveillance and characterization of newly emerging strains to aid pandemic preparedness efforts, particularly for the selection of candidate vaccine viruses and conducting risk assessments. Here, we performed a parallel comparison of the pathogenesis and transmission of genetically and antigenically diverse swine-origin A(H1N1) variant (v) and A(H1N2)v, and human seasonal A(H1N1)pdm09 IAVs using the ferret model. Both groups of viruses were capable of replication in the ferret upper respiratory tract; however, variant viruses were more frequently isolated from the lower respiratory tract as compared to the human-adapted viruses. Regardless of virus origin, observed clinical signs of infection differed greatly between strains, with some viruses causing nasal discharge, sneezing and, in some instances, diarrhea in ferrets. The most striking difference between the viruses was the ability to transmit through the air. Human-adapted viruses were capable of airborne transmission between all ferret pairs. In contrast, only one out of the four tested variant viruses was able to transmit via the air as efficiently as the human-adapted viruses. Overall, this work highlights the need for sustained monitoring of emerging swine IAVs to identify strains of concern such as those that are antigenically different from vaccine strains and that possess adaptations required for efficient respiratory droplet transmission in mammals. Influenza A and B viruses are important human respiratory pathogens capable of causing a substantial public health burden every year. According to the World Health Organization, annual influenza virus epidemics result in approximately 3–5 million cases of severe illness, and up to 650,000 respiratory deaths worldwide . While iIn this study, we used the ferret model to assess the pathogenesis and transmission capability of four genetically and antigenically diverse swine A(H1) IAVs isolated from humans between 2017 and 2020. Three contemporary A(H1N1)pdm09 viruses were included in the analysis for comparison. The findings of this study provide insight into the evolution of swine IAV, supporting that these viruses can emerge into genetically and antigenically distinct strains that in some cases are capable of transmission via the air in the ferret model at comparable levels of efficiency as seen for human-adapted viruses.Viruses. Stocks of A/Alberta/1/2020 A(H1N2)v, A/Ohio/24/2017 A(H1N2)v, A/California/62/2018 A(H1N2)v, A/Michigan/288/2019 A(H1N1)v, A/Michigan/45/2015 A(H1N1)pdm09, A/Idaho/7/2018 A(H1N1)pdm09, and A/Nebraska/14/2019 A(H1N1)pdm09 viruses were propagated in Madin-Darby Canine Kidney (MDCK) cells at 37°C for 48 hrs. Viral titers were determined using a standard plaque assay in MDCK cells as described previously [eviously . Each stPhylogenetic analysis. A phylogenetic tree was built for the influenza virus HA genome segment obtained from GISAID (http://platform.gisaid.org). The sequences for each strain were aligned using the ClustalW application and Muscle algorithm [www.megasoftware.net) [lgorithm . Neighboare.net) . SecondaAntigenic analysis. Ferret immune sera were harvested 14 days post intranasal inoculation with 6 log10 50% egg infectious dose (EID50) of virus. Two-way hemagglutination inhibition (HI) assay was used to assess cross-reactivity between different virus strains and pooled human sera  (6-13 months of age) were housed in Duo-Flo Bioclean mobile units  during experimentation. For each transmission experiment, groups of 3–6 ferrets per virus were anesthetized via intramuscular injection of a ketamine cocktail  in the hamstring and inoculated intranasally with 6 log10 PFU diluted in 1 ml of virus diluted in PBS. The next day the Direct Contact Transmission model (DCT) was established by placing a naïve ferret in each cage housing an inoculated ferret (3 ferret pairs per virus tested) and the Respiratory Droplet Transmission model (RDT) was established by placing naïve and inoculated ferrets in adjacent cages separated by a perforated wall (3 ferret pairs per virus tested) [ tested) . Each fe tested) . Any ani tested) .Phylogenetic analysis. Phylogenetic analysis was performed using the sequences of the mature HA proteins with the signal peptide removed. Four genetically distinct A(H1)v viruses associated with recent human infections were examined. The A(H1N2)v virus detected in Alberta, Canada in 2020  clustered with alpha (1A.1.1) lineage swine IAVs circulating in Canada and the USA . Alberta/1 and A/Ohio/24/2017 (OH/24), also an alpha (1A.1.1) lineage A(H1N2)v virus, evolved from classical swine IAVs that have circulated in North America since they were first detected in the 1930s [he 1930s . Genotyphe 1930s , while tNext, we examined antigenic profiles of the swine A(H1)v viruses. Ferret antisera raised to the North American OH/24 and the reverse genetics-derived OH/24-like CVV (IDCDC-RG59) failed to inhibit Alberta/1 in HI tests. Likewise, no cross-reactivity of ferret antisera raised to the recommended seasonal vaccine strain, A/Idaho/7/2018 A(H1N1)pdm09 (ID/7), was observed when tested with this virus. In addition, pooled post-vaccination antisera from children (0-3 years) and adults (19-49 years) also showed little to no cross-reactivity with the Alberta/1 virus . In cont10 PFU/ml for the A(H1)v viruses and between 6.1 and 7.8 log10 PFU/ml for the seasonal A(H1N1)pdm09 viruses , inoculated ferrets lost the least amount of weight, and no overt respiratory signs were observed during the time course of infection except for sneezing. In contrary, the ID/7 virus was the most pathogenic in ferrets as evidenced by 20.6% mean maximum loss of pre-inoculation body weight, diarrhea, as well as respiratory signs, including nasal discharge and sneezing. Out of the three inoculated ferrets from this group, one was humanely euthanized on day 9 p.i. due to severe weight loss. Ferrets inoculated with OH/24, CA/62, and MI/288 viruses frequently displayed respiratory signs of infection, transient increase in body temperature, and weight loss (mean maximum 9.0-14.0%), but with diarrhea and mortality were not observed (n = 3/virus) were sacrificed day 3 p.i. to assess virus dissemination in tissues (10 PFU/ml or g) were detected in nasal turbinates and soft palate samples from all tested ferrets, while the virus was found in only 1 trachea and 2 lung samples. ID/7 virus was present in the upper respiratory tract, including the nasal turbinates, soft palate, and trachea, although detection in all three tissues was limited to a single ID/7-infected animal (10 PFU/g). Brain tissue and olfactory bulb were tested for the presence of virus; NE/14 was the only virus detected in both tissues, while MI/288 and ID/7 were detected in the olfactory bulb. Spleen, liver, intestines, kidneys, and blood were also tested but no infectious virus was observed for any of the IAVs included in this study. Overall, while variant A(H1) viruses were detected throughout the ferret respiratory tract, like what was observed with A(H1N1)pdm09 viruses isolated in 2009 [Next, we used the ferret model to compare the severity of disease caused by four A(H1)v IAVs  and three contemporary seasonal A(H1N1)pdm09 viruses . Nasal washes were collected every two days p.i. to compare replication capacities and the amount of virus shed from each animal. The peak nasal wash titers ranged between 3.8-7.6 log viruses . The Albobserved . Additio tissues . Low titd animal . Detecti in 2009 , contemp in 2009 .Table 3Both DCT and RDT models were used to asses transmissibility of A(H1)v viruses, while the human adapted viruses, which are known to transmit between ferrets efficiently, were only tested using the RDT model to reduce the overall number of animals used in this study. Each of the A(H1)v viruses rapidly transmitted between all co-housed ferret pairs within the first day of exposure. MI/288 virus was also capable of airborne transmission between all ferret pairs by 5 days p.c. The other three A(H1)v viruses, Alberta/1, OH/24 and CA/62, transmitted between 1 out of the 3 ferret pairs in the RDT model; the 2 uninfected ferrets from each group were confirmed to be seronegative against the inoculum virus at day 20–21 p.c. All of the human-adapted A(H1N1)pdm09 viruses were able to transmit efficiently through the air by 3 days p.c. Similar clinical signs and disease outcomes were observed in all ferrets that were infected after exposure to an inoculated animal (data not shown). For example, 2 of the ID/7 virus contact animals displayed severe weight loss (14.5 and 25%) accompanied by nasal discharge or dyspnea and required euthanasia on day 8 and 11 p.c. . The ferSince the isolation of the first swine A(H1N1) influenza virus in 1930 , the claThe antigenic relatedness of a newly emerged influenza virus in comparison to viruses that are circulating in people as well as viruses that were used to produce CVVs and stockpiled pre-pandemic vaccines is a critical parameter used by the IRAT. A previous study suggested that population immunity to the highly transmissible in our study MI/288 A(H1N1)v virus (1A.3.3.2 lineage) is likely high, showing that immune sera obtained from children and adults vaccinated with seasonal influenza as well as ferrets infected with seasonal influenza virus effectively inhibited the MI/288 virus . In contBeyond the antigenic and genetic differences displayed by swine-origin IAVs, the scope of disease progression and the ability to transmit can also substantially differ between strains. Similar to humans, ferrets are highly susceptible to influenza virus infection, display pronounced clinical symptoms and signs of infection, and can transmit virus to other ferrets . For theUnlike human, seasonal influenza viruses, which transmit efficiently between ferrets through the air, including the A(H1N1)pdm09 viruses used in this study, transmissibility of A(H1)v viruses varies between strains . Barman Collectively, these data highlight the continued adaptation of A(H1N1) IAVs derived from the 2009 pandemic to humans and illustrate that, while infrequent, selected viruses nonetheless possess the ability to cause severe and fatal disease in mammals. Furthermore, while A(H1N1)v and A(H1N2)v viruses continue to sporadically cause human infections, their distinct antigenic profiles and the capacity for airborne transmission underscore the pandemic risk posed by these viruses. Continued assessment of antigenicity, pathogenesis, and transmission of novel and emerging influenza viruses from zoonotic reservoirs using the ferret model is necessary for pandemic preparedness.Click here for additional data file.