IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-β binding motif, prevents binding of C/EBP-β, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-β expression in myeloid cells.Interferon β (IFN-β) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene  Ifnb1 expression in myeloid cells. The most active enhancer of this locus is conserved in humans, but presents a frequent variant found in around 30% of the population worldwide. This variant prevents binding of the C/EBP-β transcription factor, and is associated with decreased expression of IFNB1 in activated monocytes. When mimicked in the murine enhancer, it abolishes its inducible enhancer activity. Our results describe the molecular link between a point mutation and a cellular phenotype that could influence clinical situations.Genome-wide association studies identify multiple genetic variants associated with higher order phenotypes. Pinpointing the causative variant and understanding its molecular mode of action is a complex task. Using a murine model of interferon-β transcriptional deregulation, we characterize a super-enhancer controlling  IFNB1, is rapidly induced upon viral infection through multiple pathways sensing virus-derived nucleic acids [The induction of type I interferons, and in particular of interferon beta (IFN-β), is an essential step of the antiviral response . The traic acids . The relic acids ,4, that ic acids . It promic acids ,6, and hic acids . Interesic acids –12.IFNB1 transcription has been the subject of intense research.The timing of interferon expression is of primary importance for the immune response. For instance, the relative timing of IFN-β stimulation and T cell receptor (TCR) activation decides the targeted T cell response . If the IFNB1 transcriptional control, including NF-κB (p50:relA), ATF2:c-jun, and IRF3/IRF7 [IFNB1 gene were described to positively or negatively regulate IFNB1 transcription by fixing factors such as NF-κB [IFNB1 gene that loops to the IFNB1 promoter, binds phospho-IRF3, and is required for IFNB1 induction upon virus infection in fibroblasts [Ifnb1 transcription at the end of activation by lipopolysaccharide (LPS) or poly(I:C), targeting Toll-like receptors 4 and 3, respectively [Ifnb1 Control Element. Knocking out Trim33 in macrophages leads to an increased binding of the acetyl-transferase CBP/p300 on the Ifnb1 promoter, an increased acetylation of histone H3, and an over-expression of Ifnb1 at late stages of activation [The pioneering work performed in the laboratories of T. Maniatis and D. Thanos allowed to identify the enhanceosome, a promoter proximal structure that serves as a docking site for the cooperative binding of several transcription factors involved in RF3/IRF7 –17. Seveas NF-κB , XBP-1 [as NF-κB , YY1/2 [as NF-κB ,21, or βas NF-κB . In partroblasts . We haveectively . We havetivation .Ifnb1 expression in myeloid cells [TRIM33 belongs to the TRIM family of proteins, that harbor at their N terminus a TRIpartite Motif composed of a Ring domain followed by 2 B boxes and a coiled-coil domain, and that generally carry an ubiquitin ligase function . TRIM33,id cells , the preIfnb1 promoter and ICE to a region overlapping the 100 kb downstream gene Ptplad2/Hacd4. We characterize this region as a myeloid super-enhancer. In addition, we identify the human orthologous region as a locus harboring several single nucleotide polymorphisms (SNPs) previously associated with quantitative differences in IFNB1 expression in human monocytes activated with lipopolysaccharide (LPS). The minor allele of the most associated SNP disrupts a conserved C/EBP-β binding site and prevents C/EBP-β binding. In a human context, or when mimicked in the murine sequence, it blocks the inducible enhancer activity of a DNA fragment encompassing the C/EBP-β binding site. Our results identify a new conserved IFNB1 enhancer, and functionally link its polymorphism to a cellular phenotype.In this manuscript, we show that the absence of TRIM33 in activated murine myeloid cells correlates with an increased looping of the Ifnb1 expression by TRIM33. We first tested whether TRIM33, CTCF and cohesin co-localized on the chromatin of myeloid cells. We performed ChIP-seq targeting CTCF and RAD21, a subunit of cohesin, in the murine macrophage cell line RAW264.7 cells, and aligned the observed peaks with TRIM33 peaks [Ifnb1 regulatory region (Ctcf-/- and wild-type (WT) bone marrow derived macrophages (BMDMs) [Ctcf-/- macrophages included ‘cellular response to interferon beta’  (Two reports have used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to identify chromatin peaks bound by both TRIM33 and the CCCTC binding factor (CTCF), a DNA looping organizing protein, in embryoid bodies  and male33 peaks . Nearly y region . We went (BMDMs) . Gene on0-5; -5) . These aIfnb1 gene was not modified in Trim33-/- macrophages [Ifnb1 in macrophages, we analyzed these data on a larger scale , we havrophages . To idenrophages B. Because latter . This suIfnb1 promoter driving transcription of the firefly luciferase in pGL3-basic [Ifnb1 promoter alone   G. Becaus.m., −2)  or LPS-i.m., −2)  activitiIFNB1 in LPS-activated monocytes [IFNB1, we precisely mapped them in a region covering 2 Mb around IFNB1. We performed this analysis jointly on individuals of African or European descent, in order to break population-specific haplotypic blocks, and allow a finer resolution of the mapping of causal variants [PTPLAD2 gene, including a set of 17 SNPs significantly correlated with the expression level of IFNB1 in LPS activated monocytes . The strongest association with IFNB1 expression was observed for variants rs12553564 and rs12551341, including the previously reported rs2275888 (r2 = 0.96) [Ensembl v80 database. Third, rs12553564 is overlapped by experimentally defined transcription factor binding sites established by the Encode consortium. Fourth, the nucleotide affected in rs12553564 is located at a position conserved across mammals as determined with the GERP++ tool (GerpRS score > 2). And fifth, rs12553564 overlaps several transcription factor binding sites that are conserved across mammals. Although these results do not exclude that other linked variants contribute to the observed phenotype, they prompted us to prioritize rs12553564 for further analysis.We and others have identified single nucleotide polymorphisms (SNPs) in the human population that are associated with differential expression of onocytes ,46. Thesvariants . The pea12553564 , suggest = 0.96) . Among tPTPLAD2. We observed that no super-enhancer was predicted in human myeloid cells over PTPLAD2 [PTPLAD2 region loops to IFNB1  and R848 (targeting the TLR7/TLR8 receptors), but not after infection with an influenza virus , in the third intron of  PTPLAD2 . We anal PTPLAD2 , and fouarrow inC. This larrow inC, a clasza virus . Upon Paed Genes . These rIFNB1 expression and a human polymorphism in FIRE suggested that a single nucleotide substitution was sufficient to affect FIRE enhancer function. We sought to determine whether this could be due to decreased binding of a transcription factor. We first assessed the impact of the rs12553564 variant on transcription factor binding motifs. The A to G substitution was predicted to change the binding of 26 transcription factors on the rs12553564 region . Again, the G allele did not modify the constitutive activity of the enhancer , together with the relatively similar haplotype length of both rs12553564 alleles in all populations  in compliance with the European Union legislation and the Ethics Committee of the CEA (CETEA agreement #A14_082). For experiments with human monocytes, buffy coats from anonymous healthy donors were obtained at the Etablissement Français du Sang (EFS) after informed consent. Compliance with relevant ethical regulation was controlled by EFS.+ monocytes was obtained from 200 individuals of either African (N = 100) or European (N = 100) ancestry, after 6h of stimulation by LPS (N = 184 samples) or rest. After correction for batch effects and technical covariates (GC content and 5’/3’ bias), log2-transformed FPKM were used for eQTL mapping. Genotypes were obtained for 9,166 common variants (Minor Allele frequency > 5% in either population) from the IFNB1 locus (<1Mb from IFNB1), based on genotyping with Illumina HumanOmni5-Quad beadchips, exome sequencing using Nextera Rapid Capture Expanded Exome Kits, and imputation with IMPUTE v.2 [IFNB1, we ran the MatrixEQTL R package [Mapping of eQTLs was performed using previously published gene expression and genotype data . BrieflyPUTE v.2 . Only vaPUTE v.2 . To map  package  on both  package .2 of nearby genetic variants with rs12553564 was computed across all individuals (100 of African-descent and 100 of Europeans descent). To annotate genetic variants, we retrieved regulatory elements predictions from the Ensembl Regulatory Build v80 [http://mendel.stanford.edu/sidowlab/downloads/gerp/hg19.GERP_scores.tar.gz).Peak eQTL was defined as the most significant SNP across 5 conditions, when combining both European and African indivudals. ruild v80 , and oveuild v80 , and sithttp://hgdownload.cse.ucsc.edu/goldenPath/hg19/multiz46way) and used to establish the regions that are orthologous to the IFNB1 locus (hg19 –chr9:20-22Mb) in mice (mm9) and 44 other verterbrate species. To detect TFBS that are conserved across species around each SNP, we used the searchSeq function from the TFBSTools package to identify TFs with a predicted binding score of >85% of their maximum PWM matrix value across all 46 orthologous sequences in a 21bp window around the SNP, and specifically retained the TFBS that overlapped the human variant. TFBS that were detected in >20 species were considered as conserved. We then assessed the impact of rs12553564 on the predicted binding of the 15 TFs with a conserved binding site at the locus, by comparing the predicted binding score of the two alleles and focusing on the 6 TFs whose binding score decreased by >10%. Of these, only CEBPB was highly expressed (FPKM>100) in our monocytes gene expression data, leading its prioritization for further experiments.All available position frequency matrices for human transcription factors (TF) were retrieved from the JASPAR CORE 2018 database , and onlTrim33 deletion in myeloid cells were maintained as Lyz2tm1(cre)Ifo/tm1(cre)Ifo, Trim33fl/fl, and Lyz2tm1(cre)Ifo/tm1(cre)Ifo were used as controls.Mice with a 2 in a humidified atmosphere. Bone Marrow Derived Macrophages (BMDM) were obtained as described [6 cells per dish (10 cm cell culture treated) in BMDM medium supplemented with 25 ng/ml mouse CSF1 (Miltenyi), and incubated for 7 days with complete medium changes at days 3 and 6. Incubation with LPS (Sigma #L4516) were performed for 24 h at 100 ng/ml in BMDM medium supplemented with 2.5% FCS. RAW 264.7, NIH3T3, and EL4 cells were grown in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FCS and 1% PS. In these cell lines, mycoplasma contamination was checked every 6 months with the MycoAlert detection kit (Lonza). Peripheral blood mononuclear cells were prepared by Ficoll density centrifugation  of buffy coats from anonymous healthy donors obtained at the Etablissement Français du Sang (EFS), and frozen in SVF supplemented with 10% dimethyl sulfoxide. Approximately 106 cells were retained for genomic DNA purification , and genotyped for rs12553564 and rs12551341 with snp genotyping taqman assays (ThermoFisher). After thawing and washing, CD14+ cells were purified by labelling with CD14 microbeads and magnetic isolation on LS columns (Miltenyi), according to manufacturer’s instructions. Purity was checked by CD14-PE (Miltenyi) labelling and analysis on a Guava easyCyte 8HT cytometer (Millipore). They were then directly processed for ChIP, or differentiated into macrophages in the presence of 50 ng/ml human M-CSF (Miltenyi) as described [All incubations were performed at 37°C in 5% COescribed . Brieflyescribed .ChIP experiments were performed as described . BrieflyIfnb1 promoter has been previously described [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) . The human IFNB1 promoter was inserted in front of the luciferase gene in pGL4.12 by Sequence and Ligation Independent Cloning (SLIC). A fragment of human genomic DNA centered on rs12553564 was amplified by PCR from THP-1 genomic DNA  and inserted in front of the promoter by SLIC, and then mutated to the G allele by SLIC. All constructs were sequenced (Eurofins), and primers can be found in The vector encoding firefly luciferase under the control of the murine escribed , and is cgi) see . They weST values, and population-wise iHS, Tajima’s D, and allele frequency correlation (β scores). iHS and Tajima’s D were computed based on 100 kb around the SNP of interest. The derived allele of each SNP was defined based on the 6-EPO alignments and only SNPs with a derived allele frequency (DAF) between 5% and 95% were analysed. iHS scores were normalized in 40 separate bins of DAF. Βeta scores for all 1000G populations were obtained from http://coruscant.itmat.upenn.edu/data/SiewertEA_Full_BetaScores.tar.gz. These β scores are based on a 1 kb window around the SNP (500 bp on each side). For each statistic, an empirical p-value measuring evidence for positive/balancing selection at the rs12553564 variant was computed. Namely, the empirical p-value was obtained as the rank of the observed value of FST, Tajima’D, |iHS| or β among all other values observed on chromosome 9. Time of most recent common ancestor (TMRCA) of haplotypes at the rs12553564 locus was estimated using ARGweaver. We downloaded phased VCF from 1000 genomes (1 kG) phase 3 and extracted all variants in a 10 kb region around the rs12553564 locus (5 kb on each side). After filtering out variants with a MAF < 1% across all populations, ARGweaver was run with default values for effective population size of (Neff = 10000), and mutation rate (mu = 2.5e-8), a total of 20 discrete time steps and a maximum time of 200,000 generations. Compression factor of 100 was used to speed up calculations. Genome wide distribution of TMRCA values was obtained by applying ARGweaver with the same parameters to 100 randomly sampled 10 kb regions along the genome.To evaluate the evolutionary mode of the locus, we focused on populations from the 1000 genomes project phase 2 and performed selection scans on the entirety of chromosome 9, considering global AMOVA-based FHigh-throughput sequencing results have been deposited to Gene Expression Omnibus (GEO) with accession number GSE137514. The accession numbers of publicly available data used in this study are detailed in S1 Fig(A) Alignment of ChIP-seq signals for CTCF, RAD21 and TRIM33 in RAW264.7 cells on the peaks bound by either of these proteins, after clustering with SeqMiner. (B) Genome view of ChIP-seq signals for TRIM33, CTCF, and RAD21 around Ifnb1. TRIM33/CTCF peaks are boxed in green. (C) Enrichment of GO term clusters for genes over-expressed (top) or under-expressed (bottom) in Ctcf-/- macrophages treated with LPS for 24 hrs. (D) Prediction of super-enhancers in murine myeloid cells (Young lab C/ZEBPa and SE_Hah T0 and T2) as compared to 4 other cell types. (E) Plasmids encoding firefly luciferase under the control of the Ifnb1 promoter alone (P) or combined with the 6 murine enhancers were transfected into EL4 cells together with a plasmid coding for NanoLuc luciferase under the control of the thymidine kinase promoter. After 30 hrs, luciferase levels were measured. Results are expressed as the ration of firefly to NanoLuc luciferase, normalized to the mean value of P, and presented as mean +/- s.e.m. with individual experiments shown as open circles (n = 7), each performed in triplicate. *: p<0.05.(PDF)Click here for additional data file.S2 Fig(A) Plasmids encoding firefly luciferase under the control of the Ifnb1 promoter alone (P) or combined with the 6 murine enhancers in reverse orientation (ExrevP) were transfected into RAW264.7 cells together with a plasmid coding for NanoLuc luciferase under the control of the thymidine kinase promoter. After 30 hrs, luciferase levels were measured. Results are expressed as the ratio of firefly to NanoLuc luciferase, normalized to the mean value of P, and presented as mean +/- s.e.m. with individual experiments shown as open circles (n = 5), each performed in triplicate. (B) Same as (A) except that cells were treated with 100 ng/ml LPS for the last 8 hrs. Results are expressed as the ration of the values obtained with LPS to the values without LPS (n = 5). *: p<0.05; **: p<0.01; ***: p<0.001 (ratio paired t test).(PDF)Click here for additional data file.S3 Fig(A) Association of SNPs within 1Mb of IFNB1 with IFNB1 expression in non-stimulated (grey) and LPS-stimulated (pink) monocytes after conditioning on rs12553564. Dotted line indicates the 1% Family wise error rate obtained by permutation. (B) Expression of IFNB1 in monocytes from healthy donors with different rs12553564 genotypes, as indicated, activated or not by different inducers as depicted above the graph. (C) Expression of 3 IFNB1 targets  in monocytes from healthy donors with different rs12553564 genotypes, as indicated, activated or not by different inducers as depicted above the graph.(PDF)Click here for additional data file.S4 FigResults were analyzed from RNA-seq data described in Quach et al., 2016 .(PDF)Click here for additional data file.S5 Fig(A) Plasmids encoding firefly luciferase under the control of the murine Ifnb1 promoter alone (P) or combined with FIRE (5P) or the mutated FIRE mimicking rs12553564 (5mP) were transfected into RAW264.7 cells together with a plasmid coding for NanoLuc luciferase under the control of the tymidine kinase promoter. After 30 hrs, luciferase levels were measured. Results are expressed as the ratio of firefly to NanoLuc luciferase, normalized to P, and presented as mean +/- sem with individual values shown as open circles (n = 5). *: p<0.05; ns: not significant. (B) Same experiment with plasmids encoding firefly luciferase under the control of the human IFNB1 promoter alone (P) or combined with a human genomic fragment centered on rs12553564 and carrying the A allele (AP) or the G allele (GP).(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed. Supported by remarkable advances in biotechnology and bioinformatics, the primary digital codes recorded in the genomes in creatures on Earth have been rapidly deciphered. As a result, a number of novel features of mammalian genomes were uncovered, most of which were against the expectation of scientists. For example, the number of protein-coding genes in the human genome turned out to be approximately 20,000 , which iExpansion of repetitive sequences can be achieved at the level of DNA replication. However, the contribution of such a process at a DNA level to human evolution seems to be limited, since most of the repetitive sequences in the human genome originate from mobile genetic elements entitled retrotransposons multiplying through the reverse-transcription process a 2]..2].Retrotransposons are endogenous genetic elements that produce their copies and insert them randomly in the host genome. Following regular transcription to RNA, their DNA copies are produced by reverse transcription using the RNA as a template. This “DNA–RNA–DNA” replication cycle may not appear sophisticated, but it is a fundamental process that formed the present DNA world. Given that the original self-replicating genetic material on Earth was RNA ,4, any RReverse transcriptase was first discovered in the Rous sarcoma virus (RSV), an avian oncogenic retrovirus, in 1972 . At thatAlu family that was actually regarded as a hallmark of the human genome . The orf2 encodes a protein (ORF2) with reverse transcriptase activity. Polyadenylated LINE transcripts are reverse transcribed by ORF2 from the poly (A) tail, while ORF2 also recognizes an AT-rich tract in the host genome and cuts it. As such, reverse transcription and integration of LINESs are both conducted by ORF2 protein  . D. D15]. Dv-src under the control of a strong viral promoter in the LTR, which transforms infected cells into sarcoma. The v-src gene is homologous to the host c-src gene and located downstream of the retroviral gag–pol–env cistron related to avian leukosis virus (ALV). This genomic organization suggests that RSV genomic RNA originated from a chimeric product of the parental ALV provirus with the cellular c-src gene located immediately downstream . Th. ThFGF4 FGF4 retrogenes in chromosomes 12 and 18 are distinct, these events were exerted independently by LINE-1 ORF2 capturing the same host mRNA, suggesting its remarkable activity in germ cells, or more possibly, in embryonic cells. Collectively, these two genetic events conducted by LINE-1 represent an awful property of a retrotransposon as a creator of endogenous pathogenic agents, using host mRNA as a template. Such an endogenous pathogen could even initiate hereditary diseases, as well as malignant neoplasms.Since the TSD sequences of these Alu retrotransposon was believed to be a hallmark of the human genome. However, comparative molecular genomics revealed that the Alu SINEs are widely shared by other primate species, suggesting its contribution to the evolution of primates rather than that of Homo sapiens. Among a number of genes evolved with Alu elements, the zinc finger and broad complex/tram track/bric-a-brac 38 (ZBTB38) gene is known to be expressed in chondrocytes and involved in the determination of height in humans. This gene encodes a transcriptional repressor and is conserved among vertebrates, and its murine orthologue, C-terminal binding protein-interacting BTB zinc finger protein (CIBZ) gene, is shown to regulate apoptosis. A strong association between height and ZBTB38 locus was reported both in European and Korean populations . S. SAlu inAs introduced in a previous subsection, the human genome is characterized by the involvement of a vast number of lncRNA genes that are even more abundant than the protein-coding classical genes. To date, although nearly 20,000 lncRNAs out of 27,000 in total are anticipated to play critical biological roles in the human body , the actgag and pol genes flanked by LTRs at both ends, lacking the env gene. This provirus is classified as part of the HERV-H subfamily and is located in the short arm of chromosome 19 in the human genome [As is often the case with research regarding a novel class of molecules, an early advance in lncRNA research was also conducted in relation to cancer biology. Thus, a significant number of lncRNAs were found through cancer research and were defined as cancer-associated lncRNAs. Urothelial cancer-associated 1 (UCA1) is the most typical representative of such lncRNAs. Until today, UCA1 has been reported to be associated not only with bladder cancers ,29,30, bn genome . This HEgag and pol genes, the UCA1 gene expresses lncRNAs only. This gene is able to produce several lncRNA variants, which are represented by two major ones of approximately 1400 and 2300 bases in length  is a multifactorial degenerative disease of the synovial joints and, currently, is a major medical issue in advanced countries with highly aged societies. Although OA itself is not life-threatening, progressive loss of locomotive function in patients severely impairs their quality of life. Unlike rheumatoid arthritis , OA is initiated from the loss of the integrity of articular cartilage in joints, most frequently in body weight-bearing knee and hip joints . The facenv mRNA of HERV-WE2 was found expressed in 88% of advanced OA chondrocytes, while it was absent in normal and early OA chondrocytes. The etiological role of the activation of this particular subgroup of HERV is not yet clear. Since HERV expression is occasionally induced during the development of normal tissues, this finding may represent a regenerative response of cartilage after OA-inducing damage. In this context, it is of interest that the production of syncytin 1, which is the env gene product of HERV-W, was confirmed in these OA chondrocytes. Syncytin 1 is primarily a viral envelope glycoprotein that plays a role in infection, but it is expressed in the placenta and involved in trophoblast fusion. This protein is able to induce cell fusion; hence, its pathogenic activity may be suspected. Activation of HERV in OA chondrocyte was further supported by the viral particle formation confirmed by electron microscopy, even though its pathological role is questionable. Of note, recent studies suggest the involvement of lncRNAs, including the HERV-derived UCA1, in OA pathogenesis [In 2014, a Norwegian group attempted to determine if a virus was associated with OA, since a previous report  suggesteogenesis ,91.Rheumatoid arthritis (RA) is another major disorder in synovial joints, with progressive destruction of joint tissues, including articular cartilage. In this systemic inflammatory disease with an autoimmune background, cartilage is degenerated as a result of inflammatory responses, in which synovial cells play a major role . The etiIt is now obvious that retrotransposons have made a significant contribution to the generation of mammalian species by modifying and enlarging the genome. Particularly, considering their extraordinary genome occupancy, no humans with present shape, which is determined mostly by endochondral ossification, were present without HERV, LINEs, and SINEs. In addition, the fact that the genomes of crustacean and insects contain a number of retrotransposons suggests their contribution to intramembranous ossification—the older form of bone formation ,98. ThusOur cells, particularly germ cells, are armed by a protection system against the expansion of retrotransposons in the genome. Typically, using fossils of retrotransposons, small RNAs are produced that specifically recognize them, which lead to the silencing of relevant elements. These small RNAs consist of endogenous small interfering RNA (esiRNA) , miRNA, In addition, we should stay alert to exogenous retroviruses as well, especially to human immunodeficiency viruses (HIVs). Considering the property of HIVs as lentiviruses, their genome-editing activity is evident. Although their primary targets are T-lymphocytes and macrophages, possible infection to other types of cells may not be ruled out. Indeed, direct interaction with sperm cells and HIV-1 virions via mannose receptor was observed , suggest


Drosophila TRs phosphorylated in an IIS-dependent manner. To conduct the screen, we built a library of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as the Drosophila ortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of the dPA2G4 gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. Here we report the results of an unbiased screen for  Drosophila melanogaster serves as an important genetically tractable model system for the discovery of novel pathway components and targets as well as their physiological roles  pathway is a central regulator of these processes in multicellular animals . Due to al roles .Drosophila IIS is the Forkhead transcription factor ‘O’ (FoxO), which is phosphorylated by protein kinase AKT, leading to inhibition of FoxO function through cytoplasmic retention  pathway is another important regulator of nutrient-responsive cell physiology. The mTOR pathway integrates several nutrient derived signals, including IIS activity, which promotes mTOR activity . mTOR coderstood . TORC1 aDrosophila S2R+ cells  vectors by ligase independent cloning (Drosophila Genomics Resource Center (DGRC) gold collection library when available. When not in the DGRC collection, TR cDNAs were amplified from 5′RACE (Invitrogen) cDNA library made from adult female flies. Cloning was verified by digestion, the constructs of hits were confirmed by sequencing. Drosophila S2 cells were cultured in M3 medium (Sigma) supplemented with 1X Insect Medium Supplement (Sigma), 2% fetal bovine serum (LifeTechnologies) and penicillin/streptomycin (LifeTechnologies). Transfections were performed with Effectene Transfection Reagent kit (Qiagen) according to the manufacturer’s instructions. To analyze phosphorylation, S2 cells were incubated 10 min with 10 μg/ml insulin  alone, or after inhibition for 2 hr with 1 μM Rapamycin  ( cloning . A C-terPhos-tag SDS-PAGE and Western blotting were performed according to previous studies (w1118 (BDSC 6326), NURF38 RNAi (BDSC 31341), UAS-Rheb , mTOR delP mutant (BDSC 7014), myc dm2 mutant , malt 6.5% (w/v), semolina 3.2% (w/v), baker’s yeast 1.8% (w/v), nipagin 2.4%, propionic acid 0.7%. In starvation experiments, larvae were kept on medium contain agar 0.5% (w/v), nipagin 2.5%, and propionic acid 0.7% in PBS, supplemented with or without 5% sucrose (w/v). Fly stocks used in this study were: Tub-Gal4 (RNA was extracted using a Nucleospin RNA kit (Macherey-Nagel). cDNA was synthesized using SensiFAST cDNA Synthesis kit (Bioline) according to the manufacturer’s protocol. qPCR was performed with a Light cycler 480 Real-Time PCR System (Roche) using SensiFAST SYBR No-ROX qPCR Master Mix (Bioline). The primers used in this research are listed below:CDK7-F: GGGTCAGTTTGCCACAGTTTCDK7-R: GATCACCTCCAGATCCGTGNURF38-F: GTGCCACGTTGGACCAACGCGNURF38-R: GTAGCCCTTGTGCGGGAAGCAGNURF55-F: GGACTCTCTTGGAATCCCAACCTCNURF55-R: GGCCGGTGAAGATGTTCTTGGCdPA2G4-F: AAGGGCATTGCCTTTCCCACdPA2G4-R: GCCTTTAACGTGTAGTCAGCATCGt-tests were performed for statistical analyses. n indicates the number of biological replicates and is detailed in the figure legends. Error bars indicate standard deviation.Student’s https://doi.org/10.25387/g3.12498020.Plasmids are available upon request. Information of the plasmid library is listed in Supplemental table 1. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental material available at figshare: Drosophila TR library, based on the annotation by Harvard medical school Drosophila RNAi Screening Center (DRSC) ‘Transcription Factors and Related’ RNAi Sub-Library (TRXN) (https://fgr.hms.harvard.edu/drsc-focused-sub-libraries). TRXN includes a set of 963 Drosophila genes which are annotated as transcription factor, nuclear protein, and DNA binding protein. The proteins in the library include DNA binding factors, DNA remodeling factors, and histone modifying enzymes along with their associated proteins. Ligase-independent cloning (LIC) was performed to clone these genes into a modified pMt-Strep-V5-HisA expression vector . In total 857 Drosophila genes were successfully included in our library, which provides 89% coverage of the TRXN gene set  downstream of IIS through a cell-based phosphorylation screen. For this purpose, we constructed an epitope tagged n vector . This mon vector . The pMtgene set .Drosophila  and will be referred to as Drosophila proliferation-associated 2G4 (dPA2G4). Downstream targets of nutrient sensing pathways are often regulated transcriptionally in parallel to posttranslational regulation. For example, our previous data showed that PWP1 is regulated by nutrients and IIS/mTOR pathway through both phosphorylation and gene expression  promoted dPA2G4 expression  in wandering 3rd instar larvae revealed a significant enrichment of Myc chromatin binding close to the transcriptional start site of dPA2G4 , are phosphorylated upon insulin treatment, in mTORC1-dependent manner . Similart larvae . The exp of mTOR . In cont mutants  CD. ThusDrosophila mutants of NURF55 display a strong undergrowth phenotype  provide evidence that expression of dPA2G4 is regulated by IIS/mTORC1 signaling, likely through Myc; (3) identify NURF38 as a necessary gene for tissue growth in larvae; (4) construct a library of 857 V5/Strep-tagged TRs compatible for Copper-induced expression in Drosophila cells. Collectively, this study demonstrates the potential of Phos-tag SDS-PAGE for unbiased identification of new phosphorylation targets of signaling pathways. Thus, it can be used to complement other approaches, such as phosphoproteomics. Some technical limitations were also observed. >350 proteins remained undetected, including Reptor (a known target of mTORC1). This might be due to high protein turnover or cell lethality or caused by technical reasons, such as insufficient transfer of large proteins or poor transfection efficiency. It should be also noted that some of the genes included in the library might be misannotated as TRs, including some of the hits .Animals coordinate their growth rate and metabolism in a changing nutrient landscape, which requires dynamic regulation of gene expression. Therefore, it is important to understand the identity and roles of the transcriptional regulators (TRs) involved. Here, we identify several TRs with a putative role in nutrient-dependent gene regulation. Specifically, we (1) identify two components of the NURF complex, Max and dPA2G4 as phosphorylation targets of IIS/mTORC1 signaling in Drosophila nucleosome remodeling factor (NURF) complex is a conserved ATP-dependent chromatin remodeling complex with four subunits: NURF38, NURF55, NURF301 and ISWI (NURF301 do not display a strong undergrowth phenotype, but an ecdysone-dependent pupariation phenotype (The and ISWI . MoreoveDrosophila. Another downstream target of mTORC1 signaling is PWP1, which is phosphorylated in an mTORC1-dependent manner and necessary for RNA Pol I and III-dependent expression of ribosomal RNA (PWP1 gene expression is promoted by IIS/mTOR signaling through Myc transcription factor (in vivo.Ribosome biogenesis is a key process in regulating cellular growth capacity and it is tightly coordinated by the IIS/mTOR pathway. Ribosome biogenesis involves all three RNA polymerases and is likely regulated through multiple effectors. Myc is known to control ribosome biogenesis downstream of mTOR signaling, as inhibition of mTORC1 leads to downregulation of Myc protein levels (omal RNA . In addi


Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell‐based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine‐specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC‐seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell‐fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine‐specific reprogramming approaches. Canine embryonic fibroblasts are successfully reprogrammed, but adult stromal cells remain resistant, likely due to epigenetic barriers. We performed assay for transposase‐accessible chromatin using sequencing on canine stromal cells and pluripotent stem cells, and identified distinct chromatin remodeling patterns. We identify candidate genes and transcription factor binding motifs that may be impeding adult cell reprogramming, and could be targeted to improve protocols in canines. Poor understanding of canine‐specific regulators of cellular reprogramming and pluripotency maintenance impedes the use of naturally occurring disease in companion dogs for translational regenerative medicine research. In this work, the authors identify differences in global chromatin landscape that occur during cellular reprogramming and further define candidate barriers that prevent adult canine cells from reprogramming. Specifically, the genes and loci reported can be the basis of novel canine‐specific reprogramming protocols in future research efforts. The authors believe that these findings will be of great interest to the stem cell research community, especially to those seeking novel and realistic animal models for translational research.1Induced pluripotent stem cells (iPSC) are pluripotent stem cells derived from somatic cells that have adopted an embryonic stem cell‐like phenotype. Somatic cells of murine, human, and other species can be reprogrammed by forced expression of pluripotency transcription factors (TF).Time is ripe for the application of iPSC technology in regenerative medicine, although numerous translational challenges still need to be addressed before such innovative therapies can reach patients at a larger scale. The unique challenges associated with the translation of iPSC‐based cellular products to the clinic, along with the historically low approval rates of new candidate drugs that go into human clinical trials, call for a paradigm shift in translational biomedical research. Such a paradigm should be specifically designed to be able to test critical aspects such as safety,Naturally occurring diseases in companion dogs are an extremely valuable and readily available resource as a preclinical model. Similarly to humans, dogs suffer from various complex multifactorial diseases, such as cancer, diabetes mellitus, cardiomyopathies, age‐related cognitive dysfunction, and neural damage, that are target for the development of iPSC‐derived cellular therapies.To date, six groups have reported the derivation of canine embryonic stem cells (cESC) from blastocyst‐stage embryos,We have successfully used previously described canine reprogramming protocols to reprogram canine embryonic fibroblasts (CEF) into stable ciPSC lines.To test our hypothesis, we have determined global chromatin accessibility by assay for transposase‐accessible chromatin using sequencing (ATAC‐seq)2Additional materials and methods can be found in 2.1All animals and protocols in this study were approved by the Institutional Animal Care and Use Committee at the University of California (UCD) Davis, and all experiments conform to the relevant regulatory standards. CEF were derived from elective spays of pregnant uteri obtained from the Community Surgery Service at the UCD, School of Veterinary Medicine (SVM). Fetal age was determined prior to the spay by ultrasonographic evaluation by the attending clinician, or posteriorly by pulmonary maturation evaluation in hematoxylin and eosin‐stained slides, as previously described.2 sections and digested with collagenase type II  1 mg/mL, at 37°C for 1 hour, with agitation. The digested cell suspension was centrifuged and the pellet filtered through a 100‐μm cell strainer; flowthrough was plated in complete DMEM. The remnant tissue sections were also plated in complete DMEM with the dermis side down.CDF were derived from skin samples from deceased dogs obtained from the pathology service at the UCD SVM. All dog owners consented to unrestricted use of their dog's remains. With scalpel and scissors, fat and capillaries were scraped away from the dermis and skin was cut into small 2 to 4 mmcASC were a gift from Dr. Borjesson's laboratory and cultured as previously described.2.2A number of 100 000 CEF were transduced with fresh OCT4‐KLF4‐SOX2‐IRES‐MYC (OKSIM)2.3DNA constructs were electroporated into ciPSC with an Amaxa Nucleofector 2b Device . Firefly and Renilla luciferase activity was assayed with Dual‐Glo Luciferase Assay System  following manufacturer's instructions. Bioluminescence was read in a Veritas Microplate Luminometer , and ratio of Firefly over Renilla luciferase normalized to the same ratio of pGL3‐control wells.2.4See Open chromatin DNA libraries were prepared from crude nuclei extracts by cell lysis, nuclei precipitation, and transposition for 60 minutes at 37°C. Libraries were then amplified with SsoFast EvaGreen Supermix  and Nextera polymerase chain reaction (PCR) primers. To reduce guanine‐cytosine (GC) content and size bias, we monitored the PCR using quantitative Real Time PCR (qPCR) to stop amplification before saturation. Libraries were amplified for a total of 15 to 21 cycles, purified using Agencourt AMPure XP beads , and sequenced in an Illumina HiSeq4000 system  in a paired‐end 150 bp run. At least 50 000 000 raw reads per cell type were obtained, except for cESC with only one cell line, which had approximately 40 000 000 raw reads. HTStream was used for data preprocessing, and fragments were mapped to the CanFam3.1 canine genome. Biological replicates were merged before peak calling. Differential openness analyses were conducted using the limma‐voom Bioconductor pipeline and peaks were annotated using the Bioconductor package ChIPseeker, version 1.20.0. Correlation plots, read‐depth heat maps, and profile plots were generated with deeptools. Hierarchical clustering was performed by distance calculation with Cluster 3.0 and visualized in Java TreeView. Gene ontology (GO) term classification was performed with PANTHER. TF motif enrichment analysis was performed with HOMER.2.5t tests and analysis of variance (ANOVA) tests were used to analyze statistical differences in all cases, except for the differential openness analysis . GraphPad Prism v8P value of <.05 was considered statistically significant.Pairwise Student's 33.1OCT4, NANOG, and SOX2  into low passage CEF, cASC, and CDF. The characteristics of the dogs from which the cell lines were derived are detailed in Table 2 Figure  and, whe2 Figure  and S1. 2 Figure .OCT4 locus with eGFP in CEF. Upon OSKM transduction of the modified CEF line, eGFP expression was observed starting on day 4 PT, indicating endogenous OCT4 expression, and was consistently visualized in formed ciPSC colonies on p0 and in further passages  in naïve stem cells or the proximal enhancer (PE) in primed stem cells.OCT4 locus in ciPSC we cloned the canine DE and PE in a reporter plasmid upstream of a minimal promoter controlling the expression of luciferase (luc)  Figure . The con) Figure .cASC and CDF, as well as testicular and ovarian fibroblasts, did not yield stable colonies, even though the efficiency of infection was comparable to that of infected CEF Figure .3.2We hypothesized that resistance of canine adult stromal cells  to OSKM‐mediated cellular reprogramming is due to a failure to close somatic‐fate loci, a failure to open pluripotency loci, or both, during the reprogramming process. To test our hypothesis, we studied the chromatin accessibility landscape of canine stromal and pluripotent cells by ATAC‐seq. Specifically, we studied two adult stromal cell types , CEF, CEF‐syngeneic ciPSC, and cESC. We sequenced four different cell lines of each cell type mentioned, except for cESC of which we only had one cell line available. The following are results from the analysis of all the peaks in the complete data set. The frequency of sequenced fragment size followed the expected pattern for an ATAC‐seq library with periodical peaks corresponding to the nucleosome‐free regions  and mononucleosome, dinucleosome, and trinucleosome ,When we studied the genomic element distribution of differential peaks we found that proper regulatory areas (promoter and 2 kb upstream and downstream), exons and introns, were enriched in peaks with the highest fold change Figure , indicat3.3GAPDH or RPL4/14 (data not shown). The group of loci that were differentially open in ciPSC compared with CEF (CO) included well‐validated pluripotency associated genes such as SMAD3 and ALPL, as well as candidate novel canine pluripotency‐associated genes such as WNT5A, AVPR1, or BMP7. Finally, genes that were differentially closed in ciPSC compared with CEF (OC) included many genes that are known to enhance the somatic fate, such as SYNGR2 and VEGFA as well as candidate novel canine somatic fate genes such as GTF2A1L, GSTA4, or MMP3. In both CO and OC groups, different genomic areas suffered chromatin accessibility changes in both directions (open to closed and vice versa). First, the cluster peaks lists were converted to genes lists from annotation, in order to construct a read‐depth heat map along the gene loci from TSS to TES  as a means for cell reprogramming is a more mature and robust technology with numerous live puppies born via such an approach.Here, we demonstrate the ability to reprogram CEF to ciPSC, and report the inability of canine adult stromal cells to generate stable ciPSC colonies. Cellular age could have an effect on reprogramming efficiency, but this was not the case in the current study. CEF had a tendency to proliferate faster, and while higher proliferative rate is associated with increased reprogramming efficiency, which could explain the tendency of CEF to be reprogrammed more efficiently, there were no statistical differences in PDT among the cell types studied Figure . FurtherThrough the generation of an OCT4‐eGFP reporter system, we show that the endogenous OCT4 locus is engaged during reprogramming of CEF, and that this is controlled mainly from the PE enhancer,AFP, ANGPT1, AR, and KDR, all markers of committed lineages.IGF1, both of which have roles in embryonic development and stem cell self‐renewal.We initially studied the process of reprogramming of CEF to ciPSC, identifying major pathways that change during this process. Among them, the Wnt, VEGF, FGF, and PDGF pathways were of interest.SIRT5, are strongly related to DNA repair, intracellular homeostasis, redox homeostasis, DNA transcription regulation and microenvironment regulation, all key functions during the process of genetic reprograming.Certain genes that we have identified as candidate FO genes in the current study, such as ALCAM, BMPR1B, and DAG1 as candidate FC genes. While there is limited reports of ALCAM and no reports of dystroglycans' like DAG1 effects on reprogramming in the literature,ALCAM and DAG1 could be novel reprogramming barriers in the canine model, as well as the human and murine models. The BMP receptors or their associated pathways were previously reported to be involved in somatic cell reprogrammingOur data further identified the genes Interestingly, some TF motifs that were enriched in CEF and ciPSC as opposed to adult stromal cells were FOSL2, Jun‐AP1, and JUNB, which have been shown to be involved in identity maintenance, proliferation, immune response, and cell death.TF motif discovery for CEF vs ciPSC showed surprising results, with motifs for OCT4, SOX2, and NANOG enriched in CEF instead of ciPSC, as expected, OCT4 being one of the essential pioneering factors needed for cellular reprogramming in most models studied.Moreover, genome annotation in a non‐model species like the dog is still suboptimal. An astounding 84 Mb of canine transcribed sequence is not found in the existing Ensembl canine reference. In addition, one study reports finding most ATAC‐seq peaks in promoter areas,5Our data provide a deeper understanding of nuclear chromatin remodeling during cellular reprogramming in dog cells, and define candidate barriers for somatic cell reprogramming. Such candidate reprogramming barriers could be the target of future studies that aim to generate a better understanding of the regulatory networks that govern canine pluripotency. The uncovering of particular targets to facilitate cellular reprogramming to ciPSC will support the generation of robust ciPSC for translational research in a more efficient manner.The authors declared no potential conflicts of interest.M.Q.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; M.M., R.J.J., V.L.‐C., C.K.C., M.L.S.: collection of data, final approval of manuscript; P.J.R.: design, data analysis support, manuscript editing, final approval of manuscript; A.K.: conception and design, financial support, project overseeing, manuscript writing, final approval of manuscript.DATA S1. Supplemental dataClick here for additional data file.FIGURE S1 Canine‐induced pluripotent stem cells shut down OKSM lentiviral transgene expression. qRT‐PCR showing repression of lentiviral transgenes in ciPSC after passage 15, when compared with a passage 4. Fold change expression of lentiviral cassette was assessed by primer pairs that bridge the OCT4 and KLF4, or KLF4 and SOX2 genes of the unique lentiviral transcriptional unit. Data represented is from n = 3 independent experiments, Mean ± SEM. Asterisk indicates significant difference with P < .01, when compared with ciPSC p4, by one‐way ANOVA.Click here for additional data file.FIGURE S2 Pluripotency and differentiation marker expression in ciPSC and ciPSC‐derived differentiated EBs. qRT‐PCR showing downregulation of pluripotency markers and induction of differentiation markers upon differentiation of ciPSC. Normalization to undifferentiated ciPSC (ciPSC). EB, embryoid body differentiated ciPSC. n = 2, Mean ± SD. Asterisk indicates significance with P < .05, by Student t tests for each marker. Pluripotency markers: OCT4, SOX2, NANOG. Differentiation markers: KDR (mesoderm), AFP (endoderm), TUJ1 (ectoderm).Click here for additional data file.FIGURE S3 OCT4‐eGFP reporter system construction. A, (i) Cas9‐sgRNA and (ii) cOCT4‐2A‐eGFP‐PGK‐Puro donor plasmid constructs. (iii) Schematic of the CRISPR/Cas9‐mediated editing of the canine OCT4 locus. cHA, canine homology arm. B, (i) Transfection efficiency shown as Relative mCherry Expression (count mCherry+ cells/DAPI nuclei) at day 2 PTr; and (ii) representative image of transfected CEF under puromycin selection at Day 5 PTr, showing expression of mCherry (red). C, Sequencing results of the carboxyterminal end of the canine endogenous OCT4 locus, showing insertion of the GFP sequence and downstream editions. Representative sequencing of edition results on two different CEF lines (CEF‐5 and CEF‐3). PTr, posttransfection.Click here for additional data file.FIGURE S4 OKSIM transduction efficiency in adult stromal cells CDF and cASC. Infection evaluated by OCT4 immunofluorescence at 48/72 hours post‐transduction, defined as number of OCT4+ nuclei/number of DAPI+ nuclei. n = 10 for each group. Mean ± SEM.Click here for additional data file.FIGURE S5 Population Doubling Time for CDF, cASC and CEF cell types. Population doubling time expressed in hours. Mean ± SD. NS, nonsignificant difference, as compared with CEF, P < .05, n = 4.Click here for additional data file.FIGURE S6 Chromatin accessibility of pluripotency genes in stromal cells. Selected IGV genomic views of ATAC‐seq data for stemness genes SOX2, NANOG, OCT4, KLF4, and MYC for the three stromal cells CDF, cASC and CEF. All genome view vertical scales were group autoscaled to normalize for read‐depth. Genes are oriented 5′‐3′ and graphed from 2 kb upstream of the TSS to 2 kb downstream of the TES.Click here for additional data file.FIGURE S7 Regulatory areas (promoter and 2 kb upstream and downstream), exons and introns are enriched in peaks with the highest fold‐change. Proportion of peaks over total peaks, found in different genomic areas when considering either all peaks in the data set or the highest FC peaks. FC, fold change.Click here for additional data file.TABLE S1 Breed, age and sex of canine individuals per cell line.TABLE S2. List of primers used for qRT‐PCR.TABLE S3. List of antibodies used for immunofluorescence.Click here for additional data file.


The rapid development of transcriptome sequencing technology has contributed to the discovery of numerous genes in plant; however, the role of gene expression in postharvest wheat remains largely unexplored. In this study, differentially expressed genes (DEGs) were identified by RNA‐seq in different quality wheats. The 102.6 Gb clean reads had been yielded from the nine RNA‐seq libraries. Typically, there were 1791 upregulated and 2,677 downregulated DEGs, respectively, in strong‐gluten wheat compared with weak‐gluten wheat. Specifically, a total of 4,468 DEGs were classified into 286 Gene Ontology (GO) terms and 131 Kyoto Encyclopedia of Genes and Genomes terms (KEGG). Moreover, the storage protein components, starch and sucrose metabolism, and plant hormone signal transduction‐related genes were discovered, which had involved 109 DEGs. The wet gluten proteins content was 35.24% and 17.36%, and the glutenin macropolymer content was 6.38% and 5.01% between the strong‐ and weak‐gluten wheat, respectively. The POD activities of the different quality wheats were 6,571.14, 5,341.24, and 4,851.48 U/g/min, respectively. The significant difference of starch and sucrose metabolism, hormone, POD, and CAT enzyme along with the higher ATPase activity might potentially affect gluten polymerization, which might thereby result in the different qualities of wheats. This work firstly subjected transcriptome to reveal the effect of the different quality wheat, and we found that the formation of different quality wheats may be due to the difference of GMP, storage protein, hormone, protein isomerase, antioxidase activity, and energy metabolism. The seeds were new harvested and dried naturally for about one week, and the seeds size were manually selected. All the wheat seeds were immediately stored in a −80 ℃ freezer after freezed in liquid nitrogen for 20 min, and 0.5 g of seeds was subjected to RNA isolation.2.2p‐value < .05 and fold change >2 or fold change <−2  was used to analyze GO terms enrichment of DEGs, and the KEGG database (http://www.kegg.jp/kegg) was used to identify the significantly enriched metabolic pathways. According to the functional difference, cluster analysis was used to determine the expression patterns of DEGs on different wheat varieties based on the FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) values  following the instructions. The RNA reverse transcription according to the manufacturer's procedure for the mRNA‐Seq sample preparation kit , the average fragment was 450 bp ( ±50 bp) of the paired‐end libraries, using Illumina Hiseq 4,000 at the following of the merchant's instruction . We used HISAT package  and catalase (CAT) activities were performed as previously procedure  until a clear background. Protein gels were photographed using a scanner GS‐900TM Calibrated Densitometer (Bio‐RAD).The GMP isolation from flour was performed as previously described  and subsequently for qRT‐PCR analysis. Following the reagent instructions, the reverse transcription was used a TUREscript 1st Stand cDNA SYNTHESIS Kit  for 1.0 μg RNA. The qRT‐PCR assay was processed in an analytikjena‐qTOWER2.2 (Germany) using SYBR Green Mix . The internal control was used 18S rRNA gene. All primers and their sequences are listed in Table 33.1The highest wet gluten content of 35.24% was observed in the strong‐gluten wheat, followed by the medium‐gluten wheat of 27.47, while the lowest wet gluten content in the weak‐gluten wheat was 17.36% Figure . The GMP3.2The peroxidase (POD) activities of the strong‐gluten wheat, medium‐gluten wheat, and weak‐gluten wheat were found 6,571.14, 5,341.24, and 4,851.48 U/g/min, respectively. The POD activity of strong‐gluten wheat was 1.35‐fold higher than that of weak‐gluten wheat, while it was almost the same level between medium‐gluten wheat and weak‐gluten wheat Figure . There w3.3GSE122699). Finally, 4,914, 4,717, and 4,468 DEGs were obtained from the medium‐gluten versus weak‐gluten, strong‐gluten versus medium‐gluten, and strong‐gluten versus weak‐gluten wheat transcriptome libraries, respectively. All assemble DEGs were first searched against the GO and KEGG databases using the DIAMOND  prepared from different quality wheat, and sequencing platform is the Illumina HiSeq 4,000. There were 103.69 Gb raw data were generated, and 102.6 Gb clean reads were obtained from the nine different quality wheat libraries by removing adapters and low quality sequences  . The 4 HMW‐GS and 7 LMW‐GS genes were upregulated in strong‐gluten wheat and medium‐gluten wheat than in the weak‐gluten wheat. A large number of gliadin proteins, including α‐, γ‐, and ω‐gliadin, were upregulated in strong‐gluten wheat than in the weak‐gluten wheat  and LMW‐GS (high molecular weight gene subunit) of gluten was significantly higher in the strong‐gluten and medium‐gluten wheat than in the weak‐gluten wheat. Alpha‐, beta‐, and omega‐gliadin had the same expression tendency. The globulin expression was the highest in the medium‐gluten wheat than the other quality wheat. The results suggest that the difference among the different quality of wheat was mainly manifested in the gene expression of stored protein Figure .3.6There were 34 genes annotated as starch and sucrose metabolism are found in the wheat genome, including 4 alpha‐amylase and 7 beta‐amylase that an enzyme catalyzing starch to maltose, 3 callose synthase, and 14 1, 3‐beta‐glucosidase catalyzing UDP‐Glucose to glucose, 1 beta‐fructofuranosidase catalyzing sucrose to D‐fructose, 3 fructokinase catalyzing D‐fructose D‐fructose‐6P, and, finally, 3 glucose‐6‐phosphate isomerase (GPI) catalyzing D‐fructose‐6P to D‐glucose. All of these genes exhibited differently expression among the three quality wheat. Our results suggest that the decomposition in weak‐gluten wheat is higher than that in strong‐gluten wheat Figure .3.7Transport inhibitor response 1 (TIR1), 3 auxin response factor (ARF), 6 auxin/IAA‐responsive protein (AUX/IAA), and Gretchen Hagen 3 (GH3) belong to auxin signal pathway, and upregulated in weak‐gluten wheat than in strong‐gluten wheat. PYRABACTIN RESISTANCE and PYR1‐Like (PYR/PYL) is a receptor of ABA (Abscisic acid), which suppressed the ABA signal factor phosphatase type 2C (PP2C). The PP2C increases the expression of SNF1‐related kinase 2 (SnRK2) (a receptor of ABA). Interestingly, the PYR/PYL, PP2C, and SnRK2 were upregulation in weak‐gluten wheat than in the strong‐gluten wheat. Ethylene response (ETR) and constitutive triple response (CTR1) as ethylene receptor were located in the endoplasmic reticulum, and upregulated in weak‐gluten wheat and downregulated in strong‐gluten wheat. 5 MPK6 (mitogen‐activated protein kinase 6), 1 EIN3 (ethylene‐insensitive protein 3), and 2 ERF (ethylene‐responsive transcription factor) genes exhibited upregulation in weak‐gluten wheat relative to strong‐gluten wheat Figure .3.8We applied real‐time fluorescent quantitative PCR (qRT‐PCR) assays to verify the RNA‐Seq data, and the template using was the wheat seeds used for RNA‐Seq analysis (one of three duplicated samples RNA), randomly select 10 genes for validation, including 9 downregulated genes and one upregulated genes from the different quality wheat Table . The exp42O2 or catechol into poor baking quality, the flour quality can be improved, and the quality of doughs and breads made from the above‐mentioned flours have been improved (Kieffer, Matheis, Hofmann, & Belitz, 2O2 could affect the viscosity of flour fraction in the existence of native POX (Hoseney & Faubion, The transcriptome data revealed that the HMW‐GS expression and LMW‐GS expression were upregulated in the strong‐ and medium‐gluten than in the weak‐gluten wheat. Meanwhile, compared with the weak‐gluten wheat, numerous peptidyl‐prolyl isomerase genes in strong‐gluten wheat had been upregulated, which might promote the cross‐linking of low‐molecular‐weight proteins to high‐molecular‐weight proteins (Don, Mann, Bekes, & Hamer, Moreover, reports from barley, maize, and legumes have indicated that ATP levels are correlated with the storage‐related processes, and the storage product might be divided into different category according to the energy state (Borisjuk et al., 5In summary, this is the first study investigating the effect of gene expression profiles in different quality wheats. It is proposed that the different qualities among the three kinds of wheats can be ascribed to multiple factors, including storage protein gene expression, hormone signal transduction, protein isomerase, antioxidase activity, and energy metabolism. In strong‐gluten wheat, the content of HMW‐GS and LMW‐GS is higher than that in weak‐gluten wheat, and the activities of cysteine synthase and isomerase are also enhanced, which may promote the cross‐linking of low‐molecular‐weight protein to high‐molecular‐weight protein. Meanwhile, POD enzyme can strengthen the gluten network and CAT enzyme will affect gluten polymerization, along with higher ATPase activity, which can provide energy for protein polymerization reaction, meanwhile in consideration of the wheat varietal differences, eventually leading to significant differences among the different quality wheats.The authors declare that they have no conflicts of interest with the contents of this article. This study does not involve any human testing.Jinshui Wang conceived the idea, designed the experiments, and precisely revised the manuscript. Qi Wang analyzed the data and prepared the manuscript. Feng Jia helped in data interpretation and software using. Xia Zhang analyzed the data and carry out qRT‐PCR analysis. Xiaohua Wang and Jinhe Li collected plant materials. All authors read and approved the final manuscript.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


The spatio-temporal expression pattern of ecdysteroidogenic genes is strictly controlled, resulting in a proper fluctuation of the 20E titer during insect development. However, their transcriptional regulatory mechanism is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. Based on this finding, we speculated whether any other poly(A)-related protein also regulates spok expression. In this study, we reported that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. We next examined which transcription factors are responsible for Pabp-dependent transcriptional regulation. Among the transcription factors acting in the PG, we primarily focused on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm of pabp-RNAi PG cells. In contrast, pabp-RNAi did not affect the nuclear localization of other transcription factors, including ventral vein lacking (Vvl) and POU domain motif 3 (Pdm3), in PG cells. From these results, we propose that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis.Steroid hormone signaling contributes to the development of multicellular organisms. In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG),  The 20- enzymes . The enz enzymes . Therefo enzymes . HoweverDrosophila melanogaster (phosphoglycerate kinase promoter directed over production (Pop2), which encodes a vital component of the CCR4-NOT complex, specifically in the PG, D. melanogaster animals show a larval-arrested phenotype. Furthermore, the expression levels of some ecdysteroidogenic genes are strongly decreased in these animals. Based on this finding, we hypothesized that other poly(A) related protein(s) may also contribute to the expression of ecdysteroidogenic genes.Previously, we have reported that polyadenylated tail [poly(A)] degradation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) is involved in the regulation of ecdysteroidogenic gene expression in the fruit fly nogaster . By knocpabp gene in the PG caused first instar-arrest and a decrease in ecdysteroidogenic gene expression, especially spookier (spok). Interestingly, the nuclear localization of the transcription factor molting defective (Mld), a transcriptional activator of spok, was disrupted in PG cells of pabp-RNAi larvae. Our results suggest that Pabp positively regulates ecdysteroidogenic gene expression by regulating ecdysteroidogenic transcription factors.In this study, we revealed that poly(A) binding protein (Pabp) contributes to the expression of ecdysteroidogenic genes. The knockdown of the D. melanogaster flies were reared on a standard agar-cornmeal medium at 25 or 17°C under a 12:12 h light/dark cycle. w1118 served as a control strain. phm-GAL4#22  was obtained from the Bloomington Drosophila Stock Center. UAS-pabp-IR (#22007) and UAS-mld-IR (#17329) were obtained from the Vienna Drosophila Resource Center. Transgenic RNAi experiments were conducted by crossing these UAS RNAi lines with w, UAS-dicer2, and phm-GAL4#22/TM6 Ubi-GFP. A strain carrying the 1.45 kb spok promoter-fused GFP cassette (spok>GFP) was previously described  in the same sample. Primers amplifying noppera-bo (nobo), neverland (nvd), shroud (sro), spok, phm, disembodied (dib), shadow (sad), and rp49 have been described previously . Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit . Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the THUNDERBIRD SYBR qPCR Mix  with a Thermal Cycler Dic TP800 System . Serial dilutions of a plasmid containing the open reading frame (ORF) of each gene were used as a standard. The expression levels of the target genes were normalized to an endogenous control eviously .spok promoter-driven GFP reporter assay was performed as described previously  and then immunostained.A eviously . Brieflypabp-RNAi larvae, for which we used a PG-specific driver  to knock down pabp expression by transgenic RNAi. We found that PG-specific pabp-RNAi caused a larval-arrest phenotype. Eighty eight percent of phm>pabp-RNAi animals were arrested at the second larval instar and even 132 h AEL or later, while only few animals molted into the third larval instar or pupariated at the same time point  containing C2H2 zinc-finger proteins : TK, NT, and RN. Investigation : TK, WS, NT, AN, and RN. Writing/manuscript preparation : TK and RN. Writing/manuscript preparation : TK, WS, NT, AN, and RN. Funding acquisition: AN and RN. 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.