Doxorubicin is one of the most effective chemotherapeutic drugs for breast cancer while its common drug resistance leads to poor patient prognosis and survival. Growing evidence indicate dynamically reorganized chromatin allows rapid access of the gene regulatory machinery to open genomic regions facilitating subsequent gene expression through direct transcription factor (TF) activation and regulatory element binding.To better understand the regulatory network underlying doxorubicin resistance in breast cancer cells, we explored the systematic alterations of chromatin accessibility and gene expression by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with RNA sequencing, followed by integrative analysis to identify potential regulators and their targets associated with differentially accessible regions (DARs) in doxorubicin-resistant MCF7 (MCF7-DR) cells.MT1E, GSTP1, LDHB, significantly down-regulated TFF1, UBB, DSCAM-AS1, and histone-modifying enzyme coding genes HDAC2, EZH2, PRMT5, etc. By integrating with transcriptomic datasets, we identified 18,228 DARs in MCF7-DR cells compared to control, which were positively correlated with their nearest DEGs (r = 0.6). There were 11,686 increased chromatin-accessible regions, which were enriched in up-regulated genes related to diverse KEGG pathways, such as the cell cycle, regulation of actin cytoskeleton, signaling pathways of MAPK, PI3K/Akt and Hippo, which play essential roles in regulating cell apoptosis, proliferation, metabolism, and inflammatory responses. The 6,542 decreased chromatin-accessible regions were identified for the declined doxorubicin-associated biological processes, for instance, endocrine and insulin resistance, central carbon metabolism, signaling pathways of TGF-beta and P53. Combining data from TCGA, analyses of the DAR sequences associated with the DNA-binding motifs of significantly enriched TF families including AP-1, TEAD and FOX, indicated that the loss-function of FOXA1 might play a critical role in doxorubicin-resistant breast cancer cells (DOX-R BCCs).A total of 3,963 differentially expressed genes (DEGs) related to doxorubicin resistance were identified, including dramatically up-regulated cis-regulatory elements-based putative therapeutic targets.These data exhibit the non-genetic landscape of chromatin accessibility and transcript levels in the DOX-R BCCs, and provide clear insights and resources for the detection of critical TFs and potential  Breast cancer has the highest morbidity and mortality rates of all cancers in the female population, and its incidence has gradually increased over the past few decades . TreatmeDoxorubicin induces cell death and cell growth arrest primarily by inhibition of the topoisomerase II activity, DNA intercalation and production of free radicals, leading to apoptosis, autophagy, senescence and necrosis of the fast-growing cancer cells . In spitcis-regulatory elements and activate gene expression, while closed chromatin regions impede the accessibility of promoters and enhancers to transcription machinery components leading to gene silencing  allowed us to identify the regulatory DNA sequences, putative TFs, and landscape of TF binding events that may be responsible for these changes.L-glutamine , 10% fetal bovine serum , 0.1 M sodium pyruvate , 50 units/mL penicillin and 50 μg/mL streptomycin . The MCF7 and MCF7-DR cells were grown in 10-cm culture dishes at 37°C in a humidified 5% CO2 atmosphere, and the medium was renewed every 2–3 days. Whereas parental MCF7 cells were grown in a drug-free medium, resistant-derived cells were maintained in doxorubicin hydrochloride  at a concentration of 1 μM. Doxorubicin hydrochloride was stored at a concentration of 2 mM  in DMSO  at −20°C.The MCF7 and doxorubicin-resistant MCF7 (MCF7-DR) cell lines originally obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences Committee , were kindly provided by the key laboratory of breast cancer in Shanghai, Fudan University. All cells were cultured in DMEM medium , supplemented with 2 mM Before any further experiments, the MCF7-DR cells were maintained in doxorubicin free medium for 3 days. Parental MCF7 cells were cultured in parallel with the MCF7-DR cells for comparison.Total RNA was isolated from MCF7 and MCF7-DR cells using TRIzol Reagent  and purified using RNeasy Mini Kit . Agilent Bioanalyzer 2100 was used to assess RNA integrity. The RNA-seq library preparation from 1 μg of total RNA was performed with TruSeq RNA Sample Prep Kit  according to the manufacturer’s instructions. Finally, PCR products were purified to remove adaptor dimers with VAHTS DNA Clean Beads . Library quantity was assessed using a Qubit 2.0 Fluorometer . 150 bp pair-end sequencing was performed on the Illumina HiSeq X Ten platform .2] for 10 min on ice to prepare the nuclei. Immediately after lysis, the suspension of nuclei was spun at 500 g at 4°C for 5 min to remove the supernatant. Nuclei were then incubated with the 50 μl of Tn5 transposition reaction mix  at 37°C for 30 min. The stop buffer was then added directly into the reaction to end the tagmentation. DNA was purified using the QIAquick PCR Purification Kit . PCR was performed to amplify the library for 12 cycles using the following PCR conditions: 72°C for 3 min; 98°C for 30 s; and thermocycling at 98°C for 15 s, 60°C for 30 s and 72°C for 3 min; following by 72°C 5 min. Libraries were purified with VAHTS DNA Clean Beads  to remove contaminating primer dimmers. Library quantity was checked using Qubit 2.0 Fluorimeter . Last, all libraries were sequenced on the Illumina HiSeq X Ten with 150 bp paired-end reads .The ATAC-seq libraries were prepared as previously described with minor modifications . Briefly1. The sequences were aligned to the human genome build hg19 using STAR  > 1.5 and the average FPKM > 10 in at least one of the two groups. The “ggplot2”  and ggplot2 . All sequencing data were mapped onto the hg19 human genome assembly using Bowtie2  with par ggplot2  were use ggplot2  with RPK ggplot2 . To anno ggplot2  with defTo assess the chromatin accessibility changes on a genome-wide scale, deepTools  was usedP value in R. Venn diagrams, boxplots and scatter plots were produced using “Venn Diagram”    was empl (v4.10)  was usedBased on RNA-seq and ATAC-seq data, differentially expressed transcription factors (DETFs) positively correlated with DARs were analyzed using the STRING database to identify groups of TFs that have known and predicted protein-protein associations . The net+; n = 300) and the other group . The Kaplan–Meier survival curves and log-rank test were performed by utilizing the “survival” and “survminer” packages in R to analyze survival differences for critical DEGs. The mRNA expression levels of representative genes were compared between the Dox+ and Dox– subgroups using the Wilcoxon signed rank test in R software (version 4.0.5). Pearson’s correlation coefficients and P values were also calculated to assess the associations between critical genes using the “Hmisc” R package. The P values less than 0.05 were regarded as statistically significant.Based on the genomic and clinical data from the Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA), the breast cancer samples were divided into two subgroups based on whether the Doxorubicin (a.k.a. Doxorubicin) was used alone, namely the doxorubicin alone group  were calculated in analysis. Significant differences for all quantitative data were considered when ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ****P < 0.0001. The genomic signal tracks were extracted and visualized using the WashU Epigenome Browser5.If not specified, R platform2(fold change) > 4, average FPKM > 150 in at least one group, we obtained 42 critical DEGs associated with doxorubicin resistance, including dramatically up-regulated metallothionein 1 family , GSTP1 (glutathione S-transferase pi 1), LDHB (lactate dehydrogenase B), and significantly down-regulated TFF1 (trefoil factor 1), UBB (ubiquitin B), DSCAM-AS1 (DSCAM Antisense RNA 1).To investigate potential genes associated with the resistance to doxorubicin in MCF7 breast cancer cells, RNA-seq was performed with two biological replicates for each sample . A totalGO analysis was applied to elucidate biological processes, which indicated that the up-regulated DEGs were mainly involved in the mitotic cell cycle process, cytoskeleton organization, regulation of protein kinase activity. Meanwhile, the down-regulated DEGs were significantly enriched in epithelial cell differentiation, oxidation-reduction process, lipid biosynthetic process and Ras protein signal transduction . In addiELP3, EP300 and KAT7, were significantly up-regulated; Except for EZH2 and PRMT5, most of the differentially expressed histone methyltransferase and demethylase genes were down-regulated  is the most current method for probing accessible chromatin regions, based on the ability of hyperactive Tn5 transposase to insert Illumina sequencing adaptors into active regulatory regions . To geneIn order to determine whether the alteration of chromatin accessibility was consistent with transcription changes, we assessed differential accessibility between the parental and doxorubicin-resistant MCF7 breast cancer cells (MCF7-DR) across the genome. In total, 11,686 accessible regions were > 1.5-fold more accessible in MCF7-DR cells , 3A–C. Cr = 0.6), suggesting that the alterations in chromatin accessibility contribute to differential expression of the genes associated with doxorubicin in MCF7-DR cells, such as MT1E, GPX1, LDHB and TPM2  > 3  > 3 , includiand MT1E . This stcis-regulatory specific sequences, known as motifs, in the promoters or enhancers of their target genes (P < 0.01) in hyper- and hypo-accessible regions, respectively  were intersected with the DEGs  that might act coordinately to control their gene expression, we constructed a protein-protein interaction (PPI) network using the STRING database  and without  doxorubicin alone. Remarkably, the results showed that lower expression of several significantly down-regulated genes in MCF7-DR cells was verified to be significantly (P < 0.05) associated with the poor prognosis of the Dox+ patients when compared to the Dox– patients, including FOXA1, GATA3, RARA, and SLC27A2 (GSTP1 and LDHB was related with the poor prognosis of Dox+ patients (FOXA1 and GATA3 were significantly (P < 0.05) lower in the Dox+ patients than Dox–, while GSTP1 and LDHB expression levels were higher in the Dox+ patients , GSTP1 and LDHB (r = 0.614), FOXA1 and SPDEF (r = 0.857), while FOXA1 expression was negatively correlated with GSTP1 (r = −0.653) and LDHB (r = −0.7) as expected  and analyzed expression levels of the DEGs and their impact on the prognosis of breast cancer patients treated with , metallothionein gene family  and glutathione peroxidase 1 (GPX1). It has been reported that overexpression of metallothionein confers resistance to anti-cancer drugs , ubiquitin B (UBB), and SAM pointed domain containing ETS TF (SPDEF). Furthermore, from a perspective of epigenetic regulation, we analyzed the changes in the expression of known histone-modifying enzymes especially. The results suggested that the up-regulation of histone deacetylase 2 (HDAC2) and histone methyltransferase EZH2 was potentially involved in the down-regulation of certain doxorubicin-resistant genes.Based on the analysis of differential expression level of mRNA, we identified several dramatically up-regulated genes associated with doxorubicin resistance, such as glutathione S-transferases P1 was found between DARs and DEGs, validating our expectation that increased chromatin accessibility results in higher expression of critical genes associated with doxorubicin resistance, such as MT1E, GPX1, and TPM2. In the same way, condensed chromatin, known as closed chromatin, restricts the binding of TFs and transcriptional regulators to the promoter-TSS and/or enhancer, which leads to gene silencing, including TFF1, UBB and CDH1. Furthermore, we screened the DEGs that were significantly positively correlated with chromatin accessibility changes, and then, Cytoscape (version 3.7.1) software was used to display their enriched functional pathways.Genome-wide analysis of chromatin accessibility revealed that ATAC-Seq signal was significantly enriched in promoter-TSS and distal regulatory elements such as enhancers. Combined with RNA-seq data, a significant positive correlation (P < 10–10) associated with the hyper- and hypo-accessible regions, respectively, such as TEAD1, RUNX1, GRHL2 and FOXA1. It has been previously reported that the pioneers TFs FOXA1 and GATA3 mediated ESR1 binding by shaping enhancer accessibility (GATA3/FOXA1/GRHL2 expression might play an important role in the down-regulation of key genes in MCF7-DR cells, such as SLC44A2, BATF, and PRR15L. Moreover, the analysis of genomic and clinical data from TCGA-BRCA further revealed that low expression levels of FOXA1 and GATA3 in patients treated with doxorubicin alone experienced significantly worse clinical outcomes, while GSTP1 and LDHB had opposite prognostic effects. As was expected, all of them exhibited similar gene expression patterns in both DOX+ breast cancer patients and MCF7-DR cells.Transcription factors play an important role in cancer drug resistance . By analsibility ; FOXA1 csibility . The PPIBATF, SLC44A2, and GPNMB. Besides, the opposite changes in gene expression of FOXA1 and GSTP1 might be potential therapeutic targets for overcoming or reversing doxorubicin resistance in breast cancer.Most of the previous research on drug resistance has focused on the up-regulated genes but paid relatively little attention to the down-regulated genes. Our findings suggest that down-regulation or loss of certain regulators may also play an important role in promoting the development of cancer drug resistance. According to previous studies and our data analysis, we concluded that chromatin accessibility changes could promote or inhibit the expression of critical genes associated with doxorubicin resistance by affecting the DNA-binding ability of TFs and transcriptional regulators in promoter and/or enhancer. It is probably that, in doxorubicin-resistant breast cancer cells, loss of the pioneer TF FOXA1 leads to a significant decrease in chromatin openness, which impairs the ability of co-regulators GATA3 and/or GRHL2 to occupy their specific binding sites in the promoter and/or enhancer region of specific genes, such as The genome-wide profiling of open chromatin by ATAC-seq represents a novel strategy to study accessibility dynamics and transcriptional regulation in cancer drug resistance. The application of this technique allows us to identify the accessible regulatory elements and TFs that are probably responsible for the acquired resistance to doxorubicin in breast cancer cells. Our ATAC-seq data contribute to close the loop for the association between TF binding, transcriptional and chromatin states, providing new insights into non-genetic mechanisms of doxorubicin resistance and putative targets for epigenetic therapy.All relevant datasets supporting the key findings of this study are available within the article and its Supplementary Material or from the corresponding author on reasonable request. All sequencing data have been deposited in the GEO repository: GSE174152.GW and XW conceived the idea. XW prepared samples and generated the data. XW analyzed the data with assistance from GW and JY. XW wrote the manuscript. GW and BS supervised the study and revised the manuscript. JY provided helpful comments on this study. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Organ transplantation is the most preferred treatment option for end-stage organ diseases; however, allograft rejection is the major hurdle in successful long-term transplant survival. In spite of developing better HLA matching and more effective immunosuppressive regimen, one-year graft survival has been increased by nearly 90% and the incidence of acute rejection by one-year post-transplantation has been decreased by 12.2% in the last decades, chronic allograft rejection has remained as one of the major obstacles to the long-lasting survival of the transplanted allograft. Therefore, seemingly preventing the allograft rejection and inducing immunological tolerance against transplanted allografts is one of the primary goals in transplantation research to enable long-lasting graft survival. Various mechanisms such as long noncoding RNAs (lncRNAs) have been proposed that induce immune tolerance by modulating the gene expression and regulating innate and adaptive immune responses during transplantation. Besides, because of involvement in regulating epigenetic, transcriptional, and post-translational mechanisms, lncRNAs could affect allograft status. Therefore, these molecules could be considered as the potential targets for prediction, prognosis, diagnosis, and treatment of graft rejection. It is suggested that the noninvasive predictive biomarkers hold promise to overcome the current limitations of conventional tissue biopsy in the diagnosis of rejection. Hence, this review aims to provide a comprehensive overview of lncRNAs and their function to facilitate diagnosis, prognosis, and prediction of the risk of graft rejection, and the suggestive therapeutic choices after transplantation. Solid-organ transplantation is the most preferred treatment option for end-stage organ failure and could ameliorate life expectancy and quality, but allograft rejection is still the main barrier to long-term graft survival. Although widespread use of conventional immunosuppressive drug regimens and improvement in surgical techniques, HLA matching and cross-matching technologies have significantly decreased the rate of allograft rejection, induction of the immunological tolerance and the long-lasting survival of transplanted organs have remained as the main challenges. Based on the 2019 annual data report of organ procurement and transplantation network (OPTN), the incidence of acute rejection by one year post-transplantation were 7% , 25.4%  for KidnA growing body of evidence suggests that long non-coding RNAs (lncRNAs) have been participated in developmental, biological, and pathological processes through affecting various molecular pathways, such as transcription, post-transcription, and translation levels. In the rejection processes, the pathologic process could be detected at molecular level before occurrence of histological or clinical manifestations. Several investigations were carried out for profiling of lncRNAs in serum, plasma and urine samples of transplant rejected patients to find potential biomarkers for prognosis, diagnosis, and treatment of graft rejection –16.To the best of our knowledge, no secondary study has assessed the role of lncRNAs in organ transplantation. Therefore, we aimed to conduct a comprehensive literature search and to summarize important results that coming from the original research which have evaluated the lncRNAs and their performance as predictive and/or diagnostic biomarkers for organ transplantation.ENCODE (Encyclopedia of DNA Element) project has demonstrated that about 80% of the human genome is actively transcribed to at least one RNA, while only a small fraction (less than 3%) of them translate to the protein, and the remaining are non-protein-coding genes which transcribed but do not translate to the protein, consist of non-coding RNAs (ncRNAs) . ncRNA’sIt has been revealed that the human genome is highly dynamic, and just 2.2% of the DNA sequence is conserved. Interestingly, more than 80% of lncRNAs are among the least conserved genes. The lncRNA H19 is the first non-coding gene discovered in 1989; however, it was classified as mRNA at that time.de novo origin of lncRNAs is difficult to prove; there are a few examples for it, such as Polidi that is a testis-specific lncRNA , sense or exonic, intronic, natural antisense transcript (NATs), and bidirectional or divergent lncRNAs , 27, or lncRNAs are typical RNA molecules transcribed from exonic, intergenic, or distal eukaryotic genome regions by the RNA polymerase II, III enzymes or mitochondrial RNA polymerase IV enzyme. These molecules are processed to the mature RNA, similar to the protein-coding mRNAs. The immature RNA transcripts often undergo several processing steps for reaching the mature forms during and after transcription, such as 3’-polyadenylation, 5′-capping, splicing, and chemical base modification , 32.via relocalization of regulatory factors. Decoy lncRNAs act as an endogenous sponge for other molecules such as microRNAs and alter the bioavailability of the molecule, changing cellular function. Scaffold lncRNAs participate in the formation of ribonucleoprotein complexes. As a signal, lncRNAs could regulate gene expression , migration (H19), epigenetic regulation (HOTAIR), cell cycle (MALAT), cell death (MEG3), invasion (lncRNA-ATB), angiogenesis (ANRIL), autophagy (H19), apoptosis (PTENP1) and metastasis (HULC); as well as are found to be associated with several diseases such as rheumatoid arthritis, atherosclerotic coronary artery disease, diabetes, neurodegenerative disorders, and many types of cancers –35. As mpression , 37. The(I) Regulating histone modifications; Important histone-modifying complexes that interact with lncRNAs include two repressive complexes: PRC1 and PRC2. X-inactive specific transcript (XIST) is a highly expressed lncRNA in females that silences gene expression by recruiting PRC2. Moreover, HOX transcript antisense RNA (HOTAIR), another lncRNA, can repress a locus named HoxD by interacting with PRC2; it can also interact with a histone demethylase and lead to developing repressive effect on chromatin by removing a histone mark. (II) Modulating DNA methylation; Tcf21 antisense RNA reducing DNA methylation (TARID) is a lncRNA involved in the mechanisms of DNA demethylation. (III) Regulating chromatin remodeling; some lncRNAs play a role in controlling chromatin remodeling complexes which can change nucleosome spacing, such as embryonic ventral forebrain-1 (evf2) and myosin heavy chain associated RNA transcripts (MHRT) . (IV) InThe functions of lncRNAs in transplantation have attracted wide attentions. A growing body of literatures have investigated about the functions of several lncRNA categories in cell or organ transplantation. As the studies utilized mice/rat models or human samples, different aspects of lncRNAs activity have been assessed. Therefore, according to the design of studies, we categorized them into two categories: studies that have mainly evaluated mice/rat model of transplantation  and immune tolerance induction. The evidence from previous study suggests that silencing some of the lncRNAs, such as nuclear enriched abundant transcript 1 (NEAT1), can induce tolerogenic phenotype in DCs (tol-DCs) by reducing the expression of the costimulatory molecules such as, CD80, CD86, and MHC-II through the interaction with miR-3076-3p . Moreover, TUG-1 overexpression in mice led to massive reduction of serum levels of hepatic enzymes, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) after transplantation. Therefore, overexpression of TUG1 appears to confer hepatic tissue protection and ameliorates hepatocyte functions following hepatic injury in a murine liver transplant model  expression, whereas TUG1 overexpression attenuates cold-mediated apoptosis nt model . Similarnt model .via FOXO1 TF. Myc/Max and FOXO1 TFs might play pivotal roles in the development of OB in the murine models following lung transplantation by regulating downstream lncRNAs, mRNAs, and miRNAs , primary graft dysfunction (PGD), and ischemia-reperfusion injury (IRI). For example, an integrated analysis for TFs and lncRNAs showed that eight lncRNAs such as Snhg1, Gm15290, Gm16853, Gm16761, Gm15834, AI480526, A530013C23Rik, and 5430416N02Rik had upregulated; whereas six lncRNAs including Gm10425, Gm5091, AI854517, F630040L22Rik, D130017N08Rik, and 1600020E01Rik were downregulated under the effect of Myc/Max transcription factor in mice with OB. Additionally, the expression of 9530082P21Rik and 23100001H17Rik lncRNAs were modulated d miRNAs . Furthervia siMEG-3 delivery promoted and reduced miR‐181b‐5p levels and TNF-α expression, respectively. Consequently, these changes protect kidney allografts from tubular damage (Acute kidney injury (AKI) induced by prolonged cold ischemia could lead to acute rejection by enhancing the recruitment of various immune cells, like T cells. Interaction of CXCR-3 and its ligand, CXCL-10, has been depicted to be involved in the T cell immigration to the allograft in a rat model of renal transplantation . It has r damage . These r+ (A2B5+iPSC) cells were transplanted into the injured site of rat brain. They found 83 lncRNAs and 360 mRNAs between A2B5+iPSC and TBI groups differently expressed. A2B5+iPSC transplantation could effectively ameliorate neurological dysfunction in the brain tissue of rats in the TBI group. The mechanism of improvement is mainly related to the alteration of lncRNA and mRNA expression, specially ENSRNOT00000052577 lncRNA and its target mRNA, kinesin family member 2C (Kif2C). This study showed that ENSRNOT00000052577 and Kif2C were downregulated in the A2B5+iPSC group versus the TBI group  . Induced pluripotent stem cells (iPSCs) -derived A2B5ctively) . Anotherctively) .The first study about the function of lncRNAs in corneal transplantation was explored by Wen et al. in the rat models of corneal allograft and autograft. They identified 162 differentially expressed lncRNAs in the allograft compared to the autograft group, among which 142 were upregulated, and 20 were downregulated, and it seems these dysregulated lncRNAs involved in the occurrence of allograft rejection 14 days after corneal transplantation .via interfering with protein activity, stability, and localization. As an important prerequisite to figure out signaling pathways in acute renal rejection, several studies have investigated the interaction between transcription factors (TFs), miRNAs, and lncRNAs by an integrated bioinformatic analysis of related datasets as the first step toward better comprehending the regulation of gene expression in acute rejection (AR) following renal transplantation . Lorenzen et al. found that three intergenic lncRNAs, RP11-354P17.15-001 (L-328), RP11-395P13.3-001 (L-327), and LNC-MYH13-3:1 (L-321), were most strongly changed in the urine of patients with acute rejection. L-328 and L-327 have upregulated in patients who experienced acute rejection compared with stable subjects. Moreover, only the expression level of L-328 decreased in patients with successful anti-rejection therapy and was also associated with a higher reduction in glomerular filtration rate (GFR) one-year after transplantation. Surprisingly, 83.61% of patients experienced subclinical rejection  that were identifiable by L-328 expression level. Thus, in addition to aTCMR, the subclinical rejection which would have been missed by routine serum creatinine measurements could also be evaluated by L-328 level . In lineAdditionally, multiple transcriptomic studies have reported a large number of dysregulated lncRNAs in whole blood (AB209021 and AF264622) , peripheXIST lncRNA was highly expressed in the bronchoalveolar lavage fluid (BALF) and BAL cells of the primary graft dysfunction (PGD) cohort. XIST acts as a regulatory sponge for miR-21 and thereby increases levels of neutrophil-infiltrating factor, such as IL-12A, a target gene for miR-21. Ultimately, by NET formation, neutrophils accelerate PGD occurrence after lung transplantation. Furthermore, reduced expression levels of miR-21 in BAL cells of PGD patients than those of non-PGD ones confirmed the ceRNA function of XIST. Additionally, the expression level of miR-21 was also negatively correlated with the grade of PGD. Moreover, the protein levels of pro-inflammatory mediators, including IL-6, IL-8, CCL-2, and CXCL10, were elevated in the PGD patients compared to non-PGD. Whilst macrophages were the main leukocyte population in the BALF of non-PGD patients, the major leukocyte population in BALF of the PGD patients were PMNs .It has been discovered that polymorphism in ANRIL lncRNA is associated with the overall survival in adult patients with hematologic malignancies after HSCT. rs2151280 in ANRIL was related to the poorer overall survival in these patients. Conversely, none of the four investigated SNPs rs564398, rs1063192, rs2151280, and rs2157719 were associated with clinical outcomes, including the incidence of neurotoxicity, acute kidney injury (AKI), and graft versus host disease (GVHD), in these patients. The median time to death for patients who harbored rs2151280 TT was 379 days; whereas, it’s more than 1300 days for those having rs2151280 TC/CC. The underlying mechanism could be attributed to ANRIL-dependent transcription suppression on the ARF-INK4 gene. Furthermore, rs2151280 might serve as a potential prognostic biomarker for overall survival in patients suffering from hematologic malignancies after HSCT. .Many studies have widely explored the noninvasive biomarkers for allograft rejection. For example, urinary/circulating blood concentrations of soluble adhesion molecules, chemokines, cytokines, the lymphocyte expression of granzyme B, perforin, and Fas ligand (FasL), as well as the urokinase plasminogen activator receptor (uPAR) have been assessed previously as the potential biomarkers for the allograft rejection . HoweverAmong various diagnostics parameters, tissue biopsy is considered a gold standard for diagnosing allograft rejection. Nevertheless, it is highly invasive and a risky procedure. Moreover, sampling errors and interobserver variations can further complicate the diagnosis of graft rejection. Therefore, identifying accurate, sensitive, and noninvasive biomarkers is essentially needed to diagnose subclinical transplant rejection. Ideally, such biomarkers could also become the cornerstone of personalized therapy. They would allow clinicians to decrease immunosuppression in patients with a low rejection risk and/or high risk of neoplastic complications or infection and also adapt the immunosuppressive protocol in high rejection risk patients. Over the past few years, many of the ncRNAs have been demonstrated to play a major function in determining the allograft status and thus have shown promise as potential therapeutic targets. Among ncRNA families, the subfamily of lncRNAs have recently emerged as the lead targets in the area of transplant research, and widespread efforts have been made towards the clinical application of these RNA-based diagnostics for monitoring the allograft loss after transplantation. It seems that these molecules can overcome the need for diagnostic biopsy and identify patients with a poor prognostic outcome of the allograft. As mentioned previously, several lncRNAs in different types of organ transplantation have been studied so far, but the transition from discovery to validation status is a major challenge, and large multi-center prospective clinical trials are now significantly required for accelerating the implementation of lncRNAs into the clinic. However, according to the recent studies ,14,53, aNonetheless, it is necessary to note that the findings described in recent studies are merely a starting point for the comprehension of functions that lncRNAs may play in transplant rejection episodes. In this context, it is worth noting that only the function of a minority of lncRNAs is been shown presently and further studies are required to characterize the exact role of lncRNAs for clinical use in transplantation.SS: The design and conception of the study and revising the manuscript critically for important intellectual content. AS: The design and conception of the study and revising the manuscript critically for important intellectual content. SA: The design and conception of the study and revising the manuscript critically for important intellectual content. AA: The design and conception of the study and revising the manuscript critically for important intellectual content. YM: The design and conception of the study and revising the manuscript critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Pathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis. From there, ATP7B facilitates its transport in two directions: (1) into the trans-Golgi network for loading copper into proteins that require it, like the serum copper redox protein ceruloplasmin, which is unstable in the absence of copper; (2) from the liver into the bile canaliculus for the purpose of excretion1.The ATP7B gene encodes a copper transmembrane transporter vital for copper homeostasis. It is primarily expressed in liver and brain. In cells of these tissues, Cu2, while higher fractions of 1:7000 with 2.5% individuals carrying a causative allele have been reported in more recent analyses3. Untreated, the syndrome is fatal, but responds well to copper-reducing therapy if diagnosis occurs in time.Wilson disease is an autosomal recessive disorder of copper metabolism caused by inactivating mutations in the ATP7B gene (Omim # 277900). Deficiency in ATP7B causes accumulation of copper in liver and brain due to hampered excretion of excess copper, causing hepatic or neurological clinical symptoms. The global prevalence has originally been estimated to be 1 case in 30.000 individuals4. Phenotypic variation does not correlate well with genotype; inactivating mutations like nonsense and frameshift mutations confer a more severe phenotype than the missense alteration His1069Gln in terms of age at disease onset and ceruloplasmin plasma concentration8. This is congruent with the observation that His1069Gln does not confer a complete loss of transport activity9. His1069Gln and Met769Val, another partially active ATP7B variant, are common in European Wilson patients, making up almost half of all detected variants10.Timely diagnosis is frequently hampered by the phenotypical variability of Wilson disease. Patients present with either hepatic pathology or neurological dysfunction. The age of disease onset is mostly in the 2nd or 3rd decade of life but can vary broadly and may occur in infancy as well as in old age11. It is a bidirectional promoter located between the ATP7B and ALG11 genes  have been described to reduce promoter activity in reporter assays13. Some potential transcription factor binding sites for metal-responsive transcription have been identified and been shown to affect ATP7B transcription16.The majority of causative Wilson disease mutations has been identified in the protein coding sequence of ATP7B or its splicing sites, while data on a role of promoter alterations on Wilson disease are comparatively rare. The 1.3 kbp promotor of ATP7B has been cloned in 199917. Both are also supported by experimental data listed in the Database of Transcriptional Start Sites (DBTSS). Further evidence exists for a TSS at − 500 (DBTSS) and between − 600 and − 700 (Fantom5). Moreover, a TSS at − 330 has been suggested as well11. Transcription initiation in core promoters usually occurs from one focused TSS or several dispersed TSS, or, as seen in most genes, shows a mixture of both patterns18. Within the core promotor, the TSS is typically defined by DNA sequence, while the strength of transcription depends on additional factors19. For improving the understanding of the ATP7B promoter and its regulation, identification of its core promoter and TSS is of interest.Despite these investigations, data on the core promoter of ATP7B, as defined by the transcription start site (TSS), are heterogeneous Fig. A: some eWe therefore aimed at defining the core promoter of ATP7B that is most relevant for transcription initiation. We also characterized human genetic alterations located within or in proximity of this core promoter. These included two frequent polymorphisms whose impact on transcriptional activity was assessed with the idea that they may be able to modify Wilson disease phenotype in carriers of ATP7B mutations that are not fully inactivating.21  as compared to the whole amount of transcript (measured at the junction of exons 4 and 5 of ATP7B)  and likely uses two closely spaced TSS for regular ATP7B mRNA transcript formation.Analysis of the core promoter area revealed two spots of elevated average sequence conservation Fig. B. Three We introduced these alterations into a luciferase reporter plasmid pGL4.10 containing the 1kbp ATP7B promoter sequence. As negative control, the reporter plasmid without promoter was used. Linearity of the reporter assay measurements and homogeneity of the applied plasmid DNA amounts were extensively verified  reduction of transcriptional activity, which was 62% of the wildtype sequence  and demonstrated that all one single base substitutions (D2) conferred a significant , either polymorphisms individually (RP and PR) or both polymorphic sites (PP) : His1069Gln (Europe), Met769Val (UK), Arg969Gln (Greece), Met645Arg (Spain), Gly710Ser  and Arg778Leu (Asia)19. While direct effects of DNA sequence alterations on transcription are readily detectable in luciferase reporter assays, epigenetic effects are not reflected in this experimental approach. We therefore tested the biological consequences of the polymorphisms on copper household in 24 healthy probands by determining their polymorphic status and blood copper and ceruloplasmin serum levels.Transcriptional activity is on one hand directly conferred by the (core promoter and enhancer) DNA sequence, on the other hand indirectly by epigenetic effects like histone modifications that alter accessibility of the DNA sequenceAllele frequencies were 48% and 65% for rs148013251 and rs2277448, respectively, with 50% being homozygous carriers of the rs2277448  and assess the effects of human genetic variants located in this core promoter area. While experimental results from previous investigations and promoter database data have suggested numerous potential TSS for ATP7B dispersed over almost the whole 1 kbp promoter region  sequences -site that binds RNA polymerase II bp Fig. . Consequ35, suggesting no relevance for disease. The other two alterations (rs762339422 and rs759260854) are very rare, and clinical carrier information is missing, therefore clinical conclusions are not possible. rs762339422 (D2) displayed the most pronounced effect on expression. D2 has been observed in two Chinese Wilson disease patients without providing clear evidence for clinical relevance36 and has been previously suggested to affect gene expression based on prediction37. However, since the expression repression was rather moderate, it is unlikely to be causative.Of three single-nucleotide alterations located in conserved sequences within the ATP7B core promoter, one (D2) caused a significant repression of transcription. D1  is rare in most populations but occurs in 2% of AfricansThese observations suggest that small alterations in the core promoter do not suffice to abolish transcription enough to cause Wilson disease. This is consistent with the general observation that no causative genetic variants have as yet been reported in the core promoter area.38. Both are present in approximately half of the human population with a specifically high prevalence in Eurasians; our analysis suggests this may reflect their occurrence during migration of Homo sapiens out of Africa. While neither polymorphism can be causative for Wilson disease due to their high prevalence, their location within the ATP7B core promoter potentially enables them to alter ATP7B transcription to some degree, similarly as has been observed for the D2 sequence variant. The majority of Wilson disease patients carry missense alterations that can confer variable, often incomplete effects on protein functionality which consequently show a less severe clinical phenotype in contrast to the more rarely observed truncating mutations42. Therefore, the two frequent core promoter polymorphisms are candidate genetic modifiers of Wilson disease phenotype. The marked variability of the Wilson disease clinical phenotype that is even observed in carriers of the same mutation(s) has not yet been explained; several modifying influences have been suggested to contribute: besides environmental factors, epigenetic and transgenetic effectors and protein interactions of different missense variants are in the list44.Two frequent genetic polymorphisms are located in the core promoter area. These represent the majority of ATP7B 5’ UTR entries in the Wilson mutation database since their observation has been reported in many publications19, as has been described for ATP7B as well15. It has not been determined in this study if the polymorphisms may modulate distant promoter regulation. Moreover, we only investigated copper household clinical parameters as surrogate markers since direct examination of the association of the polymorphisms with ATP7B gene expression would have required a liver biopsy to assay ATP7B mRNA levels of carriers of different polymorphic status, which was not within the scope of ethical approval. The ultimate clinical clarification of the question if these two frequent polymorphisms modify Wilson disease phenotype would require a polymorphism-phenotype analysis in a large cohort of Wilson disease patients with two  causative mutations that have residual activity. However, the current study does not give evidence in favor of a modifying influence.However, neither luciferase reporter measurements suggested a significant functional impact of the polymorphisms, nor could a modulation of clinical copper household parameters detected. Consequently, a potential modification of Wilson disease phenotype could not be substantiated. However, some restrictions remain: for example, core promoters usually serve the general transcription from a gene, while more distant promoter elements may modify transcription strengthIn summary, we localized the transcriptional start site of the biologically relevant ATP7B core promoter and showed that one human genetic variant located in conserved areas of this core promoter has a reducing effect on ATP7B transcription, presumably without being of clinical effect. Moreover, we clarified the effect of two frequent polymorphisms in the core promoter and confirmed that they are unlikely to contribute to Wilson disease phenotypical variability.The investigation of the ATP7B genotype and the serum levels of copper and ceruloplasmine in 30 healthy volunteers was approved by the ethics committee of the University Hospital of Frankfurt (Goethe-University), #88/19 in March 2019. All volunteers gave written, informed consent. Peripheral EDTA blood was taken and gDNA was extracted using the DNA mini kit from Qiagen . Copper (photometric assay) and ceruloplasmine  serum levels were determined in the central laboratory of the hospital with a Roche Cobas 8000 analyzer.HepG2 hepatocellular carcinoma cells were purchased from DSMZ  in December 2019. Huh7 cells were kindly provided by Albrecht Piiper . Both cell lines were maintained in DMEM with 10% fetal bovine serum and antibiotics.pGL4.10 (firefly-luciferase without promoter) and pGL4.74 (renilla luciferase control vector using an HSV-TK promoter) were purchased from Promega, Wisconsin, U.S.A., and the pCR2.1 TOPO cloning vector was from Invitrogen, Carlsbad, U.S.A). The ATP7B gene luciferase promoter was amplified by PCR from Huh7 cell genomic DNA using standard conditions with the primers ATP7B-Promo-XhoI-F (5’-CTCGAGCTgctcacctcaacaacttgca-3’) and ATP7B-Promo-HindIII-R (5’-AAGCTTACggacaccgaattcttctctga-3’), which allowed to generate a fragment comprising 1126 bp of the promoter (NG_008806:4024-5149) with simultaneous introduction of XhoI and HindIII restriction sites appropriate for cloning the fragment into pGL4.10 in frame. The PCR product was subcloned in pCR2.1, resulting in pCR2.1-ATP7B-p. The ATP7B fragment was cut from this plasmid using XhoI and HindIII and transferred to pGL4.10. All constructs were verified by direct sequencing to be correct: the resulting pGL4.10-ATP7B-0 vector contained wildtype ATP7B genetic sequence (NG_008806:4024-5149) except for the presence of the two polymorphisms under investigation  and a single nucleotide variant (rs1427836170) located deep into the ATP7B promoter, 842 5’ of the transcriptional start, within an unconserved region. Further variants of the promoter were subsequently prepared by site-directed mutagenesis using appropriate primers  using the primers 5’-agctCTCGAGttctctccgt gttggttgcc-3’ and 5’-agctCTCGAGaataattttgataatatcccgtgg-3’ which introduced two terminal XhoI sites in the product. This product was inserted into the XhoI restriction site of the pCR2.1-ATP7B-p plasmid (see above).45. RNA acquisition and cDNA synthesis. Total RNA was extracted from growing Huh7 and HepG2 cells using the Trizol reagent  according to the manufacturers’ recommendations. Total RNA was dissolved in 30 µl RNAse-free water after ethanol precipitation. Cell harvesting and RNA extraction were performed within two hours, and RNA was subsequently either used directly for cDNA preparation or immediately stored at − 80 °C. In order to reduce handling times, residual genomic DNA was not removed since the reverse transcription was preceded by a DNA digestion (see below). RNA content was quantified on a Beckman DU-800 spectrophotometer. cDNA was created from 2 µg total RNA. This RNA had either been prepared fresh or from samples stored at − 80 °C for less than one month and not thawed more than twice. Since most qPCR probes tested ATP7B in the promoter, they were sensitive for gDNA contamination. Therefore, RNA samples (2.5 µg) were incubated with TurboDNAse I in a total volume of 20 µl at 37 °C for 30 min. Subsequently, DNAse I enzyme was removed with 2 µl DNAse inactivation reagent according to the manufacturers’ instructions . For assessment of the transcript levels in liver, we used a human liver RNA preparation . All DNA-free RNA preparations (2 µg) were incubated with 1 µg random hexamer primers  in a total volume of 28 µl at 70 °C for 5 min. After cooling on ice, reverse transcription was performed with 100 U M-MLV Reverse transcriptase  for 50 min at 55 °C. cDNA samples were stored at − 20 °C. qPCR quantification. For quantification of total ATP7B transcript, a predesigned TaqMan qPCR probe spanning exons 4–5 was used . For testing various locations (− 70/− 130/− 300/− 550 bp) of the ATP7B promoter, we used the gene expression custom assay design tool with the appropriate sequence fragments of the ATP7B genomic reference sequence of the promoter region for generating TaqMan probes. qPCR context sequences are shown in Supplementary Table Acquisition of samples, reverse transcription and qPCR have been documented according to the MIQE guidelinesFor assessing the relative abundance of transcript containing the different sequence motifs from the 5’-untranslated region of ATP7B, all Cq values were first normalized to the signal of the internal calibration plasmid ). In a second step, they were related to the signal of the ATP7B exon 4–5 area (ΔΔCq(e45)). Factors were determined using the standard formula 2-ΔΔCq.For transfection, cells were seeded at approximately 70% density into 96-well-plates (24.000 cells per well with 100 µl medium without antibiotics). Cells were transfected using Lipofectamine 2000 . Co-transfections were performed using pGL4.74 together with pGL4.10 with or without insertions of appropriate promoter sequences of ATP7B, each with 50 ng per well. Moreover, one co-transfection with an EGFP vector was performed in parallel for quick visual assessment of transfection efficiency. All plasmid preparations used were diluted with water to a final DNA concentration of 100 ng/µl to enhance precision of pipetted volumes. Master mixes were prepared of DNA and Lipofectamine, each diluted in Optimem according to the manufacturers’ recommendations. After incubation times as specified by the manufacturer’s instructions, both solutions were combined to achieve another master mix corresponding to a multiplicity of a final amount of 100 ng DNA and 0.2 µl Lipofectamine in 10 µl per well. Transfections were then performed with these mastermixes in triple or quadruple.48 h after transfection, luciferase expression was assessed using the Dual Glow Luciferase Assay System . Culture medium in the wells was replaced by 30 µl fresh medium. Firefly-luciferase reagent was added (25 µl), rocked and incubated for 10 min. Firefly luciferase activity was measured in a Perkin Elmer Envision analyzer equipped with luminescence filter (700 nm) and optical assembly. After detection of the firefly signal, 25 µl Stop-and-glo reagent was added, rocked and incubated for 10 min. Renilla signal was then measured. Reporter signal was calculated by dividing firefly signal by renilla signal, multiplicated with 100. In each experiment, 4–8 single, identically transfected wells were measured. Experiments were performed 2–10 times for each variant. Averages and standard deviations were calculated for each variant. Measured luciferase activities correlated well with the amount of used plasmid in test transfections  to 9 (highly conserved). For clarity in a graphical presentation, the moving average of 10 periods was created for these conservation scores 25, the Fantom5 database and the Eukaryotic Promotor Database (EPD) were used for annotating potential transcription initiation sites.For analyzing the promoter conservation of ATP7B, the genetic alignment of 91 mammals from the Ensembl platform was used. This alignment was applied to a ConSeq analysisSupplementary Figures and Tables


Mgl2 with increased H3K4me3 and H3K27ac in sperm were up-regulated in embryos, and vice versa for genes such as Dcn. Additionally, differentially occupied H3K4me3 and H3K27ac in sperm were linked to gene expression changes in both paternal and maternal alleles of 4-cell embryos. In conclusion, GLA-induced changes in sperm H3K4me3 and H3K27ac are concordant with gene expression in preimplantation embryos, which might further affect embryo development and offspring health.Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its male reproductive toxicity. Abnormalities in sperm histone modification induced by GLA exposure observed in our previous study aroused our interest in whether such alterations could further affect embryonic gene expression. Here we administered adult male mice with 0.2 mg/kg⋅day of GLA for 5 weeks to collect their sperm or 4-cell embryos after copulation. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing showed alterations of sperm H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are active histone modification marks involved in embryo development, while RNA sequencing identified differentially expressed genes in 4-cell embryos. Differentially H3K4me3 and H3K27ac occupied regions were mainly distributed at the gene promoters and putative enhancers, and were enriched in pathways related to the immune system and nervous system. Integrative analysis of these sequencing data showed that genes such as  Unlike ertility  and 3D certility . TherefoSperm histone modifications are susceptible to environmental factors, such as diet  and chemin vivo study on lizards observed GLA-induced severe testis lesions by oxidative damage , one of the most widely applied broad-spectrum herbicides, is highly hydrophilic and considered safe when properly used . Howevere damage , while aficiency , which ificiency  also shoIn this study, after administration of GLA at a dose of 0.2 mg/kg⋅day to adult male mice for 5 weeks, we performed Cleavage Under Targets & Tagmentation (CUT&Tag) to demonstrate genome-wide H3K4me3 and H3K27ac profiles in sperm, and RNA sequencing (RNA-seq) to display transcriptome in 4-cell embryos derived from GLA exposed male mice. By integrating these sequencing data, we tried to answer whether GLA exposure affects H3K4me3 and H3K27ac profiles of sperm and transcriptome of preimplantation embryos.C57BL/6J male mice aged 6–8 weeks  were housed in a constant environment, and they were randomly divided into two groups either for the control (CON) or GLA treatment after 1-week acclimation. The CON group had free access to ultrapure water, while the GLA group was treated with GLA  through drinking water at a dose of 0.2 mg/kg⋅day, which was equivalent to the acceptable daily intake (ADI) of 0.01 mg/kg  after con = 10 for each group) were sacrificed to collect mature sperm from bilateral cauda epididymidis as previously described . Embryos in M2 medium (at least 30 embryos in each replicate) were immediately used in the subsequent experiment.To induce superovulation, 3–4-week-old DBA/2 female mice were intraperitoneally administered 5 IU of PMSG, followed by injection of 5 IU of hCG 48 h later. After injection of hCG, each female mouse was mated with one CON or GLA mice, and sacrificed 56 h after hCG administration to dissect its oviduct. Then, we flushed 4-cell embryos from oviducts with M2 medium. Embryos from three female mice were pooled as a biological replicate, and three biological replicates were contained in each group  following manufacturer’s instructions. In brief, embryos in M2 medium were washed in PBS and transferred to sample buffer for lysis. Then, reverse transcription of mRNA transcripts was conducted to obtain full length cDNA. Products of cDNA amplification were purified with Ampure XP beads . Library preparation was performed with TruePrep™ DNA Library Prep Kit V2 for Illumina  following manufacturer’s instructions. After DNA quantification and qualification, all libraries were sequenced using NovaSeq, 6000  by Beijing Novogene Bioinformatics Technology Co., Ltd., China.After trimming, all RNA-seq reads were aligned to the mm10 genome using STAR v2.7.6a . All unmP-value < 0.05, and FoldChange > 1.5 were considered as differentially expressed.Then, we determined parent-of-origin of uniquely aligned reads with SNPsplit v0.5.0 , and theWe merged replicates of each group for downstream analysis, normalized read counts into RPKM by counting the numbers of reads in each 100-bp bin and then summed these counts within each 2-kb window for the entire genome using deeptools2 v3.4.3 . Genome-At first, we conducted CUT&Tag on sperm of the CON group and integrated H3K4me3 and H3K27ac data to display their physiological patterns . We perfTo enable functional interpretation of distinct patterns of H3K4me3 and H3K27ac, we annotated peaks in Cluster 1–3 to their nearest genes and identified biological pathways by GO enrichment analysis . ResultsPhlpp1 promoter of GLA sperm.Next, we integrated H3K4me3 of CON and GLA sperm into one track, as well as H3K27ac of the two groups, and then zoomed in representative regions of these three clusters . We founP-value < 0.05  at its promoter, which was concordant with increased mRNA transcripts on the gene body  and H3K27ac  were distributed at the promoter region of Hsd17b11, which might account for the significant decline of mRNA transcripts , and H3K4me3 signals at the promoter were also reduced in sperm. Overall, these overlapping regions indicated that paternal GLA exposure caused differential gene expression in 4-cell embryos, which might be associated with H3K4me3 and H3K27ac alterations in GLA sperm.Considering the contribution of paternal chromosome to the embryonic development, we hypothesized that GLA exposure induced sperm H3K4me3 and H3K27ac alterations might associate with aberrant gene expression in preimplantation embryos. Thus, we collected 4-cell embryos as a critical period after fertilization and performed RNA-seq to measure transcriptomic profiles in 4-cell embryos. It was revealed that 37 genes were significantly up-regulated and 53 genes were significantly down-regulated in GLA embryos . GO analMgl2 -left,E. gl2  and almost unchanged on maternal allele in GLA 4-cell embryos, which might be regulated by decreased H3K4me3 and H3K27ac at their promoters in sperm , which were concordant with decreased H3K4me3 and H3K27ac at their promoters in sperm  derived from C57BL/6J male mice or DBA/2 female mice. As shown in in sperm . As for in sperm . Taken tAs an emerging herbicide widespread in the environment, GLA and its metabolites have been detected in human biofluids such as serum  and urinGlobal profiles of sperm H3K4me3 and H3K27ac in CON mouse showed distinct distribution in genomic loci and biological pathways in each cluster. Sperm H3K4me3 is mainly distributed at developmental promoters , while HDespite of some constantly changed genes in both sperm and embryos, most genes marked with active promoters in sperm are not expressed until the embryonic stage. For example, monovalent genes marked with H3K4me3 are mainly active in late spermatogenesis, while bivalent genes marked with H3K4me3 and H3K27me3 keep silence until embryonic development . Though When it comes to sperm H3K27ac, evidence becomes more limited. By immunostaining, the protein level of H3K27ac was similarly elevated both in sperm and embryos of zebrafish after paternal exposure to bisphenol A (BPA) . Thus, iZfp81  can be found below: Genome Sequence Archive ; GSA: The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (1811056–2).XM: investigation, writing—original draft, reviewing, and editing. YF: software, formal analysis, and data curation. WX: investigation and validation. XD: investigation. WH: conceptualization, methodology, and supervision. YX: resources provision, funding acquisition, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


KRAB-ZFP) transcriptional repressors recruit TRIM28/KAP1 to heterochromatinize the mammalian genome while also guarding the host by silencing invading foreign genomes. However, how a KRAB-ZFP recognizes target sequences in the natural context of its own or foreign genomes is unclear. Our studies on B-lymphocytes permanently harboring the cancer-causing Epstein-Barr virus (EBV) have shown that SZF1, a KRAB-ZFP, binds to several lytic/replicative phase genes to silence them, thereby promoting the latent/quiescent phase of the virus. As a result, unless SZF1 and its binding partners are displaced from target regions on the viral genome, EBV remains dormant, i.e. refractory to lytic phase-inducing triggers. As SZF1 also heterochromatinizes the cellular genome, we performed in situ footprint mapping on both viral and host genomes in physically separated B-lymphocytes bearing latent or replicative/active EBV genomes. By analyzing footprints, we learned that SZF1 recognizes the host genome through a repeat sequence-bearing motif near centromeres. Remarkably, SZF1 does not use this motif to recognize the EBV genome. Instead, it uses distinct binding sites that lack obvious similarities to each other or the above motif, to silence the viral genome. Virus mutagenesis studies show that these distinct binding sites are not only key to maintaining the established latent phase but also silencing the lytic phase in newly-infected cells, thus enabling the virus to establish latency and transform cells. Notably, these binding sites on the viral genome, when also present on the human genome, are not used by SZF1 to silence host genes during latency. This differential approach towards target site recognition may reflect a strategy by which the host silences and regulates genomes of persistent invaders without jeopardizing its own homeostasis.Krüppel-associated box-domain zinc finger protein  a mechanism for directing constitutive heterochromatin and ii) a strategy that allows the host to use the same heterochromatin machinery to regulate an invader without deregulating itself. Heterochromatin mediates functions ranging from gene regulation and regulation of imprinting control regions and endogenous retroviruses to maintenance of stem cell pluripotency –4. HeterThough TRIM28/KRAB-ZFP complexes engage both self and foreign genomes, how KRAB-ZFPs recognize target sequences on DNA in the natural context of the genome remains poorly understood. Further, whether KRAB-ZFPs like SZF1 target host and foreign genomes in a similar manner is also not known. While binding sites of some KRAB-ZFPs have been informatically inferred from ChIP-seq experiments utilizing overexpressed and tagged KRAB-ZFPs , definedUsing ChIP-exo, a high-resolution in situ strategy on isolated pure subpopulations of EBV-infected cells harboring latent versus lytic virus, we mapped SZF1 footprints on both self and viral genomes. Our analysis of the footprints on cellular genomes revealed a motif that was able to regulate gene expression. Footprints bearing this motif contained a repeat sequence and were enriched on pericentromeric regions of chromosomes. In contrast, viral genomes demonstrated no footprints bearing this motif. Experiments using virus mutants confirmed that SZF1 instead uses distinct non-consensus sequences to silence lytic genes on viral genomes, thereby shifting the balance towards latency. We found that such lytic silencing via interactions between SZF1 and non-consensus binding sites on the viral genomes is also needed to establish viral latency and transform newly infected B cells. Overall, the number of SZF1 footprints on the cell genome did not differ greatly between cells harboring latent or lytic viral genomes. That said, we found SZF1 to be enriched at the latent origin of replication (oriP) in lytic cells, though not contributing actively to viral genome replication arising from the lytic origins. The abovementioned non-consensus SZF1 binding sites identified on the viral genome were preferentially enriched in latent versus lytic cells, pointing towards SZF1-mediated differential host gene programming during latent and lytic states. Remarkably, although these non-consensus sites also mapped to the cell genome, they were not used by SZF1 to silence the host during latency. Thus, compared to self-genomes, the SZF1-TRIM28 machinery silences foreign herpesviral genomes using a different targeting strategy that may allow the host to shield itself from collateral damage.+ tumor/transformed B cell lines that were available, we chose HH514-16, a well-studied Burkitt lymphoma (BL)-derived cell line because these cells are tightly latent at baseline but can be readily triggered to support the lytic phase. As shown in To identify SZF1’s footprints in the genomic context, we combined two approaches. The first is a cell-separation strategy that we developed to isolate latent cells in which EBV is refractory to lytic triggers from those that support the EBV lytic phase ,22–this BZLF1 promoter , BGLF4 (early lytic) gene, and BDLF2 (late lytic) gene, respectively, that demonstrated between 44% and 99% repression of GFP. In contrast, the others did not silence GFP; an example of one such site associated with BALF1 is also shown  can transcriptionally activate kinetically downstream lytic genes Cell extracts were electrophoresed in 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and blocked using 5% milk. Immunoblotting was performed using indicated antibodies at concentrations recommended by the manufacturer.+ and GFP- gates based on parallel untransfected control samples. As a transfection control in these experiments, cells were co-transfected with SignalSilence Control siRNA Cy5 Conjugate . FACS of HH514-16 cells for ChIP-exo was performed using a BD Aria II FACS instrument; again, staining and gate placement were performed as described in ChIP-Exo.HEK-293T cells were washed twice with 1x PBS and then subjected to flow cytometry. Flow cytometry data were acquired on a ThermoFisher Attune NxT Flow and analyzed using FlowJo software (Tree Star) with placement of GFPTotal RNA was isolated from HH514-16 cells by using an RNeasy kit (Qiagen) followed by DNase digestion (Promega). RNA was quantitated by using a NanoDrop instrument (Thermo Scientific). RNA (1μg) was converted to cDNA by using MuLV Reverse Transcriptase (New England Biolabs). Relative transcript levels of selected cellular genes were determined with gene-specific primers  and then Proteinase K overnight at 37°C. Absolute EBV genome copy number was determined with a standard curve obtained through qPCR using serially diluted BACmid p2089 as template. Primers targeting the EBV BALF5 gene were used to calculate relative released virus compared to wild type 293-BAC cells.Viral DNA in supernatant was measured using quantitative-PCR (qPCR) by amplifying EBV 6 primary B cells from three individual donors were infected at an MOI of 1  with each respective virus. Live cell counts were performed beginning on day 14 post infection.To assess changes in growth of primary B cells infected with wild type p2089 virus or SZF1-binding site mutant viruses, 5x10P values were calculated by comparing the means of two groups of interest using unpaired Student t test.S1 FigBZLF1p, BGLF4, and BDLF2. Coverage of the reads from lytic and refractory genomes were determined with Bedtools software (v2.30.0) and plotted in R. The lytic reads were normalized to refractory EBV genome copy number. Rightward and leftward strands are indicated by blue and red, respectively. Genome position numbers corresponding to the reference genome NC_007605 are indicated and validated SZF1 binding sites are indicated with brackets.Plots show read distributions at SZF1-binding sites and +/- 60 bp mapping to (TIF)Click here for additional data file.S1 TableTotal SZF1 ChIP-exo reads mapping to the human genome reference (Hg38/GRCh38) or EBV genome reference (NCBI Accession: NC_007605) for each experimental condition . Mapped and unique lytic reads were normalized based on EBV copy number in refractory cells.(XLSX)Click here for additional data file.S2 TablePeaks: overlapping clusters of reads with sigma = 5 exclusion zone = 10 (s5e10F1), or sigma = 20 exclusion zone = 40 (s20e40F1), excluding singletons. Singletons: peaks wherein all associated reads are located at a single chromosomal coordinate. Peak-median: median read count for all peaks, excluding singletons. Peak-mean: average read count for all peaks, excluding singletons. Median-std: median standard deviation for all peaks with standard deviation greater than zero. The standard deviation reflects the variance of read positions within each peak. A peak with a standard deviation = 0 defines a singleton peak and is excluded from further analysis. Mean-std: average standard deviation for all peaks with standard deviations greater than zero.SZF1 ChIP-exo sequence reads were mapped to either the EBV (NC_007605) or human (GRCh38) genome. Total indexed reads and unique read counts are shown for ChIP-exo sequence reads contributing to peak calling. (XLSX)Click here for additional data file.S3 TablePeak midpoint positions mapped to the EBV reference genome (NC_007605), listed in descending order based on number of contributing midpoint sequences. Immediate early, early, and late refer to kinetic classes of lytic genes; CDS, coding sequence. Sites corresponding to the bolded genes were analyzed via GFP repression assay in (XLSX)Click here for additional data file.S4 TableSZF1 binding sites within the EBV genome were mapped to the human genome with the requirement of 12 contiguous nucleotides sharing 100% identity. Neighboring genes within 100 kb of the potential binding sites, with the potential to be regulated via SZF1 binding, are shown. Sites with validated presence in peak-pair sequences, i.e. footprints determined from the SZF1 ChIP-exo data for each condition  are also shown.(XLSX)Click here for additional data file.S5 TableSequences derived from the top 27 motifs identified from Meme-suite analysis of SZF1 ChIP-exo read libraries mapped to Human (GRCh38) or EBV (NC_007605) genomes. Note: Fine grain peak-calling when sigma (s) = 5, exclusion zone (e) = 10; Coarse grain peak-calling sigma (s) = 20, exclusion zone (e) = 40; Untreated Human s5e10 and Lytic EBV s5e10 resulted in no motif hits. e values in bold indicate validated Motifs 1 and 2 while e value highlighted in grey indicates Motif 3 depicted in (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 TableStart and end positions of motif 1 consensus (ATGGAATCGAATGGAATC) clustered on chromosomes 16 and 5 of the human genome (GRCh38) are shown along with any genes found within 100 kb upstream or downstream of the motif clusters. Additional SZF1 motif 1 clusters are shown in (XLSX)Click here for additional data file.


Strongylocentrotus purpuratus. The most prevalent among these elements were the unknown elements—in total, they contributed 78.5% of REs . It was revealed that the transcription pattern of genes and REs changes significantly during gastrulation. Using the de novo transcriptome assembly, we showed that the expression of RE is independent of its copy number in the genome. We also identified copies that are expressed. Only active RE copies were used for mapping and quantification of RE expression in the single-cell RNA sequencing data. REs expression was observed in all cell lineages and they were detected as population markers. Moreover, the primary mesenchyme cell (PMC) line had the greatest diversity of REs among the markers. Our data suggest a role for RE in the organization of developmental domains during the sea urchin embryogenesis at the single-cell resolution level.Repetitive elements (REs) occupy a significant part of eukaryotic genomes and are shown to play diverse roles in genome regulation. During embryogenesis of the sea urchin, a large number of REs are expressed, but the role of these elements in the regulation of biological processes remains unknown. The aim of this study was to identify the RE expression at different stages of embryogenesis. REs occupied 44% of genomic DNA of  Eukaryotic genomes are larger and more complex than those of prokaryotes, and they also significantly differ in size. However, there is no correlation between the genome size and the organism complexity. For example, the genome size of some amphibians, bony fish, and some arthropods exceeds by twice or more the size of the human genome, however, we can hardly claim the same order of complexity for these organisms ,2,3,4. IAll repetitive DNA in the genome is grouped into two large classes according to the genomic organization: tandem repeats and dispersed repeats or transposable elements  ,9. AlthoIt is known that most of the mammalian and other studied eukaryotic genomes are transcribed during normal embryonic development ,15. The A deeper understanding of RE function has been limited by difficulties in analyzing the RE transcription in RNA-seq data. Analysis of RE transcription is challenging for short-read RNA sequencing due to their highly similar sequences and repetitive nature. RNA-seq reads that originate from one locus can be ambiguously aligned to many TEs sharing similar sequences dispersed throughout the genome. Due to these barriers, conventional RNA-seq analyses of REs have either discarded multi-mapping alignments  or combiDrosophila melanogaster brain cells. The authors looked at chimeric transcripts containing transposons and mRNA and concluded that transposon expression was driven by neighboring genes, and this diversified the neural transcriptome [Caenorhabditis elegans early embryo have shown that LTR elements are mostly expressed in undifferentiated cells and might play a role in pluripotency maintenance and activation of the innate immune response. Meanwhile, non-LTR are expressed in differentiated cells, in particular in neurons and nervous system-associated tissues, and DNA TE are homogenously expressed throughout the C. elegans early embryo development [Moreover, the analysis of RE expression in scRNA-seq data becomes even more complex. Several studies have made decent attempts to analyze RE expression at the level of single cells. For example, RE expression at the single-cell resolution was detected in criptome . TE exprelopment . It shouHe and co-authors managed to capture specific RE types that were expressed in subpopulations of human embryonic stem cells and were dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. In addition, RE expression was detected in somatic cells, including human disease-specific REs that are undetectable in bulk analyses . A constin vitro cultivation of an embryo for more than two weeks according to the “14-Day Rule”. In our opinion, the purple sea urchin Strongylocentrotus purpuratus is a convenient model for studying epigenetic regulation based on noncoding RNAs. Sea urchins have been used by biologists as a model organism for over 150 years [Experimental research on human embryonic development is complicated by ethical issues and the impossibility of 50 years . In addi50 years ,30. TherUsing this model organism, Davidson and colleagues were able to present the first experimental validation and systematic description of the gene regulatory network that controls the specification of endoderm and mesoderm for sea urchin embryogenesis . The netS. purpuratus genome build. Using bulk transcriptome data at different timepoints, we revealed the transcription pattern of those REs from the unfertilized egg to the late gastrula stage. Considering the bulk RNA-seq-derived data on the activity of each of the RE instances and scRNA-seq data, we also discovered several RE-derived transcripts that are important for the formation of main developmental domains of the sea urchin embryo. This data may be later used to identify new mechanisms of ncRNA-dependent modulation of transcription during the embryogenesis of sea urchin and other deuterostomes.The evidence concerning gene regulatory network (GRN) in single-cell resolution is only starting to emerge, and none of the existing experiments have been made while taking into account the highly repetitive nature of deuterostomes’ genomes. In this study, we refined the repertoire of the REs of the latest S. purpuratus embryos were downloaded from the SRA database under accession number PRJNA531297. The data contained RNA-sequencing reads of unfertilized eggs, embryos from 2, 4, 8, 18, 32, 64, and 128 cell stage (blastula stage) and 24, 40, 41, and 48 h after fertilization (gastrula stage), which were maintained at 15 °C.The publicly available transcriptome data from S. purpuratus, from the eight-cell stage to late gastrula stage.Raw reads of the single-cell RNA-sequencing data were downloaded from the SRA database stored under accession number GSE149221 . The datS. purpuratus genome version 5.0 were identified and classified by RepeatModeler 2.0.1 using the Dfam 3.1 database. Consensus sequences of repetitive elements (consensi.fa output file) were used for downstream analysis of differentially expressed genes. The RepeatMasker was used for locating the discovered REs in the genome.Repetitive elements in For the RNA-seq data, gene expression abundances were computed using the Kallisto software (0.46.0) with accompanying sleuth R package (v0.30.0). Transcriptome reference files for pseudoaligment were prepared by concatenating reference transcriptome (GCF_000002235.5_Spur_5.0_rna.fna) of protein-coding genes with consensus sequences of repetitive elements, identified by RepeatModeler (consensi.fa output file).Identification of transcriptionally active RE instances was performed using TEcandidates v.2.0.5., which assesses the active RE instances based on de novo transcriptome assembly with Trinity. TEcandidates takes as input the RNA-seq data (fastq.gz), the genome sequence (Spur_5.0.fa), and the RE annotation file (Spur_5.0_genome.fna.out.gff). RE annotation file was produced by RepeatModeler and contained coordinates of all RE copies. TEcandidates returns a list of coordinates of the candidate REs being expressed, the REs that have been removed, and the genome sequence with removed REs as masked. This masked genome is suited to include REs in downstream expression analysis, as the ambiguity of reads coming from REs is significantly reduced in the mapping step of the analysis. We used the list of expressed RE for constructing reference transcriptome for the further steps. The RE instance was included in the resulting annotation only if it had been expressed in at least one embryo stage.Considering the repetitive nature of non-coding DNA, we chose the pseudoalignment approach with expectation-maximization RE resolution. This approach was implemented in the Kallisto Bustools suite and was used for single-cell primary analysis . The strhttps://github.com/larisaok/Repeats_in_sea_urchin_genome, accessed on 30 September 2021.The computer code used to generate the results presented in this manuscript is available at Helitron transposons. Repeats in total occupied 33.75% of the S. purpuratus genome. The most prevalent were the unknown elements, which in total contributed 78.5% of repeats   . The secnome see . InteresS. purpuratus species origination. As can be clearly seen, the maximum genome portion was shifted and laid at 5–6% of Kimura substitution level.The Kimura plot depicted in S. purpuratus.As previously unknown repeats occupy most of the genome of sea urchin, their portion in any of the bars would be the largest one. From the shape of the bar plot, we can conclude that the majority of the repeats do not actively replicate in the genome. If the TEs were actively jumping, the distribution peak would shift to the left. In this species, it is highly likely that repeats have undergone active expansion in the near past. This assumption is correct for the Class II TEs and for those unknown. In contrast, DNA transposons have maximum abundance at the 1% level, and this can indicate that they are expanding in modern Analysis of the bulk RNA-seq data revealed that 404 REs significantly changed their expression at the gastrulation stage. It is worth noting that the number of upregulated REs (344) was significantly higher than the downregulated REs (60) b. Here, Next, we assessed the pattern of RE expression. As can be seen in z-value = 160.98, p-value < 2.2 × 10−16). Furthermore, there was no correlation between the sequence length of the RE and copy number . Taken together, this result may indicate that there are additional mechanisms controlling high-copy RE transcription.Subsequently, we evaluated the association of expression rate with the copy number of RE in the sea urchin genome with TEcandidates. We pooled together all the available stages in RNA-seq data and extracted RE instances that were being expressed in at least one of the assayed stages. The scatter plot illustrating the connection between the copy number of the RE in the genome and the number of their transcribing copies c shows tHistone H1   .The 5th cluster was highly likely to have originated from the endomesoderm cell lineage because of the specific marker, which marks the secondary mesenchyme domain . There wLisitsa and hydraulic elements were expressed in all clusters except for the 3rd, and Lariok RE was not expressed in the 5th cluster. Named RE sequences were queried against the RNAcentral database, and they were shown to match several previously described sea urchin lncRNAs sequences  expression, which has a tropism to ectoderm. Both of these clusters had the rnd-4/349 element as the marker; however, in cluster 1, it is the negative marker, while in the 2nd cluster, it is the positive one  of FoxA and Endo16 expressed cells. Both of these genes were the specific markers of the endoderm . Taken together, these facts exclude the oral ectoderm as the affiliation for this population in favor of endoderm. This population had a few REs among the markers—two positive and one negative, and four positive and two negative in the 2nd and 3rd clusters, respectively . They form the skeletal spicules of the larvae. The ingression starts at the hatched blastula stage, and these cells are shown to originate from the large micromere cells.COLP4alpha, Alx1, C-lectin protein, MSP130-2, SM37, and SM50. Approximately ~50% of this cluster expressed MSP130-1 and P16. A small percent of cells also expressed SM30. All of these genes were defined as confident markers. Furthermore, this cluster comprised seven REs as the positive markers and nine as the negative. The largest portion of the cells of this cluster expressed the Kotleta element (~25%).At the HB stage, cluster 7 represented the PMC with ~75% of its cells expressing several specific markers: At the MB stage, the 10th cluster obviously represented the PMC d. It hadCompared with this population at the previous embryo stage, the 11th cluster in EG had more spicule-specific genes among the markers. The number of REs among significant markers also dramatically increased—13 downregulated REs and 24 upregulated REs.Although the transcription of genes was similar to the EG stage, the repertoire of the RE expression dramatically shrunk—only six REs were upregulated and one was downregulated in the 10th cluster of the LG stage .At the latest stage of archenteron invagination, the secondary mesenchyme cells (SMC) form the tip of the archenteron and provide the contact of the archenteron and inner blastocoel wall .rnd-6/1309 served as a marker of this cluster and was expressed in the largest portion of cells when compared with other clusters.At the HB stage, it is highly likely cluster 3 represented the precursors of the SMC c. This aCol4Alpha. The combination of these markers suggests an endomesoderm cell population, which is most likely to be the SMC. There were also three upregulated REs in this cluster .The izations . TherefoGRNs play the role of a conductor of molecular processes of cell differentiation and morphogenesis during embryogenesis ,46. TheyTranscription of the zygotic genome in sea urchin embryos begins soon after egg fertilization ,48. AccoPrior to the sequencing of the sea urchin genome, a preliminary endomesoderm GRN was assembled based on several transcription factors and signaling molecules . A detaiThe early embryonic development of the sea urchin is of particular interest as an in vivo model of the epithelial–mesenchymal transition (EMT), which is an evolutionary-conservative cellular program ,54. The pmar1, alx1, ets1, hesC, tbr, dri, hnf6, and snail [In view of the widespread interest in the EMT mechanisms, GRN that controls the differentiation micromeres—PMCs—has been well documented ,55. Prognd snail ,57,58,59As mentioned above, the GRN’s design is based on the generalization and analysis of protein-coding gene transcription data. However, for many organisms, including humans and other mammals, it is shown that noncoding RNAs are the major players in the gene transcription regulation system .S.purpuratus [In the course of embryogenesis, transcription of the total genome occurs , which rrpuratus ,61, the It has been shown that repetitive sequences of genome, include transposons, can serve as a source for noncoding RNAs in evolution .S. purpuratus, while 2588 and 1915 miRNA sequences were described in humans and mice, respectively [The TE-derived miRNAs regulate numerous biological processes and are present in all bilaterian animals. According to the miRNA online database (miRBase.org), there are 70 sequences ,63,64 inectively . It was ectively . No suchThe TE–derived long non-coding RNA in sea urchin, LncRNA and lincRNA , are involved in many cellular processes ,68, inclLncRNAs are less conservative than miRNAs. It has been shown that most of the vertebrate lncRNAs are sufficiently specific only within taxon. This can be explained by their variability and fairly rapid evolution . It was About one thousand lncRNAs described for humans have homologues in other mammals, but only hundreds of lncRNAs have homologues outside the taxon . Humans Most lncRNAs, including lincRNA, contain TE sequences . AccordiThe new repetitive sequences described in our study range in size from 75 to 494 bp, but, at the same time, they are not independent transcripts and are parts of longer molecules in the form of a domain see  Table S2Since the gene regulatory network responsible for the differentiation of primary mesenchymal cells has been described in sufficient detail, we can assume that a systematic overlay of the regulation of the transcriptional level of these lncRNAs will provide insight and a new perspective on the combined effect of transcriptional and post-transcriptional regulation during the isolation of the PMC population.In the source paper, 22 cell types were identified in the merged dataset at the gastrula stage; this corresponds well to the established fate maps of urchin embryos ,42,50. TIt turned out that the expression of RE can be specific to cell populations and persists between stages. If they expressed transcriptional noise, they would not be defined approximately anywhere as cluster markers . HoweverNevertheless, thanks to our approach we were able to show in principle that REs are important in embryonic development and, on a par with protein-coding transcripts, can determine the fate of cells in embryogenesis.As forspecific features, different numbers of REs among the markers were also found depending on the population and the stage of embryonic development. The most interesting phenomenon in this case was the population of mesenchymal cells—they had the highest number of REs. There can be two opposite explanations for this.The first explanation is that these are terminally differentiated cells, which means that there is no need to preserve multipotency, and the mechanisms involved in suppressing transcription do not work. This is supported by the existence of mechanisms to suppress TE activity, which are particularly active in germline cells.This idea is opposed by another explanation, suggesting that in the cells of this population, on the contrary, there is a modulation of transcription due to these REs. For example, their transcription is associated with some regulatory elements or, on the contrary, with the suppression of gene transcription. It may also happen that one copy of the RE is expressed suppressing the others. Meanwhile, this hypothesis can only be tested experimentally, which was not provided within the framework of this paper.S. purpuratus 5.0 genome assembly. Using the alternative WindowMasker pipeline, they masked 39% of the genome. It is noteworthy that a masked portion in the genome is not a good subject to compare between different taxa because the percentage of ncDNA varies greatly even between neighboring species [As a result of the RepeatModeler + RepeatMasker pipeline run, we found that 44% of the urchin genome was occupied by repeats, and most of them (78.74%) belonged to previously unknown families. Currently, the classification of RE is too complicated due to the limited number of well-assembled genomes. The DFAM consortium continuously develops new approaches to classification and updates consensus sequences of REs. Our result agrees in general with the result of the authors of the latest  species Drosophila) and to “tame” such repeats . In our study, we revealed the co-option of the REs in embryo development. According to our assumptions based on the analysis of the RE repertoire, we can say that a massive expansion of REs occurred in sea urchin in the recent evolutionary past. Perhaps this continues today within a narrow group of DNA transposons. It is worth noting that a careful classification of the other families may shift the estimates of the timing of this expansion, but it is unlikely to dramatically change the overall picture. Summarizing the resulting data, we can say that it is possible that further investigations will reveal new mechanisms of REs in the genome and modulation of transcription.RE expansion is a phenomenon that can work as an evolutionary factor. Without control, REs can expand and disrupt gene functions. On the other hand, organisms have learned both to resist expansion  REs significantly change their expression during the gastrulation process; (2) the expression of an RE is independent of the number of its copies in the genome; (3) RE expression distinguishes cell lines from the morula stage (4) expression of RE is observed in all cell lineages and they are detected as population markers at a single-cell resolution level. The pattern of RE expression can vary between cells of different lineages.