Taking all four time points together, 662 target genes were identified and segregated either by time of differential gene expression into 179 primary and 483 secondary targets or by driver of expression change into 293 direct and 369 indirect targets. The latter classification revealed that more than 50% of target genes were primarily driven by the cells' response to ex vivo exposure than by the nuclear hormone and largely explained its down-regulatory effect. Functional analysis indicated vitamin D’s role in the suppression of the inflammatory and adaptive immune response by down-regulating ten major histocompatibility complex class II genes, five alarmins of the S100 calcium binding protein A family and by affecting six chemokines of the C-X-C motif ligand family. Taken together, studying time-resolved responses allows to better contextualize the effects of vitamin D on the immune system.Peripheral blood mononuclear cells (PBMCs) belong to the innate and adaptive immune system and are highly sensitive and responsive to changes in their systemic environment. In this study, we focused on the time course of transcriptional changes in freshly isolated human PBMCs 4, 8, 24 and 48 h after onset of stimulation with the active vitamin D metabolite 1α,25-dihydroxyvitamin D The evosed skin . In addised skin . This issed skin  or COVIDsed skin , as wellsed skin ,9 or in sed skin .3 metabolite 1,25(OH)2D3 binds and activates the transcription factor VDR (vitamin D receptor) [3 in health and disease is therefore directly linked to 1,25(OH)2D3-dependent changes of the transcriptome in VDR expressing cells [The vitamin Deceptor) ,12, i.e.eceptor) . VDR beleceptor)  and has eceptor) ,16. The ng cells . The vitng cells ,18,19,20ng cells . Since png cells . PBMCs ang cells .2D3 and coordinate its spatio-temporal response to the nuclear hormone. The chromatin model of vitamin D signaling [2D3. The model suggests that a primary vitamin D target gene is modulated in its expression, when the topologically associated domain (TAD), in which the gene is located, carries a prominent VDR binding site. Since TADs have a size between 100 kb and 2 Mb, this determines the maximal distance between an enhancer containing a VDR binding site and the transcription start site of the respective vitamin D target gene.The genome-wide binding pattern of VDR is known for a number of human in vitro cell culture systems representing macrophage-like cells , monocytignaling ,31 had b2D3. The analysis of time-resolved transcriptional responses allows a wider view on the effects of vitamin D on these immune cells, such as a segregation into primary and secondary target genes as well as direct and indirect targets, in the context of inflammatory responses of the innate immune system and its crosstalk with the adaptive immune system.This study describes changes of the transcriptome of freshly isolated human PBMCs that had been stimulated for 4, 8, 24 and 48 h with 1,25(OH)2D3 or solvent (0.1% ethanol (EtOH)). Total RNA was extracted and RNA-sequencing (RNA-seq) applied. Classical pairwise comparison of time-matched treated versus control cells detected 10 and 179 differentially expressed genes at the early time points 4 and 8 h (fold change (FC) > 1.5, false discovery rate (FDR) < 0.05, glmTreat test) as well as 466 and 341 genes at time points 24 and 48 h   < 0.05) . The 466from one  or five from one  treated from one   (ACVR1B (activin A receptor type 1B), CLMN , ENTPD7 (ectonucleoside triphosphate diphosphohydrolase 7), G0S2 (G0/G1 switch 2), HBEGF (heparin binding EGF like growth factor) and SEMA6B (semaphorin 6B) passed the statistical testing threshold (FC > 1.5 and > 2 at FDR < 0.05) at all four time points (FFAR2 (free fatty acid receptor 2), LAMB3 (laminin subunit beta 3) and SLC37A2 (solute carrier family 37 member 2) were also identified as very early targets of vitamin D, but missed the statistical threshold at time point 24 h. A similar discontinuous pattern was observed for CD14 (CD14 molecule) and 13 other genes, which were significantly differentially expressed at 8 and 48 h but their absolute FC dropped below 1.5 at 24 h  , ACVR1B e points B. The ge at 24 h . By cont2D3 were considered as primary targets, while the remaining 483 genes were classified as secondary targets  targets B. The la targets C. Moreov primary , 130 per primary , two tra primary , four pe primary , seven d primary  and eigh primary ) and 409 primary , 208 per primary , 27 tran primary  and 98 p primary  and two  primary ). In con primary  and 33 s primary ), i.e., 2D3 and 483 secondary targets in PBMCs. The majority of these genes are down-regulated.In summary, classical differential expression analysis over four time points identified 179 primary targets of 1,25(OH)k-means was applied, which segregated the expression pattern information of all 662 vitamin D target genes into four clusters  of differentially expressed genes. In particular, down-regulated genes are 3.1-times more likely indirect vitamin D targets than up-regulated genes ). By contrast, primary vitamin D responding genes are 2.2-times more often found to be direct targets . The latter tendency is also visible in multidimensional scaling (MDS) plot 2D3 are very likely mediated by VDR binding to the regulatory regions of the respective direct vitamin D target genes, while indirect target genes may be the consequence of more global effects, such as changes in the epigenetic landscape  plot , which dandscape B.2D3 and solvent using clustering analysis allows a dissection of the vitamin D responding genes into 293 direct and 369 indirect targets.Taken together, the analysis PBMC gene expression after treatment with 1,25(OH)2D3 down-regulates “neutrophil degranulation”, “inflammatory response” as well as “cytokine-mediated signaling pathway” and affects “extracellular matrix organization” as well as “positive regulation of angiogenesis”. The 179 primary vitamin D target genes influence “inflammatory response”, “extracellular matrix organization” as well as “cytokine-mediated signaling pathway” and down-regulate “positive regulation of cytosolic Ca2+ concentration” as well as “PLC (phospholipase C)-activating GPCR (G protein-coupled receptor) signaling pathway”  term enrichment A. Accordpathway” B. Since pathway” C. Of thipathway” . The mospathway” , which rIn contrast, the segregation of the proteins encoded by all target genes , primaryIn summary, GO term enrichment analysis indicated that vitamin D majorly down-regulates “inflammatory response”, “neutrophil degranulation” and “adaptive immune response”.2D3 in PBMCs , AIF1 , LST1 (leukocyte specific transcript 1), HSPA1A (heat shock protein family A (Hsp70) member 1A), HSPA1B and C2 (complement C2). Within this genomic region of approximately 2 Mb in chromosome 6, four persistent VDR binding sites are known [CXCL1, CXCL5, CXCL7 ), CXCL8 and EREG (epiregulin) as well as the down-regulated genes CXCL9, CXCL10 and PARM1 (prostate androgen-regulated mucin-like protein 1)  genes of the alarmin family in chromosome 2 S100A4, S100A6, S100A8, S100A9 and S100A12 as well as the genes SLC27A3 (solute carrier family 27 member 3), RAB13  and PMVK  are down-regulated by 1,25(OH)2D3 in PBMCs  highlighet genes A. Intereotein 1) B. In thi5(OH)2D3 C. In thi2D3 stimulation of PBMCs is prominently mediated via the regulation of clusters of HLA, CXCL and S100A gene families.Taken together, the functional impact of 1,25(OH)2D3 analyzed by classical differential gene expression as well as via clustering analysis. Compared to traditional analysis of a single treatment time, such as 24 h [2D3 in relation to a time-matched solvent control, i.e., the VDR ligand affects gene expression over time, while the control sample does not change. The opposite applies for indirect target genes, which are primarily responding to environmental conditions, such as cellular stress due to isolation and ex vivo culture of PBMCs but not to 1,25(OH)2D3. In this context, 1,25(OH)2D3 has a stabilizing effect on the transcriptome, i.e., vitamin D counteracts transcriptional changes triggered by other factors. Thus, more than half of the 662 target genes are responding indirectly to vitamin D treatment.This study describes the temporal changes of the transcriptome of human PBMCs in response to 1,25(OH) as 24 h ,35, the  as 24 h . In cont2D3. Nevertheless, the list of these 58 persistently up-regulated primary vitamin D targets contains a number of well-known genes, such as CYP24A1 and FBP1 (fructose bisphosphatase 1), which have been reported in numerous studies [2D3 rather counteracts their up-regulation than prominently down-regulates their expression. Since most of the 662 vitamin D responding genes are indirect targets, this also implies that their regulation is context dependent, such as the health and lifestyle situation of the individual. Moreover, 192 genes show unusual transcriptional dynamics, such as transient, discontinuous or mixed FC profiles. Most these genes are indirect vitamin D targets, i.e., their unusual behavior is primarily due to changes of the reference samples. Thus, the analysis of gene expression over time allows a far more differential description and understanding of vitamin D responding genes than snapshots of vitamin D effects at a single time point.Persistently up-regulated primary vitamin D target genes are mechanistically well understood via VDR actively changing the epigenetic landscape and regulating gene transcription as described in the chromatin model of vitamin D signaling ,31. This studies ,37. In cCXCL1, CXCL5, CXCL8 and PPBP (CXCL7) encode for CXCL chemokine family members that act as neutrophil chemoattractants [IL18, CXCL9 and CXCL10, that mediate the cytokine storm associated with the severe form of the disease [S100A8, S100A9 and S100A12, that are highly expressed and released by neutrophils and inflammatory monocytes/macrophages during tissue damage or cellular stress reactions [S100A8 and S100A9 are prominently up-regulated in inflammatory diseases being associated with phagocyte activation, like autoimmune and autoinflammatory disorders, infections, allergies, cardiovascular diseases and cancer [S100A8 and S100A9 gene expression may be important for the treatment of various inflammation-associated disorders. Interestingly, S100A8, S100A9 and S100A12 protein levels are used as sensitive biomarkers for monitoring disease activity in juvenile idiopathic arthritis, inflammatory bowel disease and psoriasis.Functional analysis of all 662 targets identified in this study in comparison to the subgroups of primary, secondary, direct and indirect genes suggests that the overall effect of vitamin D treatment is the down-regulation of inflammatory response, neutrophil degranulation and adaptive immune response. Since neutrophil degranulation involves the release of anti-microbial and inflammatory proteins , this prractants . Since i disease ,41. Thiseactions ,43. The d cancer . Given tHLA genes encoding for MHC-II proteins are direct down-regulated vitamin D targets. MHC-II proteins are expressed on the surface of antigen-presenting cells, such as dendritic cells, monocytes/macrophages and B cells, through which they present CD4+ T helper (TH) cells peptide antigens [ICAM1 (intercellular adhesion molecule 1), CIITA (class II major histocompatibility complex transactivator) as well as CD4, which encode for a co-stimulator of T cell activation, the master regulator of HLA gene transcription and the main co-receptor of the T cell receptor, respectively [H cells and lowers the risk of overboarding adaptive immune responses in this way. This may provide a mechanistic explanation of vitamin D's beneficial role in the context of autoimmune diseases [HLA family, HLA-DRB1, is known as the major risk gene for multiple sclerosis. Since the risk variant HLA-DRB1*15:01 leads to high HLA-DRB1 protein levels, its down-regulation by vitamin D may serve as a therapeutic target [HLA-DRB1 has been observed in PBMCs from different individuals [The regulation of adaptive immune response is another important function of vitamin D in PBMCs. It is mediated by later responding but direct target genes, many of which many are also associated with IFNγ-mediated signaling. Importantly, this study demonstrated that 10 of 12 antigens . This isectively . Thus, vdiseases . Interesc target ,49. Notaividuals  and thusHLA, CXCL and S100A genes, which have four, one and two persistent VDR binding sites in their neighborhood, respectively. Thus, it can be assumed that ligand-activated VDR modulates the epigenetic landscape at the respective genomic regions and explains the response of the gene clusters to vitamin D.Persistent VDR binding sites are reasonably conserved between comparable cellular models, such as PBMCs and THP-1 cells ,25. TherG0S2, which display significant differential expression already after 4 h but their expression levels are not changing at later time points. Thus, some very early responding genes may have been mislabeled as indirect targets.The focus of this study was on the effects of vitamin D stimulation of PBMCs immediately after their isolation. Since cell sorting induces stress to cells , the RNAHLA, CXCL and S100A genes, which mediate inflammatory processes of the innate immune system as well as the response of the adaptive immune system.In conclusion, this study describes the time-resolved transcriptional response of human PBMCs to vitamin D stimulation. This experimental design allows the segregation of the 662 vitamin D responding genes into primary, secondary, direct and indirect target genes. Prominent vitamin D targets are the clusters of the Peripheral blood was collected from a single healthy individual  that participated in the VitDHiD trial (NCT03537027) .2 incubator and treated for 4, 8, 24 or 48 h with either 10 nM 1,25(OH)2D3 (Sigma-Aldrich) or solvent (0.1% EtOH). All experiments were performed in three repeats. Deconvolution of RNA-seq data using the algorithm CIBERSORTx [+ T cells (32%), CD4+ T cells (20%), natural killer cells (21%) and monocytes/macrophages (20%) within the PBMC pool.PBMCs were isolated immediately after collecting 20 mL peripheral blood using Vacutainer CPT Cell Preparation Tubes with sodium citrate (Becton Dickinson) according to manufacturer’s instructions. After washing with phosphate-buffered saline the cells were grown at a density of 0.5 million/mL in 5 mL RPMI 1640 medium supplemented with 10% charcoal-depleted fetal calf serum, 2 mM L-glutamine, 0.1 mg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified 95% air/5% COBERSORTx  and its Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) according to manufacturer’s instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer system (RNA integrity number ≥ 8). rRNA depletion and cDNA library preparation were performed using the New England Biolabs kits NEBNext rRNA Depletion, NEBNext Ultra II Directional RNA Library Prep for Illumina and NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2) according to manufacturer’s protocols. RNA-seq libraries went through quality control on an Agilent 2100 Bioanalyzer and were sequenced on a NextSeq 500 system (Illumina) at 75 bp read length using standard protocols at the Gene Core facility of the EMBL . All samples were prepared and sequenced as one batch.http://doi.org/10.5281/zenodo.4323183, accessed on 25 June 2021) [BiomaRt (version 2.46.0) [Single-end, reverse-stranded cDNA sequence reads were aligned to the reference genome (version GRCh38) with Ensembl annotation (version 103) by using default settings of the nf-core/rnaseq STAR-Salmon pipeline (version 3.0) (ne 2021) . The num 2.46.0) . EnsemblEdgeR (version 3.32.1) [FilterByExpr, in order to mitigate the multiple testing problem and to not interfere with the statistical approximations of the EdgeR pipeline. This requirement was fulfilled by 12,305 genes. After filtering, library sizes were recomputed and trimmed mean of M-value normalization was applied. The transcriptome data structure was explored via the dimensionality reduction method MDS  between the samples. This distance was calculated as the root mean square deviation (Euclidean distance) of the largest 500 log2FCs between a given pair of samples, i.e., for each pair a different set of top genes was selected. The inspection of the MDS plot showed a time-dependent divergence from the native transcriptome state and its modulation by 1,25(OH)2D3 treatment as the principal two factors driving differences in PBMCs gene expression profiles. The gene-wise statistical test for differential expression was computed using the generalized linear model quasi-likelihood pipeline [glmTreat approach was used to test for differential expression relative to FC > 1.5 at the early time points 4 and 8 h and FC > 2 at 24 and 48 h. A classical pairwise comparison was chosen for its superior performance in time course RNA-seq data analysis [p-value, i.e., false discovery rate (FDR) < 0.05 and a total trimmed mean of M value-normalized CPM count > 25  were considered as significant vitamin D target genes . Gene symbols from external datasets were updated with HGNC helper (version 0.8.1).Differential gene expression analysis was computed in R (version 4.0.5) in the CentOS 7 Linux operating system using the tool  3.32.1) . The anathod MDS . MDS waspipeline . Trendedpipeline . The glmanalysis . Genes wet genes . MA plotk-means Hartigan–Wong method with 25 random sets and a maximum of 1000 iterations implemented in R stats . No further clustering was applied, i.e., genes within each cluster are displayed in alphabetical order. Clusters were assessed for over-representation using a two-sided FET with a Benjamini–Hochberg corrected p-value applying rstatix (0.7.0). GO term enrichment for biological processes was performed using topGO (version 2.42.0) with the default weight01 algorithm and FET (The 662 vitamin D target genes  were cluode from ) and vis and FET . As refeBiomaRt. Functional attributes of all vitamin D target genes were retrieved through programmatic access (REST API) of the UniProtKB (Uniprot Knowledge Base) website using requests (version 2.25.1) and io in Python (version 3.8.8). Only manually reviewed (Swiss-Prot) entries were used for annotation. Gene product locations and functions were preferentially assigned using UniProtKB/Swiss-Prot Keywords and complemented with GO annotation (cellular component and molecular function domains). The total list of functional annotations was manually inspected and simplified into overarching terms, by which the genes were assigned with . Custom code of the analysis can be found at https://github.com/andreahanel/2021_Timecourse, accessed on 23 December 2021.Gene names were mapped to UniProtKB identifiers via ned with . UnmappakaryoploteR (version 1.12.4). VDR chromatin immunoprecipitation sequencing (ChIP-seq) data [CrossMap (version 0.2.6) and visualized using the IGV browser [GenomicRanges (1.38.2) and rtracklayer (1.46.0).Location of genes along chromosomes was plotted using eq) data  were con browser  together


We have determined the effect of seven serine- and arginine-rich (SR) proteins and 15 heterogenous nuclear ribonucleoproteins (hnRNPs) on human papillomavirus type 16 (HPV16) late gene expression. Of the seven SR proteins analyzed here, SRSF1, SRSF3, and SRSF9 induced HPV16 late gene expression, and five of the SR proteins affected HPV16 L1 mRNA splicing. Of the 15 hnRNP proteins analyzed here, hnRNP A2, hnRNP F, and hnRNP H efficiently induced HPV16 late gene expression, and all of the hnRNPs affected HPV16 L1 mRNA levels or mRNA splicing. Thus, the majority of SR proteins and hnRNPs have the potential to regulate HPV16 L1 mRNA splicing. Strict control of the expression of the immunogenic L1 and L2 capsid proteins may contribute to the ability of HPV16 to cause persistence.The online version contains supplementary material available at 10.1007/s00705-021-05317-2. Persistent infections with a subset of human papillomaviruses (HPVs) defined as high-risk HPVs may cause cervical cancer , 21. HPVHeLa cells were transfected with plasmids encoding seven serine- and arginine-rich (SR) proteins together with an HPV16-derived subgenomic reporter plasmid named pC97ELsL . The use of HeLa cells in this study is justified by the fact that they are HPV18 positive and as such represent cells that are targeted by HPV. Methods are detailed in the Supplementary Information file. HeLa cells were cultured in Dulbecco’s modified Eagle medium  with 10% bovine calf serum and 100 U of penicillin and 100 µg of streptomycin (Gibco Thermo Fisher Science) per ml. The plasmid pC97ELsL has a secreted luciferase (sLuc) reporter gene inserted after a poliovirus 2A internal ribosome entry site (IRES) in the L1 coding region that serves as a marker for HPV16 late gene expression  , 13. TraThe deletion also did not affect the ability of SRSF9 to promote skipping of the central exon between SA3358 and SD3632 on the L1 mRNAs Fig. . FinallyWe also performed RT-PCR on RNA extracted from HeLa cells transfected with pC97ELsL and serial dilutions of expression plasmids encoding SR proteins. Of the three proteins that induced HPV16 late gene expression, monitored by measuring sLuc as shown in Figure Next, we compared the effect of various hnRNPs on HPV16 late gene expression and HPV16 late L1 mRNA splicing. pC97ELsL was individually co-transfected with each of fifteen hnRNP expression plasmids, and sLuc levels were determined Fig. A and B. hnRNP A1 and A2 have previously been shown to affect splicing of the E6 and E7 mRNAs in an opposite manner . We therThe hnRNP D gene gives rise to four alternatively spliced mRNAs encoding hnRNP D proteins with molecular weights of 37, 40, 42, and 45 kDa  . These aFinally, transfection of serially diluted plasmids encoding hnRNP Q or hnRNP R with pC97ELsL confirmed that overexpression of either of the proteins induced HPV16 late gene expression, albeit at relatively high levels of expression plasmid . RNA analysis by RT-PCR confirmed that hnRNP Q and hnRNP R acted by enhancing inclusion of the central exon between SA3358 and SD3632 on the HPV16 L1 mRNAs . Quantitation of the levels of the alternatively spliced L1 mRNA revealed that the L1 mRNAs with the SA3358-SD3632 exon retained (L1) increased to a higher degree than L1 mRNAs with the SA3358-SD3632 exon excluded (L1i) in response to hnRNP Q or hnRNP R . L1i represents the L1 mRNA spliced directly from SD880 to SA5639. We also investigated if hnRNP Q or hnRNP R affected alternative splicing of early HPV16 mRNAs, including mRNAs utilizing the HPV16 splice sites SD226 and SA409, SA529, or SA742  to SA3358 (E4 mRNAs) or SA2709 (E2 mRNAs) . However, we were unable to detect significant effects of hnRNP Q or hnRNP R on any of the alternatively spliced HPV16 early mRNAs . In conclusion, hnRNP Q and hnRNP R primarily affected alternative splicing of the HPV16 late L1 mRNAs.Taken together, our results confirm that SRSF3 and SRSF9 control HPV16 late gene expression . It has Regarding hnRNP proteins, we have previously shown that hnRNP A2 can induce HPV16 late gene expression , while hSupplementary file1 (PDF 4098 KB)Below is the link to the electronic supplementary material.


Then, we demonstrated that upregulation of S100A8 and S100A9 alone or together enhanced characteristics of EMT in normal keratinocytes, manifested by excessive proliferation rate, abnormal ability of cell invasion, and high expression levels of EMT marker proteins. The transcription factor PU box-binding protein (PU.1) bound to the promoter regions and transcriptionally promoted the expression of S100A8 and S100A9 both in the human and mice, and it had strong positive correlations with both S100A8 and S100A9 protein levels in burned skin in vivo. Moreover, PU.1 positively regulated expression of S100A8 and S100A9 in a dose-dependent manner, and enhanced EMT of keratinocytes in vitro. Finally, through the burn mouse model, we found that PU.1-/- mice displayed a lower ability of scar formation, manifested by smaller scar volume, thickness, and collagen content, which could be enhanced by S100A8 and S100A9. In conclusion, PU.1 transcriptionally promotes expression of S100A8 and S100A9, thus positively regulating epithelial-mesenchymal transformation (EMT) and invasive growth of dermal keratinocytes during scar formation post burn.S100 calcium-binding protein A8 (S100A8) and S100A9 are small molecular weight calcium-binding regulatory proteins that have been involved in multiple chronic inflammatory diseases. However, the role of S100A8 and S100A9 in keratinocytes in wounded skin and how they are regulated during this process are still unclear. Here, we found that S100A8 and S100A9 were both upregulated in burn-wounded skins  Scar formation is a frequently occurring problem in plastic surgery, which is a skin fibroplasia disorder characterized by abnormal proliferation of dermal and epidermal cell components and excessive deposition of extracellular matrix (ECM) . Scar leS100 calcium-binding protein A8 (S100A8) and S100A9 are small molecular weight calcium-binding regulatory proteins, belonging to the S100 protein family. S100A8 and S100A9, mainly expressed in myeloid cells, including neutrophils, monocytes, and macrophages, and some other cell types with secretory functions, including tissue epithelial cells, endothelial cells, and keratinocytes, have been involved in multiple chronic inflammatory diseases, such as cancer, thrombosis and arthritis –9. Severin vitro and in vivo. Moreover, we explored how S100A8/A9 expression was regulated during this process.In a previous study, using proteomics and microarray techniques, we identified that S100A8/A9 were robustly upregulated in the keratinocytes derived from wounded skin tissues. Our elementary study revealed that S100A8/A9 overexpression increased, while silencing S100A8/A9 decreased, the migration capacity of human keratinocytes, accompanied by upregulation of several EMT marker proteins, hinting their potential promoting effect on EMT and invasive growth. In this study, we investigated their exact role in regulating EMT of keratinocytes and scar formation in vitro for simulating a state of burn injury. Western blotting and double immunofluorescence analyses showed that S100A8 and S100A9 were also significantly upregulated in response to heat stimulation in a time-dependent manner  were incubated with epidermal keratinocytes feration  and invaferation , and expferation , while tferation , 1B, 1D.feration , 1B, 1D.proteins . These dS100A8 and S100A9 usually function synergistically, whereas, it is not clear that how S100A8 and S100A9 expressions were regulated. We searched co-expressed regulators with both S100A8 and S100A9 with the online bioinformatics tool ChIPBase. The outputs showed that the transcription factor PU.1 displayed a strong positive correlation with both S100A8 and S100A9 in various human tissues , 2. SequLike expression patterns of S100A8 and S100A9, PU.1 was significantly upregulated in burned skin tissues and thermal-stimulated keratinocytes –4C. Morein vivo. Quite lower expression levels of S100A8 and S100A9 were observed in PU.1-/- mice  or spleen focus forming virus (SFFV) proviral integration oncogene (SPI1), is an ETS-domain transcription factor and master gene in regulating myeloid and B-lymphoid cell development. A couple of recent studies revealed that PU.1 played an important role in wound healing and tissue fibrosis –27. HoweIn conclusion, S100A8 and S100A9 promoted the phosphorylation and nuclear translocation of Snail protein, EMT of epidermal keratinocytes and enhanced scar development. Moreover, they display high synergy in expression, which are transcriptionally regulated by PU.1.A total of 38 tissue specimens were collected from 21 male and 17 female patients (aged 30.1 ± 5.9 years), with second- and third-degree burns admitted within 24 h post burn injury, at the Department of Dermatology, the Second Affiliated Hospital of Xi’an Jiaotong University  between Feb, 2017 and May, 2018. Patients with the following exclusion criteria were excluded: severe complications of burns, long-term hormone intake or drug addiction, malignant tumors, and receipt of chemoradiotherapy. During dealing with wounds, burned skin tissues and adjacent normal control skin tissues were respectively obtained. The protocols were approved by the Ethics Committees of the Second Affiliated Hospital of Xi’an Jiaotong University, and all the subjects gave informed consent.2. For thermal stimulus, cell cultures were placed into a water heater tank and incubated at 52° C for 30 s, and then cultured in the normal conditions for different time.Primary epidermal keratinocytes were derived from normal human skin collected from patients who had undergone plastic surgery procedures. The derived tissues were dissected and cut into small pieces, washed with phosphate-buffered saline, and then digested with 0.25% Dispase II  overnight at 4° C. Then, the suspension was digested with 0.25% trypsin (Sigma-Aldrich) for 30 min. The isolated cells were screened with 70-μm filters to obtain keratinocytes, which were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  at 37° C in an atmosphere with 5% CO5 cells/mL. On reaching about 70% confluence, the vectors or siRNAs were transfected into dermal keratinocytes by using the Lipofectamine®3000 reagent (Thermo Fisher Scientific). Recombinant proteins were transfected with BioTrekTM .Adenoviral expression vector containing human PU.1 (Ad-PU.1), mouse s100a8/a9 (Ad-s100a8 and Ad-s100a9), and their negative control  were purchased from Sangon Biotech . Short interfering RNA (siRNAs) were designed and synthesized and validated by RiboBio Co., LTD . Human recombinant S100A8 and S100A9 proteins (rS100A8 and rS100A9) were purchased form Abnova Corporation (Chinese Taipei). Epidermal keratinocytes were subgrown in 6-well plates at a density of 2 × 104 cells/well into 96-well plates. Following incubation, cell number was counted with a Cell Counting Instrument .The epidermal keratinocytes were seeded at a density of 105 cells were plated in the wells above in 200 μL serum-free medium, and 800 μL of medium supplemented with 10% FBS was added to the bottom wells. After incubation at 37° C with 5% CO2 for 48 h, the Transwell membrane was fixed with methanol for 20 min, stained with 0.1% crystal violet, and counted the cell numbers of 5 fields  to obtain the mean value via light microscopy.Cell invasion was detected with Transwell Cell assay using Matrigel-coated Transwell chambers , respectively. After 48 h of transfection, 105 system (Bio-Rad). The reaction system was incubated under the following conditions: 95° C for 3 min, followed by denaturation at 94° C for 15 s, annealing at 55° C for 25 s and extension at 72° C for 15 s for 35 cycles. 18S RNA was used as an internal reference gene control.Total RNA from the tissues and keratinocytes was extracted by using the Total RNA Purification Kit containing DNase I , and then applied in cDNA synthesis. RNA abundances were detected using a final volume of 25-μL reaction system containing SYBR ExScript qPCR kit  in an IQTotal Protein Extraction Kit (Promega) was used to extract total protein. Twenty-five micrograms of protein sample were separated by polyacrylamide gel at 130 V for 2 h and then electro-transferred onto the polyvinylidene fluoride membrane . The following primary antibodies  were respectively used to incubate with the membrane at 4° C overnight: Rabbit polyclonal antibody to phosphorylated SNAIL , Rabbit monoclonal antibody [E184] to α-SMA , Rabbit polyclonal antibody to Vimentin , Rabbit monoclonal antibody [EPR22624-20] to PU.1 , Rabbit monoclonal antibody [EPR3554] to MRP8/S100A8 , Rabbit monoclonal antibody [EPR22332-75] to S100A9 , and Rabbit monoclonal antibody [EPR16769] to β-Actin . After incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated Goat-anti-Rabbit IgG) at 37° C for 1 h. After elution, the abundances of the proteins were detected using a ChemiDoc XRS Imaging System (Bio-Rad).http://rna.sysu.edu.cn/chipbase/). PU.1 binding motif was also searched on the promoter regions of S100A8/S100A9 using ChIPBase 2.0.The co-expression of PU.1 and S100A8/S100A9 was evaluated by the online bioinformatics tool ChIPBase version 2.0 . In brief, cells were fixed with 4% formaldehyde for 10 min. 100 ~500 bp DNA fragments were obtained from lysed cells by ultrasonic crushing. The immuno-complexes were precipitated using PU.1 antibody and normal serum IgG through rotational incubation at 4° C overnight. The same amount of chromatin not incubated with antibodies was used as the input control. Enrichment of promoter sequences of S100A8 or S100A9 was then detected with qPCR in the precipitated DNA.Luciferase reporter gene assay was used to evaluate the transcription regulatory activity of PU.1 on S100A8/S100A9 genes. Vectors containing different truncated promoter sequences were cloned and inserted into pGL3-basic vector  with PU.1 at the upstream. The assay was performed according to the manufacturer’s instructions.-/- or ΔPU.1) BALB/c mice (about 20 g) were purchased from Laboratory Animal Center, Shanghai Institutes for Biological Sciences . For the generation of ΔPU.1 mouse, in brief, keratin14-CRE transgenic tool mice and PU. 1 conditional knockout (PU.1loxp/loxp) mice mated two generations to obtain the mice with PU. 1 gene specific deletion in keratinocytes from embryonic day 14.5. After adaptive feeding for two weeks, deep second-degree experimental burn model was established: mice were intraperitoneally injected with 7% chloral hydrate, and the injection volume was 5 mL/kg. After successful anesthesia, hair on the back of the mice was removed to expose the skin, and then sterilized with 75% ethanol. Then the back of the mice was contacted with a 92° C -copper cylinder with 1.5 cm bottom diameter for 4 s, resulting in burn wound. Adeno-associated-viral vectors delivering S100A8 or/and Ad-S100A9 were locally injected into the epidermis at 24 h before burn. After the burn wound of the mice was healed, the formation of scar on the back of the mice were observed regularly. Until the scar height increases no more, the mice were sacrificed, and the keloids were carefully obtained for measurement of the scar thickness at the lowest and highest sites, the scar volume, and the content of total collagens. And skin tissues (with an area of ~1 cm2) were collected for histological examination with HE staining) and expression of related proteins with Western blotting.Clean-grade wild type (WT) and skin-specific PU.1 knockout , S100A9  or Snail . The next day, the sheets were washed in PBS and incubated in ABC  for 30 min. Following washing in PBS, DAB solution  was used to incubated with the sheets for 5 min, followed by hematoxylin staining. Finally, the sheets were counterstained with hematoxylin and images were taken with a FSX100 microscope  and analyzed by Image-Pro Plus 6.0 system.+p63+ cells represent the purity of keratinocytes, and higher percentage of CD44+CD24- indicates higher EMT level of keratinocytes.FACS was used to verify the purity of isolated primary keratinocytes (based on keratinocytes specific surface marker proteins cytokeratin-14/CK-14 and p63) and EMT phenotype of primary keratinocytes treated with different recombinant proteins (based on EMT marker proteins CD44 and CD24). In brief, keratinocytes were tagged with either green or red FITC-antibodies and then analyzed with a Attune™ NxT Flow Cytometer (Invitrogen). The ratio of CK-14P < 0.05 using one-way variation analysis followed by student’s t-test.Each measurement was obtained from at least triple experiments and shown as the mean ± standard error of mean (SEM). Data analysis was performed with SPSS version 23.0. Pearson correlation coefficient assay was used to evaluate the correlation of PU.1-S100A8-S100A9. Statistical significance difference was set at Supplementary FiguresSupplementary Tables


Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics.The muscleblind RNA-binding proteins  are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (This article has an associated First Person interview with the first author of the paper. Summary: We generated a panel of single, double and triple homozygous zebrafish mbnl mutants to model myotonic dystrophy. They exhibited decreased body size, impaired movement and widespread, disease-relevant alternative splicing changes. The muscleblind (MBNL) family of RNA-binding proteins  is highly conserved in structure and function across multicellular species . VertebrRYR1 and ATP2A1 contributes to altered calcium homeostasis in DM1 muscle (Myotonic dystrophy (DM) types 1 and 2 (DM1 and DM2) are human genetic disorders caused primarily by MBNL protein loss of function. In DM, expression of expanded CTG or CCTG repeat RNAs leads to the formation of RNA stem loop structures, which contain dozens to thousands of YGCY motifs that sequester MBNL proteins, blocking their normal functions  Fig. 1AAFig. 1A.1 muscle .Fig. 1.Mbnl mutant mice have been invaluable for studying how individual Mbnl proteins contribute to DM-related phenotypes. Mouse Mbnl1 is widely expressed, while Mbnl2 expression is brain enriched, and Mbnl3 is expressed primarily during embryonic development and injury-induced adult skeletal muscle regeneration  and triple (Mbnl1/Mbnl2/Mbnl3) homozygous mutant mice are reduced in size, have dramatic and widespread alternative splicing changes, muscle weakness, and most die in the neonatal period due to respiratory distress  and triple (mbnl1/mbnl2/mbnl3) homozygous mutants, all of which were viable to adulthood. Zebrafish mbnl mutants exhibited DM-relevant physical phenotypes including decreased body size and impaired motor function. They also exhibited widespread alternative splicing changes, including many of the same changes that were present in DM patients and mouse Mbnl mutants. These alternative splicing changes occurred both in larval and adult fish, but were most dramatic in adult skeletal and heart muscle. As in mice, double and triple homozygous zebrafish mbnl mutants exhibited more severe phenotypes than single mutants. Thus, zebrafish mbnl mutants had physical and molecular phenotypes consistent with those present in DM, and are powerful new vertebrate models for studies of MBNL function.In this study, we made homozygous zebrafish MBNL loss of function, we targeted constitutively included exons in each zebrafish mbnl gene using CRISPR, and established two mbnl1 (A/A1 and B/B1), one mbnl2 (A/A2) and three mbnl3  mutant lines (Tables S1 and S2)   or unchanged  levels of the mRNA expressed from the mutated gene, and the expression of the other mbnl family members was largely unchanged , arguing against genetic compensation in these lines (To model DM-associated  and S2) . Althougable S2) . Homozygse lines .mbnl1, mbnl2 and mbnl3 mutants survived to adulthood in roughly Mendelian ratios (Table S3), were fertile, and did not exhibit the dramatic morphological phenotypes that were previously observed in mbnl2 morpholino-injected larvae  and triple  mutants were also viable, although triple mutants were present in lower than expected numbers and were unable to produce embryos except through in vitro fertilization (Table S3). This was, to our knowledge, the first time that mbnl1/mbnl3 and mbnl2/mbnl3 double homozygous mutant animals were generated. The viability of B/B;2A/A1 and B/B;2A/A;3C/C1 zebrafish was in contrast to the neonatal lethality of analogous mouse models (mbnl mRNAs were present at low or very low levels in wild-type (WT) unfertilized eggs , consistent with a previous report that only maternally deposited mRNAs of mbnl1 and mbnl2 could be detected by non-quantitative reverse transcription PCR (RT-PCR)  . HoweverMbnl mutants were significantly smaller than WT . Taken together, these results suggested that zebrafish Mbnl proteins have partially redundant functions, as the growth defect was more dramatic in compound mutants than in single mutants.We asked whether zebrafish had similar phenotypes to mice, in which compound, but not single,  than WT . We raisC/C fish C. Althounificant C. Mutatimbnl mutant larvae were similar in size to WT, except for A/A;3C/C2, which were slightly smaller than WT . These results suggest that the reduced size phenotype in zebrafish mbnl mutants arose later in development.We also measured 7 days post-fertilization (dpf) larval fish prior to exogenous feeding. All double homozygous  than WT D. 1B/B;2B/B;2A/A1 and B/B;2A/A;3C/C1, did not exhibit the dramatic myofiber atrophy or centralized nuclei that were described in compound mbnl mutant mice and in individuals with DM  . This could be an indication of muscle pathology, but could also be a consequence of the overall decrease in mutant fish size compared to WT  , 2017. Ted to WT C.mbnl mutants exhibited altered swimming behavior by introducing individual adult fish to a novel tank and tracking their position over 5 min  and the B/B;2A/A;3C/C1 mutants swam significantly decreased distances compared to WT .Given the DM-relevant size and movement phenotypes of proteins . The intmbnl1, mbnl2 and mbnl3 mRNAs were present in larvae and across adult tissues, and that levels of all three were highest in skeletal muscle and brain . Inclusion of mbnl1 exon 5 and mbnl2 exon 5, as measured by RT-PCR, was significantly elevated in zebrafish mbnl mutants across all tissues . The phenotype was most dramatic in skeletal muscle and heart, where mbnl1 and mbnl2 exon 5 were almost entirely skipped in WT and predominantly included in B/B;2A/A1 and B/B;2A/A;3C/C1 mutants . The brain and cornea exhibited mbnl splicing patterns similar to one another, but with lower magnitude changes than those observed in muscle . Modest but significant splicing changes were observed in whole larvae and intestine .We harvested total RNA from whole larval zebrafish and adult skeletal muscle, heart, brain, cornea and intestine. Quantitative RT-PCR (RT-qPCR) analyses of WT fish indicated that  tissues ; Fig. S6mbnl1 and mbnl2 splicing changes than single mutants . This is consistent with the idea that zebrafish Mbnl proteins, like mouse Mbnl proteins, have partially redundant functions when it comes to splicing regulation .In most tissues, double and triple mutants exhibited larger  mutants ; Fig. S6gulation . Strikinmbnl1 mutation alone was sufficient to increase mbnl1 exon 5 inclusion, whereas mutation of both mbnl1 and mbnl2 was required to increase mbnl2 exon 5 inclusion . Overall, these results suggest that, as in other systems, zebrafish Mbnl proteins work in concert to regulate alternative splicing across tissues.In mouse model and DM patient tissues, some regulated exons were much more sensitive than others to changes in overall levels of free MBNL proteins . Zebrafimbnl mutation on alternative splicing, we performed RNA-Seq analysis using RNA isolated from the skeletal muscle of adult WT, B/B1, A/A2, C/C3, B/B;2A/A1, B/B;3C/C1, A/A;3C/C2 and B/B;2A/A;3C/C1 zebrafish. There were no significant changes in the normalized counts of mbnl1 and mbnl3 mRNAs in mutants compared to WT, while mbnl2 mRNA levels were slightly decreased when mbnl2 was mutated . Hundreds of significantly misregulated RNA alternative splicing events were detected in all mbnl mutants, with misregulated cassette exons outnumbering alternative 3′ and 5′ splice sites, retained introns and mutually exclusive exons (Table S4). The B/B;2A/A1 and B/B;2A/A;3C/C1 fish had the largest number of significantly misregulated splicing events . The highest magnitude of splicing change across events was also observed in B/B;2A/A1 and B/B;2A/A;3C/C1 fish  between WT and mutant] for the 62 cassette exon events that were significantly misregulated in the majority of C/C fish B. These C/C fish .B/B;2A/A1 and B/B;2A/A;3C/C1 fish were more similar to each other than they were to WT fish or any of the other mbnl mutant fish . The inclusion of these alternative exons tended to be misregulated in the same direction in zebrafish DM models and in DM1 patient-derived tissues .Given the widespread changes in alternative splicing in  tissues  or in DM tissues  comparedmbnl1 exon 5 and mbnl2 exon 5 appeared on both lists of orthologous misregulated cassette exons, and PSI values were strikingly similar as determined by RNA-Seq and RT-PCR . Two other randomly selected orthologous exons, aplp2 exon 7 and atp6v1h exon6, also showed similar splicing phenotypes by RNA-Seq and RT-PCR, and contained potential YGCY Mbnl-binding motifs in their surrounding intronic sequences . Fig. S7A do not represent an exhaustive list of orthologous misregulated exons, as a manual literature search revealed several additional orthologs of disease-associated exons that were misregulated in mbnl mutant model fish, some of which were only annotated in an earlier zebrafish genome assembly . For example, the decreased inclusion of alternative exons within the human ATP2A1 and RYR1 genes contributes to altered calcium homeostasis in DM muscle, and we detected decreased inclusion of orthologous exons in zebrafish mbnl mutants    . Likewisig. S12) .B/B;2A/A;3C/C1 fish, which had the most severe alternative splicing and physical phenotypes. Over a thousand genes had significantly changed expression levels in the triple homozygous mbnl mutants compared to WT, suggesting significant molecular dysfunction in muscle (Table S6). We then compared the gene ontology (GO) classifications of dysregulated genes in the B/B;2A/A;3C/C1 mutant zebrafish skeletal muscle to those uncovered in our analysis of published RNA-Seq data from human DM1 myotubes . Taken together, these results indicate that the overall coordinated splicing regulation program of the Mbnl proteins, as well as gene expression changes, were well conserved between zebrafish and humans.We also analyzed gene expression level differences in skeletal muscle, focusing on myotubes . We founMBNL loss of function by generating a complete panel of vertebrate mbnl single, double and triple homozygous mutants. These fish exhibited molecular and physical phenotypes similar to those observed in humans with DM and mouse models, including decreased body size, impaired movement and widespread changes in alternative splicing. We propose that zebrafish B/B;2A/A1 and B/B;2A/A;3F/F1 mutants, which exhibited the most dramatic phenotypes, represent severe forms of DM1, with long CTG repeats in which most, but probably not all, MBNL proteins are sequestered, while other double and single mbnl mutants model more moderate forms of the disease, in which MBNL sequestration is less robust.For the first time, we have modeled DM-associated mbnl2 mutant zebrafish were viable to adulthood, as this contrasted with the dramatic morphological phenotypes of mbnl2 morpholino-injected larvae , but definitive null alleles would be needed to determine whether mbnl2 is required for viability in zebrafish.We were initially surprised that homozygous d larvae . We propd larvae B or morp in mice . We are B/B;2A/A1 and B/B;2A/A;3C/C1 mutants was also surprising, as this differed from previous findings in mouse models , and maybe the presence of partially functional Mbnl2 protein across tissues including skeletal muscle was sufficient for zebrafish survival. Although viable, the triple mutants had more severe physical impairments than the single or double mutants. They did not mate spontaneously, required supplemental feeding, and all died by 18 months of age.The viability of zebrafish e models . Most mudulthood . Perhapsnl genes , all thrmbnl mutant fish are nulls or hypomorphs, they are valid models of DM, as sequestration of MBNL proteins by CUG/CCUG repeats is likely not complete in the human disease state. The robust molecular and physical phenotypes, yet viability, of B/B;2A/A1 and B/B;2A/A;3C/C1 fish will make them particularly useful tools for studying how loss of mbnl function contributes to disease-relevant phenotypes. The compound Mbnl mutant mouse models that most closely mimic DM phenotypes are challenging to breed in large numbers and most die during the neonatal period, making studies of adult phenotypes difficult . We do not know the mechanism underlying the size phenotype in mbnl mutant fish, but one possibility is DM-relevant impaired movement leading to decreased ability to access food. Consistent with this idea, we found that B/B;2A/A1, B/B;3C/C1 and B/B;2A/A;3C/C1 fish swam a significantly decreased distance compared to WT fish during a 5-min swim test . We observed a subtle decrease in the absolute cross-sectional area of individual muscle fibers in B/B;2A/A1 and B/B;2A/A;3C/C1 fish compared to WT. However, it is difficult to determine whether this is an indication of muscle pathology, or is merely due to decreased body size, which is roughly associated with decreased muscle fiber size, but not through a simple linear relationship . Mis-splicing was also observed in the cornea, suggesting that zebrafish mbnl mutants may also model the genetic disorder Fuchs endothelial corneal dystrophy, a subtype of which was recently shown to be caused by an expanded CUG repeat that is expressed from an intron of the TCF4 gene and is associated with MBNL protein sequestration and mis-splicing   . Hundredgulation A. Consisgulation A,B . As observed in other organisms, YGCY putative Mbnl-binding motifs were enriched upstream of exons that were suppressed by zebrafish Mbnl proteins, and enriched downstream of exons for which inclusion was promoted .Our data indicated that, just like other model organisms, zebrafish Mbnlpromoted D . These included several that are known to contribute to disease phenotypes, such as changes in ATP2A1 and RYR1 splicing that contribute to dysregulation of calcium homeostasis . Zebrafish DM models complement other existing vertebrate model systems because hundreds of larval or adult fish can be generated easily to study subtle or variable phenotypes and to test potential therapeutics. Additionally, transparent larval zebrafish can be used to directly study disease-related phenotypes, such as altered gut motility and heart abnormalities, in live animals.These new zebrafish models, and the accompanying RNA-Seq data that we generated, will be valuable in future studies of how individual Mbnl proteins and specific alternative splicing and gene expression changes contribute to DM-relevant phenotypes. For example, exons that were misregulated in all zebrafish henotype O-Q. LikeDanio rerio) experiments were performed with the guidance and approval of the University of Oregon Institutional Animal Care and Use Committee . Guide RNAs (gRNAs) targeting zebrafish (Danio rerio) mbnl1, mbnl2 and mbnl3 were designed using the Chop Chop website (http://chopchop.cbu.uib.no). DNA templates for the gRNAs were generated by a template-free Phusion polymerase (New England Biolabs) PCR reaction using a common scaffold primer  and a gene-specific primer , then cleaned using the QIAquick PCR Purification Kit (Qiagen). gRNAs were transcribed from DNA templates using a MEGAscript kit (Ambion) and purified by phenol-chloroform extraction and isopropanol precipitation. Cas9 RNA was generated by linearizing the pT3TS-nls-zCas9-nls plasmid (in vitro transcription reaction using the T3 mMESSAGE kit (Invitrogen), and purifying the RNA using the RNeasy Mini kit (Qiagen). AB zebrafish embryos were microinjected at the one-cell stage with 1-2 nl of a mixture containing 100 ng/µl Cas9 mRNA, 50 ng/µl gRNA and Phenol Red, and raised to adulthood. Mosaic mutants were identified by PCR amplification and Sanger sequencing of fin clip DNA using primers specific to the targeted region (Tables S1 and S2), and outcrossed to WT AB zebrafish to generate heterozygotes. Fish with predicted loss-of-function mutations were identified by Sanger sequencing (Table S2), and further crossed to generate single, double and triple mbnl1, mbnl2 and mbnl3 homozygotes. Fish were genotyped by restriction fragment length polymorphism analysis using the primers and restriction enzymes indicated in Table S2. Sperm from all zebrafish mutant lines were cryopreserved and are available upon request from the corresponding author. Roughly equal numbers of males and females were used for all experiments involving adult fish.All zebrafish  were anesthetized in 168 mg/l tricaine methane sulfonate, photographed against a white background with a ruler using a tablet computer, fin clipped and genotyped. To ensure identical density and feeding conditions, fish were compared with others from the same clutch  or different clutches that had been raised together in the same tank , with the exception of the larvae in Table S5).Young adult fish (2-4 months old) . RNA was washed and eluted according to the manufacturer's instructions, and the concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific).Fish were euthanized by tricaine methane sulfonate overdose (larvae) or hypothermic shock (adults). Tissues of interest were dissected and flash frozen in 1 ml Trizol (Ambion), thawed, and homogenized with a mortar and pestle (larvae) or Bullet Blender Storm 24 (adult tissues). Chloroform (200 µl) was added to each tube followed by mixing, centrifugation at 12,000 Table S1). The resulting complementary DNA (cDNA) was amplified by 28-31 cycles of PCR using Taq polymerase and the forward (F) and reverse (R) primers indicated in Table S1. Samples were separated by electrophoresis on a 6% bis-Acrylamide (19:1) gel that was stained overnight with 1× SYBR Green I Nucleic Acid Gel Stain (Invitrogen). The gel was imaged and quantified using an AlphaImagerHP (Alpha Innotech). The background-corrected sum of each band was measured and the percentage exon inclusion was calculated using the following formula: [(exon included sum)/(exon included sum+exon excluded sum)]×100. Data were analyzed by ordinary one-way ANOVA with multiple comparison correction.RNA (20-200 ng) was reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions using gene-specific reverse (R) primers located in the exon downstream of the regulated exon of interest (RNA was extracted using the RiboPure RNA Purification Kit (Invitrogen AM1924) from epaxial skeletal muscle from the tails of three biological replicates of adult WT and mutant fish. RNA quality was examined using the Fragment Analyzer RNA Analysis DNF-471 kit  and all RNA quality number (RQN) values were >8.0. Ribosomal RNA was depleted from 200 µg of the RNA using the NEBNext rRNA Depletion Kit (NEB E6310X), and then a cDNA library was prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB E7530L). The libraries were checked for quality using the Fragment Analyzer NGS Analysis DNF-474 kit  and quantitated using the KAPA Library Quantification Kit (KAPA Code KK4824). The completed libraries were then pooled in equimolar amounts and sequenced on an Illumina Next-Seq 500. A minimum of 60 million paired-end 75×75 reads were obtained for each library.GSE86356 . The fastq files were then checked for quality using FastQC (version 0.11.8) and aligned to the GRCz11 zebrafish genome using STAR (version 2.5.1b). The alternative splicing events were then analyzed using rMATS (version 4.0.2), grouping the biological replicates of each mutant and comparing them against WT. A similar preliminary analysis of the same sequencing data was performed using GRCz10 and rMATS (version 4.0.2). Significant mis-splicing events were categorized as having a false discovery rate (FDR) <0.10. Cluster analysis was performed with R and visualized with the factoextra package (version 1.0.5) using the top 800 most variable cassette exon splicing events in PSI values. Data were first tested using the gap statistic and then K-means clustering was performed with two centers. The data for human DM1 tibialis anterior comparisons were downloaded from NCBI Gene Expression Omnibus (GEO), accession number GSE86356 . Data foRP158284 . The datS4049994 .B/B;2A/A;3C/C1 zebrafish, and for WT and DM1 myotubes were obtained using Stringtie (version 1.2.3). Differential expression analysis was performed using DESeq2 (version 1.28.1). Significantly differentially expressed genes (compared to their respective controls) were filtered at an absolute log2 value of 1.0 and a FDR value of less than 0.05, and then analyzed using PANTHER Over-representation Test . A dot plot was created using GGPlot2 (version 3.3.2) showing significantly over-represented GO terms (FDR<0.05) in B/B;2A/A;3C/C1 zebrafish compared to WT. General GO categories with >100 annotated genes were omitted from the plot, and GO categories that overlapped with those over-represented in DM1 myotubes were highlighted.For GO analysis, BCL files were demultiplexed and converted to Fastq files using BCL2Fastq (version 2.16.0.10). The Fastq files were then checked for quality using FastQC (version 0.11.8) and aligned to the GRCz10 genome using STAR (version 2.5.1b). Reads counts for WT and B/B;2A/A1 mutants, B/B;2A/A;3C/C1 mutants or in the intersection of both sets . For each set of cassette exons, the frequency of YGCY motifs  was compared to the frequency of control 4-mers with identical A+T and CpG content within the following regions: cassette exons , intronic sequences 1-250 bp immediately 5′ or 3′ of the cassette exons, intronic sequences 1-250 bp 3′ of the upstream constitutive exons, and intronic sequences 1-250 bp 5′ of the downstream constitutive exons. The log2 [(enrichment of YGCY motifs relative to control motifs in each region associated with regulated cassette exons)/(enrichment of YGCY motifs relative to control motifs in each region associated with non-regulated exons)] was plotted in heatmap form.Enrichment of putative YGCY Mbnl protein-binding motifs was analyzed for the sets of cassette exons for which inclusion was significantly increased  or decreased  compared to WT in mbnl RNA levels in zebrafish tissues. Total RNA was prepared from 5 dpf larvae and adult tissues using the same procedure as for alternative splicing analysis. RNA was treated with TURBO DNase (Ambion) according to the manufacturer's instructions, then reverse transcribed with an oligo(dT)20 primer using the Superscript III cDNA First Strand Synthesis kit (Invitrogen). The quantitative PCR reaction was set up using a KAPA SYBR FAST ABI Prism kit (KAPA Biosystems) according to the manufacturer's instructions and run on a Quant Studio 3 System (Thermo Fisher Scientific) using the default settings for SYBR Green reagents and the fast run mode in the QuantStudio Design and Analysis Software v.1.4.2. The comparative CT (ΔΔCT) method was used to calculate relative mRNA levels. Five biological samples of each genotype or tissue type were run in triplicate and data were normalized to the expression of the housekeeping gene eef1a1l1 (Table S1.RT-qPCR was used to measure eef1a1l1 . PrimersWestern blots were performed using 15-50 µg of protein lysates that were prepared in RIPA buffer (Boston Bioproducts) with complete mini EDTA-free protease inhibitor (Roche) from HeLa cells (positive control for MBNL1 and MBNL2 protein expression), whole 5 dpf zebrafish larvae, adult skeletal muscle, heart, brain, intestine and cornea, or purchased from Santa Cruz Biotechnology (human placenta extract used as a positive control for MBNL3 protein expression). The antibodies tested included Santa Cruz Biotechnology anti-MBNL1 (D-4) sc-515374 (1:250), Santa Cruz Biotechnology anti-MBNL1 (4A8) sc-136165 (1:500), Millipore anti-MBNL1 ABE-241 (1:1000), Abcam anti-MBNL2 ab105331 (1:250), Sigma-Aldrich anti-MBNL3 SAB1411751 (1:150) and Sigma-Aldrich anti-Actin (20-33) A5060 (1:1000). Santa Cruz Biotechnology goat anti-rabbit IgG-horseradish peroxidase (HRP) sc-2004 (1:2000) and Sigma-Aldrich goat anti-mouse IgG FC-specific HRP (1:2000) were used as secondary antibodies.https://github.com/stednitzs/daniopen) to measure swim distance and distance from the bottom of the tank . Following the test period, the fish were returned to their home tanks.Age-matched single adult zebrafish were placed into a novel environment consisting of a custom glass aquarium measuring 18 cm deep×25.4 cm long×7.6 cm wide A. The fithe tank . To deteTen-month-old WT and mutant adult zebrafish were euthanized by hypothermic shock and fixed in Bouin's solution (Sigma-Aldrich), then washed in 70% ethanol. Fixed fish were bisected parasagitally or transversely, processed for paraffin embedding, sectioned at 7 µm thickness, and stained with Hematoxylin and Eosin by a histologist from the University of Oregon Institute of Neuroscience. Muscle morphology was analyzed by a fish pathologist from the Zebrafish International Resource Center who was blinded to genotype. Differential interference contrast microscopy images of tail epaxial muscle transverse sections along the dorsal fin were taken using a Leica DMLB microscope and muscle fiber cross-sectional areas were measured using Fiji. For each genotype, five fish were sectioned and a total of 400-1000 muscle fibers from the left side epaxial muscle proximal to the spine were measured across five to six sections of each fish.


The purinergic P2X7 receptor (P2X7R) is a cell membrane protein whose activation has been related to a variety of cellular processes, while its dysregulation has been linked to inflammation and cancer. ATP plays a key role in numerous metabolic processes due to its abundance in the tumour microenvironment. P2X7R plays an important role in cancer by interacting with ATP. The unusual property of P2X7R is that stimulation with low ATP doses causes the opening of a permeable channel for sodium, potassium, and calcium ions, whereas continued stimulation with high ATP doses leads to the formation of a non-selective pore. The latter effect induces the cell death. This evidence suggests that P2X7R has both pro- and anti-tumour potential. In this review, we aimed to describe the most relevant characteristics of P2X7R function, activation, and its ligands, while also summarising the role of P2X7R activation in the context of inflammation and cancer. The currently used therapeutic approaches and clinical trials of P2X7R modulators is also described.The purinergic P2X7 receptor (P2X7R) is a transmembrane protein whose expression has been related to a variety of cellular processes, while its dysregulation has been linked to inflammation and cancer. P2X7R is expressed in cancer and immune system cell surfaces. ATP plays a key role in numerous metabolic processes due to its abundance in the tumour microenvironment. P2X7R plays an important role in cancer by interacting with ATP. The unusual property of P2X7R is that stimulation with low doses of ATP causes the opening of a permeable channel for sodium, potassium, and calcium ions, whereas sustained stimulation with high doses of ATP favours the formation of a non-selective pore. The latter effect induces a change in intracellular homeostasis that leads to cell death. This evidence suggests that P2X7R has both pro- and anti-tumour proprieties. P2X7R is increasingly recognised as a regulator of inflammation. In this review, we aimed to describe the most relevant characteristics of P2X7R function, activation, and its ligands, while also summarising the role of P2X7R activation in the context of inflammation and cancer. The currently used therapeutic approaches and clinical trials of P2X7R modulators are also described. These mith eATP . The opeith eATP . Howeverith eATP . Particuith eATP , whose eith eATP ,12. This+2 through the channel [+2 input through the pore becomes toxic [In low ATP concentration conditions in the TME, ranging from 0.1 to 0.5 mM, P2X7R activation promotes tumour growth due to limited entry of Ca channel . Contrares toxic ,15. P2X7In this review, we aimed to describe P2X7R activation and functions, and the effects of P2X7R interaction with its ligands on cancer and immune response, as well as its role in inflammation and tumour-associated inflammation. We also describe the currently employed therapeutic approaches and the clinical trials of P2X7R modulators.+, K+, and Ca+2. The P2XR coding genes map in different chromosome loci, whilst homologous genes have also been identified in other vertebrates [P2X7R (or P2RX7) gene maps in the 12q24.31 chromosome [2+-independent facilitation current [The P2XR family includes seven different subtypes, i.e., P2X1-7, which are typed according to their pharmacological properties and structures. These receptors are cation-permeable ligand-gated ion channels that open in response to the binding of eATP consisting of three protein subunits (homotrimers or heterotrimers) activated by ATP permeable to Natebrates . The P2Xromosome , containromosome , while tromosome  and miRNromosome , which aromosome ,23,24,25romosome ,27. The romosome . The C-tromosome . PTMs ocromosome . The cysromosome ; P2X7R Cromosome . Mutatioromosome . Likewisromosome ,34,35. Promosome ,36. For romosome . Moreoveromosome  while co current , includi current ,41.Crystallographic studies have shown that P2X7R consists of three subunits, which include the ATP binding site in the pocket structure . The ATPP2X7R coding gene is not yet completely clear. However, it is known that in the case of loss-of-function SNPs, the receptor may promote cell growth by inhibiting apoptosis, while in the case of gain-of-function SNPs, P2X7R induces cell proliferation and the release of factors, such as vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β) [+, K+, and Ca+2 ions [The role of SNPs within the  (TGF-β) . In addi (TGF-β) . Indeed, (TGF-β) ,47. The a+2 ions , but cona+2 ions ,50. The a+2 ions . Anothera+2 ions . Indeed,a+2 ions . This pra+2 ions . Sustaina+2 ions . Cell mea+2 ions . Indeed,a+2 ions . P2X7R ia+2 ions ,54,55,56a+2 ions , prolifea+2 ions ,58,59. Ra+2 ions ,46,53,60A crucial P2X7R characteristic is that prolonged receptor stimulation leads to the opening of a non-selective pore, allowing for the passage of several molecules with molecular weights of up to 900 Da. This event causes an alteration in the intracellular homeostasis, which can lead to cell death. However, as it has been reported that P2X7R activation confers an advantage for cancer cell survival and growth, the role of the receptor in cancer is enigmatic . Moreove2+ homeostasis and stimulation of mitochondrial metabolism [Overexpression of P2X7R leads to an increase in tumour growth, metastasis, production of VEGF, and release of matrix metalloproteinases (MMPs) , which atabolism . At the tabolism . Under h+2 intake [+2 influx [P2X7R induces apoptosis by activating caspases 3 and 7 following a massive Ca2 intake ,69. At l2 influx . Activat2 influx ,70,71, w2 influx ,72,73,742 influx . One mec2 influx . P2X7R a2 influx . Indeed,2 influx .+2 in the absence of the pore. This is understandable, as despite P2X7R being expressed in MPM cells, the concentration of this receptor is insufficient in detecting the effects of pore opening. Another explanation might be that the cells may express a receptor isoform that does not favour pore opening [Lung cancer animal models indicate that P2X7R inhibition leads to a dramatic reduction in the migration of transplanted tumour cells into immunocompromised mice ,79. Phar opening .MYCN) degradation. MYCN is an oncogene overexpressed in neuroblastoma [In neuroblastoma, overexpression of P2X7R is involved in maintaining an undifferentiated cell state and decreases apoptosis. It also allows cell growth, even in the absence of serum or glucose . Moreoveblastoma . PI3K acblastoma . Moreoveblastoma .The anti-proliferative/pro-apoptotic effect of activated P2X7R is associated with plasma membrane permeabilisation in several cancer types. However, in other cell types, the receptor functions as an ion channel but no pore formation occurs because a non-functional form of the receptor with a truncated C-ter is expressed. In this case, P2X7R controls other processes such as proliferation or invasiveness . AnotherATP and adenosine are present in modest amounts (nmol/L) in the interstitium of healthy tissues, while high levels of both molecules can be reached in the inflammatory microenvironment (IME), which is an essential component of the TME . In physSeveral diseases have been associated with P2X7R-mediated inflammation: diabetes gut, liver, kidney, respiratory tract, and cardiovascular diseases . As a reThe ATP release from immune and non-immune cells can increase as a response to pathogenic infections, while subsequent activation of P2X7R following ATP binding leads to the modulation of innate/adaptive immune responses ,94. For ATP-P2X7R signalling modulates immune responses against several virus types. For instance, in vitro data indicated that P2X7R, when interacting with ATP, is able to protect marrow-derived macrophages from vesicular stomatitis virus (VSV) infection. The mechanism behind this process comprise the induction of IFN-β release via P38/JNK/ATF-2 signalling activation, which leads to the reduction of VSV replication levels . SimilarChlamydia trachomatis, (ii) Mycobacterium tuberculosis and bovis, and (iii) Gram-positive bacteria. Regarding the bacterium, which is a common sexually transmitted pathogen [Chlamydia species [Chlamydia immune response via NLRP3 inflammasome activation and IL-1b release [Micobacterium tuberculosis-driven tuberculosis in mice with experimental advanced pulmonary tuberculosis [Micobacterium bovis burden in comparison with WT mice [sepsis. Data obtained with an animal model of sepsis based on an ADORA2b KO mouse indicated that P2X7R on macrophages can reduce the bacterial DNA load and increase the release of inflammatory chemokines/cytokines while also improving mouse survival [An important antimicrobial activity through the production of inflammatory mediators in phagocytic cells and the modulation of the adaptive immune response against bacterial infections has been attributed to P2X7R . The mospathogen , it has  species  while bo release . The inhrculosis , while t WT mice . Furthersurvival . An addisurvival .Candida albicans-stimulated PBMCs [Paracoccidioides, presented reduced inflammation in comparison with WT mice [Alternaria alternata-exposed mice under treatment with P2RX7 inhibitors showed an attenuated T helper 2 (TH2) response [The implication of P2X7R signalling on pathogenic fungal infections has been described. For instance, P2X7R blockade can reduce the IL-1b levels in ed PBMCs . Data ob WT mice . In anotresponse , which tresponse .Plasmodium chabaudi, Leishmania amazonensis, Toxoplasma gondii, and Trypanosoma cruzi, has been remarked upon. For instance, P2X7R overexpression/activation has been reported to be effective in counteracting several protozoal infections by (i) favouring NLRP3 inflammasome activation and stimulating IL-1b secretion, as occurs during Toxoplasma gondii infection [Leishmania amazonensis infection [The protective role of P2X7R for multiple protozoa, including nfection  and (ii)nfection .Schistosoma mansoni infection, the downregulation of P2X7R in mesenteric endothelial cells has been reported [Schistosoma mansoni infection compared to WT mice, who exhibited a complete survival [Helminth is a group of large microparasitic worms that mainly infect the gastrointestinal tract and the blood vessels . Currentreported , while trophages . Moreovesurvival . It shousurvival ,121.In summary, these studies cumulatively indicate that the P2X7R signalling pathway plays a role in numerous aspects of pathogenic infection-driven inflammation, including immunomodulatory and proinflammatory activities. P2X7R can therefore be considered a possible therapeutic target for pathogenic infections management.The TME comprises a large variety of immune cells, including monocytes, macrophages, dendritic cells, lymphocytes, and myeloid-derived suppressor cells, that mediate the induction of inflammatory processes and enable tumour growth and progression through direct interaction with cancer cells. This highly inflammatory milieu modulates the immune response against tumours, while P2X7R activation, expressed on dendritic cells, play a key role in this context by activating NLRP3 inflammasome . NLRP3 hNLRP3 inflammasome activation also encompasses a functional interplay occurring between P2X7R, Pannexin-1 channel, and P2X4R ,123. TheThe response following P2X7R activation not only affects cancer cells but also modulates the activity of cells with anti-tumour/immunosuppressive activity, or can stimulate the release of growth factors . It seemSERPINA1 gene [Understanding the intracellular signalling pathways activated by P2X7R is still in the early stages. Numerous cell-specific signal transduction pathways are associated with receptor activation, such as PKC/MEK/ERK/FOS/JUN, PI3K/AKT/Mtor, MyD88/NF-κB, MMP-2/9, and calcineurin/NFATc1 ,127. ManNA1 gene ,131. By NA1 gene . P2X7R aNA1 gene . It alsoHigh concentrations of ATP in the TME leads to an increase in the concentration of adenosine, which has immunosuppressive and anti-inflammatory effects . Under tAdditionally, a recent patent proposed a method of treating cancer by administering polymyxin B in combination with ATP as a novel tool to Treg CD4+ CD25+ lymphocytes, which prevent excessive immune responses and are known T-cell suppressors. In particular, the role of ATP in this patent is to stimulate P2X7R on Treg to promote apoptosis .P2X7R is differentially regulated depending on different ATP concentrations. At ATP concentrations above 3 mM, P2X7R can co-induce cell death. However, the receptor is also activated at low ATP concentrations, i.e., between 1 and 3 mM, which is typically found within the TME, especially near necrotic areas . These l+ appears to be involved in P2X7R activation as CD38-mediated NAD+ degradation causes a decrease in receptor activation [+ stimulates ATP secretion that, in turn, activates P2X7R [2+, release of the pro-inflammatory cytokine IL-1β, and favours cytotoxicity [2+ ions, as well as the cytotoxic effect and the permeabilisation of the plasma membrane [The most potent P2X7R agonist is 2′,3′-benzoyl-4-benzoyl-ATP (BzATP) . Althougtivation . It is aes P2X7R . Among ttoxicity . P2X7R atoxicity , which itoxicity . These emembrane . P2X7R amembrane .P2X7R blockers can be divided according to the P2X7R binding site. Some molecules share the same ATP binding site and therefore act as competitive antagonists, while other molecules bind other binding sites, thereby acting as allosteric modulators. P2X7R antagonists of the first generation include BBG and periodate-oxidised ATP. Although these compounds have been tested in several studies, they are not P2X7R-specific . Indeed,Different effects have been demonstrated on wild-type (WT) mice treated with P2X7R antagonists and P2X7R KO mice upon comparing the anti-tumour response of immune system cells . In P2X7nude/nude mice [P2X7R activation in melanoma, colon cancer, and neuroblastoma cells induces VEGF secretion . A signiude mice . The P2XWith the aim of developing novel MPM therapeutic approaches, researchers recently tested AZ10606120 in vitro in combination with an agonist of the adenosine A3 receptor (A3AR) . The A3AThe use of P2X7R antagonists induced the decrease in tumour size; the upregulation of the anti-oncogenic kinase GSK-3β; and the downregulation of MYCN, HIF-1α, and VEGF . These dwww.clinicaltrials.gov, accessed on 1 February 2022). AZD9056 is a P2X7R inhibitor capable in counteracting the positive effect of BzATP on P2X7R activation [Clinical trials have been designed to test the safety, tolerability, and clinical application of several P2X7R antagonists such as AZD9056, CE-224535, and GSK1482160 for the treatment of autoimmune and inflammatory diseases (tivation . The inhtivation . The P2Xtivation . Althougtivation  may havetivation . Regarditivation .Notably, to the best of our knowledge, no clinical trials have been started to test the use of antagonists for the treatment of cancer since P2X7R activation induces an anti-tumour response in cells in the immune system that infiltrate the tumour and therefore the use of antagonists would seem to favour tumour progression. However, the data reported from preclinical studies are very promising, and therefore further tests could be conducted to validate the use of antagonists in the treatment of cancer. The antitumor effect of P2X7R antagonists has also been reported as being greater in immune-competent mice than in immune-deficient mice. This could also be due to the blocking of receptors expressed in MDSCs, in which activation of the receptor induces the release of immunosuppressive factors .P2X7R is a purinergic receptor involved in inflammation and cancer. Its activation can have both pro- and antitumor effects, depending on the cell type and a variety of different factors. It is considered a key mediator of the antitumor immune response, but its expression on cancer cell membranes appears to mediate a dual role. It has been reported that P2X7R and its ligands play a central role in cancer development, progression, and metastasis. Previous in vitro studies and animal models have increasingly demonstrated the paramount importance of P2X7R in a variety of cancers, including lung, colon, thyroid, pancreatic, prostate, breast, lymphoma, MPM, and glioma. On the basis of these findings, P2X7R has recently emerged as an attractive potential diagnostic/prognostic marker and target for cancer therapy.Clinical trials based on P2X7R antagonists are currently ongoing for the management of inflammatory diseases. In addition, although clinical trials have not yet been initiated to test the effect of P2X7R antagonists in the treatment of cancer, the preclinical data are very encouraging. In fact, a number of ligands have already shown promising results in animal models, while their clinical efficacy requires further investigation.Investigating the role of P2X7R and its ligands in inflammation and in cancer development/progression is an important area of research. Since a considerable number of signalling pathways are involved in the stimulation of P2X7R, understanding the mechanism behind its activation is essential to deepen the role of this receptor in inflammation, tumour development, progression, and metastasis. In addition, the rationale for the dual nature of P2X7R in mediating cell proliferation and apoptosis under different pathophysiological conditions should be further investigated. The role of P2X7R in immune response is well established ,171. The


KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized.Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS.Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death.These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.The online version contains supplementary material available at 10.1186/s12860-021-00394-2. KDM6A . Therefore, 1 ng eGFP-KDM6A WT would yield an integrated fluorescence signal of 1.29E+ 03 AU. G. Data from E. and F. were used to transform the x-Axis with the fluorescent signal into the amount of KDM6A protein,. Approximated from D., the respective c50 always corresponded to 62.4 product. This correlation was used to calculate the specific activity for each variant. Given the fixed time of 240 min per assay, the specific activity under these conditions was calculated. The c50 for all available substitution variants was obtained in the same manner, fixing all parameters except c, using it as a relative measure of activity. At least four independent triplicate measurements were obtained for each substitution variant. H. absorption for truncated variants, three repeats with triplicates each. Since the truncated variants showed an unusual spectrum with a high scatter fraction at lower wavelengths (see C.), a quantification was impossible. Therefore, we qualitatively confirmed, that all truncated variants with a JmjC domain are catalytically active (green boxes) and those without JmjC domain are catalytically dead (red boxes). The distribution of the signal over the background is an evidence for activity in all truncated variants with a JmjC domain. ΔJmjC and TPR do not exceed assay background levels (zero), while WT, ΔIDR, ΔTPR and JmjC are all above background. I. Activity of T726K is dependent on post-expression time. WT activity does not change significantly between 24 h (black) to 48 h (grey), whereas in T726K activity is slightly lower than WT after 24 h (blue) and strongly reduced after 48 h (cyan) when comparing c50-values.Additional file 6: Figure S3. Control experiments for antibody staining specificity and endogenous KDM6A protein levels. SW-1710 and T-24 cells were transfected with KDM6A WT or ΔIDR variants and stained with two different antibodies  raised against epitopes within the KDM6A central region (IDR) and 2nd antibody labeled with AbStar Red (Abberior). Clearly, both antibodies (in red channel) detect transfected KDM6A WT protein , but not the ΔIDR variant. The untransfected control cells show endogenous KDM6A in both cell lines stained with both KDM6A antibodies.Additional file 7: Figure S4. Localization study of KDM6A variants in normal and invasive urothelial (cancer) cells. A. KDM6A substitution and deletion variants are localized in the cytoplasm and nucleus in T-24 cells. Substitutions cause mild effects, whereas deletion variants cause stronger cellular responses such as DNA release and mitotic errors. B. Representative KDM6A deletion variants are localized in the cytoplasm and nucleus in HBLAK. Two different phenotypes have been observed: a milder phenotype (majority of cells) and a phenotype (minority of cells) which reproducibly causes stronger cellular responses such as DNA release and mitotic errors. DAPI counterstaining of the nucleus and the respective overlay of both channels (DAPI in blue and GFP in green) is displayed. Images are taken in 2048 × 2048 resolution using a widefield microscope Olympus FX81 with 60x oil objective with post-processing using HuygensPro20.10. Effects of KDM6A variants in SW-1710 cells is shown in Fig. Additional file 8: Figure S5. Western Blot control experiments and raw blots. A. eGFP-KDM6A TPR whole cell lysate and the corresponding IP fraction was separated on a Western Blot and stained with eGFP (CST) or RBBP5 (CST) antibodies. RBBP5 precipitated with eGFP-KDM6A TPR. B. Dynabeads washing control on same membrane, washed with 3x RIPA like buffer (RLB). While KDM6A WT pulls a clearly visible RBBP5 band and a low WDR5 band, both are undetectable in eGFP-transfected cells alone (negative control). C. Raw blots from Fig. D. Raw blots from Fig. /D. Protein lanes shown in main figures are in red boxes. The PageRuler Plus Prestained protein ladder (Thermo Fisher) was used for orientation.Additional file 9: Figure S6. Pathway enrichment analysis of MS data from KDM6A-tagGFP2 cell lines. A. Data from quadruplicates of three independent experiments of two different KDM6A-tagGFP2 cell lines using the reactome database. MS data sets were applied to the reactome analysis database. The resulting output shows significantly enriched pathways. The five most interesting pathways are depicted: cell cycle, chromatin organization, DNA repair, RNA metabolism including rRNA processing and protein metabolism including ribosome biogenesis and post-translational modification of histones. B. Top ten proteins identified by MS in KDM6A-tagGFP2 stable cell lines VM-CUB1 and RT-112. Among the top ten proteins in KDM6A pull down and subsequent MS, Nucleophosmin (NPM1), Nucleolin, ribosomal subunits 40S and 60S, as well as histone variants, are detectable. Mutational KDM6A and KMT2C/D status and experiments with RT-112 and VM-CUB1 have been recently published [ublished .Additional file 10: Figure S7. Cellular back mapping identifies co-localizing protein populations at extranuclear DNA segments. A. images shown in Fig. B. Analysis of KDM6A TPR and NPM1: Cellular back mapping identifies a colocalizing population (orange) and free KDM6A TPR (green) and NPM1 (red) at cytoplasmic DNA segments. C. scatterplot analysis and cellular back mapping of KDM6A variants and DAPI as indicator of KDM6A-DNA complexes. In KDM6A WT scatterplot we identified four populations: 1 = cytoplasmic KDM6A (without DAPI) and 4 = DAPI (without KDM6A), as well as, populations #2 and #3, which resemble different ratios of KDM6A-DNA complexes. Population 2 is located at the lamina and in nucleolar puncta, and population 3 is located within the nucleoplasm. Substitution variant T726K shows minor differences in populations #2 and #4, as the lamina in #2 is less clearly visible, but strongly enriched in population #4. Contour scatterplot of JmjC or TPR with DAPI identified only three populations, with population #1 being absent. Back mapping clearly indicates compartment-wise localization of different DAPI-KDM6A JmjC or TPR compositions.Additional file 11: Figure S8. Distribution and expression levels of p-γH2A.X and RAD51 after KDM6A overexpression and endogenous localization of KDM6A during mitosis. A. p-γH2A.x protein level in untransfected and KDM6A variants transfected T-24 cells. Note, that even in strongly overexpressed KDM6A WT cells, p-γH2A.X was not increased, but strongly so in KDM6A JmjC and ΔTPR cells. B. Endogenous localization of KDM6A  as imaged with Leica STED SP8 in HBLAK cells. For STED, the sample was depleted with 35% of 775 nm laser. C. RAD51 proteins were detected at a persisting chromatin bridge of two daughter cells, with cell 1 being KDM6A ΔTPR positive. Image was taken with Abberior STED, excitation laser 640 nm 5% and depletion laser 775 nm with 40%.


Penaeus vannamei (body weight 9.56 ± 0.10 g), reared with 12L: 12D full spectrum light as background light under five UVA (peak at 400 nm) photoperiods  at a light intensity of 1 W/m2 were investigated. The results showed that the 2L: 22D and 4L: 20D UVA photoperiods enhanced the growth performance and reduced the feed conversion ratio and the shrimp mortality. Shrimp exposed to UVA (2L: 22D and 4L: 20D) also displayed higher levels of hepatopancreas catalase (CAT), superoxide dismutase (SOD), acid phosphatase (ACP), phenol oxidase (PO) and lysozyme (LZM) compared to the 8L: 16D and 12L: 12D groups. The malondialdehyde (MDA) levels increased in line with the extension of the UVA irradiation time. The mRNA expression of apoptosis-related genes in all the UVA treatments were significantly higher than with the control treatment, except for the 2L: 22D group. The results of the 2L: 22D and 4L: 20D treatments were significantly higher than those of the control group, except for LGBP. In conclusion, 2L: 22D and 4L: 20D UVA photoperiods increased growth performance and decreased FCR, improved the innate immunity and antioxidant response and reduced the mortality rate in adult shrimp.UVA is the most common type of solar UV radiation in aquatic environments; however, the effects it causes in shrimp farming in recirculating water systems (RAS) is unclear. Thus, the growth performance, immune responses, antioxidant status and apoptosis-related gene expression in Pacific white shrimp,  Girella laevifrons Mortality (%) = 100% × [(Nt − LnW0)/tSGR (%) = 100% × (LnWt − W0)FCR = F/, t is time of rearing (days) and F is total feed intake (g).Where Np < 0.05 for all the analyses. Means and standard deviations are used to represent all the data.Using SPSS 26.0 software, the results were subjected to one-way analysis of variance (ANOVA), followed by a Duncan’s test to determine significance among the shrimp groups . Unless otherwise noted, significant values were established at P. vannamei under five different UVA photoperiods in full-spectrum background light (12L:12D) were investigated. Exposure to 2L:22D and 4L:20D UVA photoperiods resulted in increased growth performance and immune responses while decreasing stress responses, whereas the opposite results were found for the long UVA photoperiod groups (8L:16D and 12L:12D). The apoptosis-related gene expression levels in the 2L:22D and 4L:20D groups were lower than with the other treatments, except for the control group (0L:24D). As a consequence, supplementing with UVA light at specific periods activated antioxidant enzyme activity and the immunological defense system, protecting cells from oxidative stress. We believe that selecting a specific UVA photoperiod is beneficial to shrimp growth and immunological function and that the effects of choosing 2L: 22D and 4L:20D UVA photoperiods are considerable, based on the results of this experiment.The growth performance, immune responses, antioxidant status and apoptosis-related gene expression of