Pioneer factors are transcription factors (TFs) that have the unique ability to recognise their target DNA sequences within closed chromatin. Whereas their interactions with cognate DNA is similar to other TFs, their ability to interact with chromatin remains poorly understood. Having previously defined the modalities of DNA interactions for the pioneer factor Pax7, we have now used natural isoforms of this pioneer as well as deletion and replacement mutants to investigate the Pax7 structural requirements for chromatin interaction and opening. We show that the GL+ natural isoform of Pax7 that has two extra amino acids within the DNA binding paired domain is unable to activate the melanotrope transcriptome and to fully activate a large subset of melanotrope-specific enhancers targeted for Pax7 pioneer action. This enhancer subset remains in the primed state rather than being fully activated, despite the GL+ isoform having similar intrinsic transcriptional activity as the GL– isoform. C-terminal deletions of Pax7 lead to the same loss of pioneer ability, with similar reduced recruitments of the cooperating TF Tpit and of the co-regulators Ash2 and BRG1. This suggests complex interrelations between the DNA binding and C-terminal domains of Pax7 that are crucial for its chromatin opening pioneer ability.  Pioneer transcription factors are unique in their ability to recognise and bind their target DNA sequences in closed chromatin . Their aThe structure of a few pioneer factors was probed through structure function studies. For the FOXA factors that specify endoderm derivatives, closed chromatin binding was shown to involve the DNA binding domain (DBD) that interacts with its target DNA sequences, as well as the C-terminal domain that interacts with histones H3 and H4 . FurtherThe pioneer factor Pax7 is expressed in a variety of tissues, but its pioneering role is clearly supported only in the pituitary gland where Pax7 opens a new enhancer repertoire to specify the intermediate pituitary tissue and the melanotrope cell fate ,19. ThisIn the present work, we have investigated the structural features of Pax7 implicated in chromatin opening of the enhancer repertoire that specifies the pituitary melanotrope fate. The ability to reprogram a corticotrope cell model, the AtT-20 cells, into melanotropes enabled us to investigate the pioneer ability of naturally occurring isoforms of Pax7, together with a series of deletion and alanine replacement mutants. The Pax7 isoforms differ by just a few amino acids within the DNA binding paired domain but these suffice to create isoforms with or without pioneer ability, but with similar inherent transcriptional activities. The pioneering-deficient Pax7 isoform is similar to the related Pax3 in their inability to activate the melanotrope transcriptome. A similar loss of pioneering ability was observed for C-terminal deletion mutants of Pax7. It is striking that similar losses of pioneering ability are produced by either introduction of two amino acids within the paired domain or by deletion of C-terminal sequences. The loss of pioneering ability is clearly linked to the loss of chromatin opening rather than to failure of chromatin recruitment. This pattern corresponds to an inability of one isoform and mutants to proceed to the second step of chromatin opening by Pax7 . To generate stable transgenic AtT-20 Pax7 cell populations, retroviral vectors (pLNCX2) containing the indicated Pax7 constructs (with 3xFlag at both N- and C-termini under control of CMV promoter) were packaged using the Platinium-E Retroviral Packaging Cell Line  and infections performed as described . Selecti5 cells per well) and transfections performed the next day using PEI 25K (Polysciences #23966–1) in triplicates. Transfected DNA  was added to the PEI 25K solution (4 μl per 150 μl DMEM) and incubated at room temperature for 30 min before adding 100 μl of the mixture to each well. The media was changed 24 h after transfection and luciferase activity assessed 48 h after transfection. Growth medium was removed from transfected cells and 200 μl of lysis buffer  added to each well. After 10 min on a vigorous shaker, 100 μl of cell lysate supernatant was used for analysis using the GloMax Navigator luminometer (Promega) using the kinetics protocol . Statistical significance was assessed by 2-way ANOVA and Tukey's multiple combinations test.AtT-20 cells were plated in 12-well dishes (4 × 10http://easeq.net) and the data exported to Microsoft Excel to create graphs. To view the ChIP-Seq and ATAC-Seq heatmaps, we used the Integrative Genome Viewer tool of Easeq . ChIP experiments were performed in AtT-20 cells as described earlier ,26. ATACof Easeq  or a cusof Easeq . All ChI6 cells) were washed and collected in PBS. The cells were resuspended in 800 μl of a cold buffer with low salt content . After 15 min of incubation on ice, 100 μl NP40 10% was added. Cytosolic fractions were removed, and nuclear proteins extracted in 100 μl of high salt cold buffer containing 20 mM HEPES pH 7.9, 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.6 μM aprotinin, 0.6 μM leupeptin and 2 μM pepstatin A. Protein concentration was quantified in the Bradford assay.To prepare nuclear extracts, AtT-20 cells from a 100 mm Petri dish , were prerun in Tris-glycine buffer (40 mM Tris and 195 mM glycine) for 60 min at 300 V at 4°C. The gel shift reaction mixture was then loaded, and the gel run for 3 h at 300 V at 4°C. Gels were dryed on 3MM Whatman paper in a gel dryer at 80°C during 60 min under vacuum. Autoradiography films  were then exposed with the dried gels for 10–48 h using an intensifier screen.RNA was extracted from AtT-20 cells using the RNeasy Plus Mini kit (Qiagen #74134), and cDNA synthesized using 5 μg RNA, oligo-dT and SuperScript III reverse transcriptase  following manufacturer's recommendations. The resulting cDNAs (5 μl of 1/50 dilution) were analyzed by qPCR using SYBR Green reagent (ThermoFisher Scientific #A25741) supplemented with 500 nM of each gene-specific primer pair . The number of Pax7 motifs present within a window of 200 bp around the summit of each pioneer site was assessed using a custom program based on Python  experiments  of enhancers that exhibit a single peak of H3K4me1  binding , and corresponding effects on Pcsk2 mRNA levels  by Pax7  show no ATAC signal  shows a reduced single peak of H3K4me1 for Pax7-431 and a diminished bimodal signal for Pax7-465Ala459-461  that loses Pax7 recruitment in ChIP-Seq experiments and other subsets  that fail to be completely opened and remain in the Primed state Figure  suggestsPax7 gene appears crucial for function: indeed, a splicing mutant resulting in loss of the OAR-containing isoform of human PAX7 is associated with a global neurodevelopmental delay   Figure . In cont) Figure  or to C-) Figure  show ove) Figure . Also, t) Figure  and whet) Figure . A simil) Figure . But it o Figure  but failo Figure , highligo Figure . Within o Figure . In summ459-461 subsets of impaired pioneer sites is similar (Figure Tpit gene results in failure of Pax7 pioneer capacity in the mouse pituitary (It is noteworthy that the GL+ isoform or the deletion mutants that are impaired in pioneering ability exhibit subsets of targets that appear blocked in the primed state rather than being fully activated by the reference Pax7 Figure . We alsor Figure  and hencr Figure ,8 and knituitary . Furtherituitary . This maituitary .Considered globally, the results present a picture of gradual loss of pioneering ability by the GL + and deletion mutants that affect the same subsets of pioneer sites suggesting that it is the intrinsic properties of those sites that underlie their sensitivity. The nature of the critical properties that are involved remains to be better defined.gkad520_Supplemental_FilesClick here for additional data file.


PLAAT4 . On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as   Host inducible immune responses triggered by pathogen recognition via pattern recognition receptors (PRRs) comprise a key aspect of the innate immune system. By activating transcription of numerous antiviral effector genes, such responses mediate early protection against viral infections prior to the onset of adaptive immunity. By contrast, accumulating evidence supports the functional importance of constitutive innate immune defense as a mechanism of protection, acting even prior to the occurrence of host innate immune responses ,2. Such PLAAT4 (a.k.a. RARRES3/RIG1/TIG3), a potent HAV restriction factor , as an important host factor modulating IRF1 binding to a DNA promoter element driving efficient transcription of 2 atmosphere. AML12 immortalized mouse hepatocytes were grown in DMEM/F-12 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 1 × Insulin-Transferrin-Selenium (Gibco), 40 ng/ml dexamethasone and 2 mM GlutaMAX-I. Primary human hepatocytes (PXB cells) were purchased from PhoenixBio Co. and maintained in Leibovitz's L-15 medium (Wako) supplemented with 26 mM NaHCO3, 100 IU/ml insulin, 1 μM hydrocortisone and 10% FBS (Gibco).PH5CH8 immortalized human primary hepatocytes, A549 human lung carcinoma cells, 293T and 293FT human embryonic kidney cells, and HepG2 and Huh-7.5 human hepatoma cells were mycoplasma-free and cultured in Dulbecco's modified Eagle's medium (DMEM), High Glucose supplemented with 10% fetal bovine serum (FBS), 1 × GlutaMAX-I and 1 × MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific) at 37°C in a 5% COPrimary antibodies to IRF1 , Src , and phospho-Src (Y416)  were from Cell Signaling Technology; DYKDDDDK  and GAPDH were from Wako ; CSNK2B  and CSNK2A2  were from Bethyl Laboratories; and CSNK2A1  and AFAP1  were from Proteintech. IRDye 680 or 800 secondary antibodies including #926-32211, #926-32212, #926-32214, #926-68020 and #926-68073 (1:20 000 dilution) were from LI-COR.CX-4945 (Silmitasertib) was purchased from Selleck. Puromycin and Hygromycin B Gold were from InvivoGen. Pyridone 6 (JAK Inhibitor I) was from Cayman Chemical. All-trans-retinoic acid was from Wako. Recombinant human IFN-γ and CSNK2B were from Pepro Tech and NKMAX, respectively. PSI-7977 (Sofosbuvir) was from ChemScene. Lambda protein phosphatase was obtained from Bio Academia. miR-122 mimics were synthesized by Dharmacon and transfected by electroporation as miRNA/miRNA* duplexes as described (A cell culture-adapted variant of HM175 strain HAV (HM175/18f) and the HAV/NLuc reporter virus, 18f/NLuc were prepared as described before . A chimeDENV/NLuc reporter virus carrying a subgenomic RNA containing NS1-5 region fused with a NLuc reporter was produced by co-transfecting with plasmids expressing capsid and prME as described .PLAAT4 promoter followed by a minimal TATA promoter, 5′-CTAGCAAAAGGAAAGTGAAAGTGAAATTCAAAAGGAAAGTGAAAGTGAAATTAAGCTTAGAGGGTATATAATGGAAGCTCGACTTCCAG-3′ and 5′-AGCTCTGGAAGTCGAGCTTCCATTATATACCCTCTAAGCTTAATTTCACTTTCACTTTCCTTTTGAATTTCACTTTCACTTTCCTTTTG-3′, and inserted into pNL2.3 plasmid (Promega) using NheI and HindIII restriction sites.The lentiviral transfer plasmids encoding AFAP1 were created by PCR amplifying the host genes using cDNA derived from PH5CH8 cell total RNA as template and primers flanked by XbaI and NheI restriction sites, and ligated into pCSII-EF-MCS vector. The NLuc reporter vector pNL-4 × IRF1 was prepared by annealing oligonucleotides containing IRF1 binding motifs derived from the In vitro transcription of HAV or HCV RNA was carried out using T7 RiboMAX™ Express Large Scale RNA Production System (Promega) as per manufacturer's protocol. Transfection of viral RNA (5 μg) was performed in a Gene Pulser Xcell Total System (Bio-Rad) or using TransIT-mRNA Transfection Kit (Mirus) for subgenomic HAV-Luc RNA as described ,12.Lentiviral transfer vector was co-transfected with standard packaging plasmids into 293T cells using PEI MAX reagent (Polysciences) and the supernatant fluids harvested at 72 h were filtered through a 0.22 μm syringe filter. Production of sgRNA CRISPR/Cas9 lentivirus was similarly carried out by co-transfecting 293T cells with sgRNA expressing vectors listed in Total RNA extraction was performed with the RNeasy mini Kit (Qiagen). Detection of HAV genome RNA was carried out by a two-step quantitative RT-PCR analysis with the ReverTra Ace qPCR RT Kit and THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) using specific primers 5′-GGTAGGCTACGGGTGAAAC-3′ and 5′-AACAACTCACCAATATCCGC-3′. Quantification of cellular genes was performed with the primer pairs listed in P-40 supplemented with EDTA-free Complete protease inhibitor cocktail (Roche) and resolved on a 10% Phos-tag SDS-PAGE gel .Western blotting was performed with standard methods. Odyssey CLx Infrared Imaging System (LI-COR Biosciences) was used for visualization. Lysates for Phos-tag gel analysis was prepared in Tris-buffered saline containing 1% Nonidet siRNA pools listed in NLuc or Firefly luciferase (FLuc) activity was measured using Nano-Glo Luciferase Assay System or Luciferase Assay System (Promega), respectively, as per the manufacture's protocol. Luminescence was analyzed on a Mithras LB940 (Berthold).3VO4 and 1% Triton X-100) supplemented with a Complete protease inhibitor cocktail (Roche). Clarified lysates were subsequently purified by binding to Anti-FLAG M2 affinity agarose gel (Sigma), followed by elution with the FLAG peptide (0.2 mg/ml). The resulting eluate was concentrated using an Amicon Ultra 10K Centrifugal filters (Millipore) and diluted in wash buffer . This procedure was repeated five times to reduce the concentration of the FLAG peptide.FLAG-tagged IRF1 proteins were ectopically expressed in 293FT cells grown on 15 cm dishes using PEI MAX reagent (Polysciences). Cells transfected with empty vector were processed in parallel. Twenty-four hours after transfection, the cells were scraped into a lysis buffer  as per manufacturer's instruction, and treated with 100 μl 2% acetonitrile/0.1% trifluoroacetic acid (TFA) in water. Samples concentrated by SpeedVac were used for LC-MS/MS analysis. To identify phosphopeptides derived from IRF1, gel lanes were sliced in 2-mm intervals, and slices were cubed and destained with 50 mM ammonium bicarbonate/30% acetonitrile. Gels were dehydrated in 50 mM ammonium bicarbonate/40% acetonitrile and dried by Speed Vac. After dried gels were immersed on ice in 12.5 ng/ml trypsin (Promega) in 50 mM ammonium bicarbonate, proteins were in-gel digested overnight at 37°C. Peptides were extracted with 70% acetonitrile/0.1% TFA, and concentrated by SpeedVac.Affinity- purified samples (15 μl) were treated with 1 μl 50 mM tris-(2-carboxyethyl) phosphine and incubated at 60°C for 2 h, followed by addition of 0.5 ml 200 mM Methylmethanethiosulfonate and incubated at RT for 15 min. Protein samples at pH 7.5–8.5 were then digested at 37°C for 2 h with Lysyl Endopeptidase, Mass Spectrometry Grade  packed with 3 cm of a C18 analytical column . Peptides were gradient-eluted using 0.1 M formic acid/80% (vol/vol) acetonitrile in water at a flow rate of 300 nL/min into an TripleTOF 5600+ (AB Sciex Instruments). Resulting data were analyzed using ProteinPilot Software 5.0.1  and searched against a database ‘uniprot_sprot_can + iso_20100622’, as well as a decoy database of reversed protein sequences.NetPhos server 3.1 was used to analyze candidate CK2-phosphorylation sites in IRF1 CSNK2B or control siRNAs were lysed in HKMG buffer  3 day post-transfection. Lysates were incubated at 4°C for 2 h with 5′-biotinylated double-stranded oligonucleotides spanning the 3 tandem repeats of IRF1 binding sequence derived from the PLAAT4 promoter, 5′-CTAGCAAAAGGAAAGTGAAAGTGAAATTCAAAAGGAAAGTGAAAGTGAAATTCAAAAGGAAAGTGAAAGTGAAATT-3′ (1 μg), in the presence of 1 μg poly(dI–dC) (Thermo Fisher Scientific), followed by addition of 25 μl Magnosphere MS160/Streptavidin (JSR Life Science). Another 1 h later, the beads were washed with HKMG buffer five times, followed by Western blotting analysis of the DNA probe-bound IRF1 protein.PH5CH8 cells transfected with either 5 PH5CH8 cells transfected with CSNK2B versus non-target control siRNAs using CUT&RUN Assay Kit  as per manufacturer's protocol. In brief, trypsinized cells were bound to Concanavalin A beads, permeabilized with digitonin, and incubated with IRF1 antibody  overnight at 4°C on a thermo shaker. On the next day, Protein A-fused micrococcal nuclease was incubated for 30 min at 4°C to bind IRF1 antibody and digest bound sites, followed by incubation at 37°C for 10 min to release digested DNA fragments. DNA was purified using NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL) and eluted in 50 μl nuclease-free water.CUT&RUN was performed on 5 × 10Integrity of DNA samples was determined with a TapeStation HS D5000 Screen Tape (Agilent). Library for next-generation sequencing was prepared using TruSeq ChIP Library kit. Paired-end 2 × 150 bp sequencing was performed on an NovaSeq6000 platform at Macrogen Japan . Sequencing quality were comparable for all samples. Raw paired-end reads were trimmed of poor quality bases and adaptors via fastp (v0.23.2)  with ada2 fold change of 5 were excluded. Read counts for this final set of peaks were normalized for library size via trimmed mean of M-values (TMM) in csaw. Differential peak abundance was tested via a quasi-likelihood F-test implemented in edgeR  . Brieflyin edgeR   6.8.n  =  3 or greater. All between-group comparisons were carried out by ANOVA or two-tailed t-test using Prism 6.0 software . The P-values were calculated from 3 biological replicates.Unless noted otherwise, the error bars represent the standard deviation and are shown for experiments with PLAAT4, in transfected 293FT cells  or C-terminal (IRF1-FLAG) FLAG epitope in 293FT cells. We confirmed that ectopically expressed FLAG-tagged IRF1 proteins were transcriptionally active and capable of driving expression of its primary target gene, FT cells  (3), andFT cells .t1/2 = 50 min in HEK293 cells) . Analysis of a control sample processed via identical procedures from empty vector-transfected lysates identified several proteins nonspecifically bound to beads and eluted by FLAG peptides . By subt3 cells) . Thus, w3 cells) .IRF1 itself was used as a positive control for inhibition of IRF1 in these functional experiments. Three days post-siRNA transfection, total RNA was extracted and analyzed for mRNA levels of PLAAT4, the gene most down-regulated upon IRF1 depletion  successfully reduced the protein expression of CSNK2B, and this decrease was paralleled by an increase in HAV RNA levels  in response to IRF1 binding , a tetrameric complex composed of two catalytic α-subunits (CSNK2A1 and CSNK2A2) and two regulatory β-subunits (CSNK2B). We found IRF1-FLAG specifically co-immunoprecipitated with endogenous CSNK2B, as well as with the catalytic subunits, implying that CSNK2B might interact with IRF1 as part of a CK2 complex   Figure . Experims Figure . Despite4 Figure , signifi4 Figure . These rPLAAT4, in both PH5CH8 cells and primary hepatocytes , which induces IRF1 transcription in a STAT1-independent manner  than control cells (8.18-fold). Together, these results attest to the functional importance of the CSNK2B-IRF1-PLAAT4 axis in the induced IRF1 response.The data presented above show CSNK2B positively regulates the basal activity of IRF1, thereby maintaining constitutive transcription of its target gene,  Figures  and 4C. t manner . Since AH8 cells , we usedd PLAAT4 . We confn Figure . ATRA do4 Figure –F. We als Figure . While aPLAAT4 transcription, it remains uncertain how it does so. To examine whether CSNK2B modulates the binding affinity of IRF1 to the PLAAT4 promoter element, we conducted in vitro pull-down experiments using a biotinylated DNA probe containing the IRF1 binding motif derived from the PLAAT4 promoter and compared the IRF1 abundance co-precipitated with the DNA probe in CSNK2B-depleted PH5CH8 cell lysates versus control. Whereas CSNK2B by itself did not bind a DNA probe containing the IRF1 binding motifs, IRF1 binding was reduced in CSNK2B-depleted lysates . Despite large variations across the biological replicates analyzed by CUT&RUN, differential binding analysis showed numerous sites where IRF1 binding affinity is significantly changed, with the majority weakened in the absence of CSNK2B NN(GAAA) sequence, those found in the peaks significantly reduced by CSNK2B depletion were highly constrained to ‘(GAAA)GT(GAAA)’  and those involved in antiviral responses , proviral factors , and immune responses  in unstimulated and IFNγ-treated PH5CH8 cells. With few exceptions, most genes were suppressed basally and less well induced by IFNγ when depleted of CSNK2B  were altered . Con. ConCSNKnhibitor . These rCSNK2B depletion resulted in impaired expression of AFAP1 (actin filament-associated protein 1) . Thus, we explored an alternative hypothesis, namely that the more rapidly migrating bands represent decreased CK2-catalyzed phosphorylation of AFAP1. In support of this, λ phosphatase treatment, as well as pharmacologic inhibition of CK2, converted the immunoreactive bands into a single faster migrating band  Figure , H, desp) Figure , F. Thus) Figure , H. Immus Figure . Whereasn Figure , G, abse1 Figure . We hypod Figure . These rAFAP1 abundance is an unfavorable prognostic marker in lung and renal cancers  replication in PH5CH8 cells  of CK2 resulted in a 50-fold increase in production of infectious DENV particles in PH5CH8 cells  protein of SARS-CoV-2 directly interacts with CK2 components (CSNK2A2 and CSNK2B) and activates its catalytic activity for productive infection  immune checkpoint, thereby promoting its expression [114]. On the other hand, genetic loss of IRF1 is frequently found in various human cancers, including leukemia and gastric cancers (AFAP1 loci is fine-tuned by CSNK2B (Figure AFAP1 transcription, CSNK2B acts as a CK2 complex to promote AFAP1 phosphorylation that subsequently activates Src signaling (Figure In addition to host anti-pathogen defense, IRF1 has been recognized to play important but opposing roles in the regulation of tumor progression. On one hand, IRF1 drives escape from anti-tumor immune surveillance by binding directly to the promoter of  cancers ,35. The  cancers . Yet, ce cancers ,37, to bB Figure . In addig Figure , E. Impog Figure ,39. Seveg Figure , such asOur results show a new role for AFAP1 as a downstream antiviral effector of IRF1 and CSNK2B/CK2 signaling that down-regulates a late step in the replication of DENV. Whereas AFAP1 is tightly crosslinked to actin filament and controls its integrity, loss- and gain-of-function experiments confirmed that expression of AFAP1 is associated with reduced permissiveness to DENV replication Figure , H. It sIn summary, our results reveal that the DNA-binding activity of IRF1 is regulated post-translationally by CSNK2B, and suggest that the transcriptional activity of IRF1 in different tissues may be influenced by the expression status of CSNK2B. While the target selectivity of IRF1-CSNK2B complex remains to be elucidated, further clarifying the mechanisms by which CSNK2B modulates IRF1 binding to its target DNA elements likely provide the basis for developing antiviral and anticancer therapies.ChIP-Seq data generated in this study are available in the SRA database under the accession code PRJNA885123.gkad298_Supplemental_FilesClick here for additional data file.


PPARγ, C/EBPβ, C/EBPα, AP2, SREBP1, FASN, ACC, HSL, and ATGL. Consistently, the knockdown of FOS, facilitated by two distinct siRNAs, significantly promoted intramuscular adipocyte lipid accumulation. Moreover, our analysis revealed multiple potential binding sites for FOS on the promoters of PPARγ, C/EBPβ, and C/EBPα. The expression changes in PPARγ, C/EBPβ, and C/EBPα during intramuscular adipogenesis were opposite to that of FOS. In summary, FOS inhibits intramuscular lipogenesis in goats and potentially negatively regulates the expressions of PPARγ, C/EBPβ, and C/EBPα genes. Our research will provide valuable data for the underlying molecular mechanism of the FOS regulation network of intramuscular lipogenesis.Goat intramuscular fat (IMF) deposition is precisely regulated by many key genes as well as transcription factors. Nevertheless, the potential of the regulators of goat IMF deposition remains undefined. In this work, we reported that the transcription factor FOS is expressed at a low level at the early differentiation stage and at a high level in late differentiation. The overexpression of FOS inhibited intramuscular adipocyte lipid accumulation and significantly downregulated the expressions of  Owing to rising consumer awareness of meat-associated quality, consumer demand for high-quality goat meat has increased significantly . Meat quc-fos. FOS is a transcription factor superfamily, including FOS, v-FOS, FOSB, Fra1, and Fra2, which together with members of the proto-oncogene JUN family  and the activating transcription factor (ATF) protein family constitute the AP-1 transcription factor complex [FOS gene reduced lipid droplet accumulation and inhibited the differentiation of 3T3-L1 adipocytes [FOS gene promoter in CGL patients, resulting in a reduced expression of FOS that interferes with preadipocyte differentiation [The transcription factor FOS is a nuclear-like protein with a basic leucine zipper encoded by the proto-oncogene  complex ,6. FOS i complex ,8,9,10.  complex . For exaipocytes . Meanwhiipocytes . Congenintiation . TherefoThis study aims to investigate the impact of FOS on the regulation of adipogenesis in goat intramuscular preadipocytes. First, the localization of FOS expression was detected in goat intramuscular precursor adipocytes. Subsequently, the expression levels of FOS were detected at various stages of lipogenesis in goat intramuscular adipocytes. Then, the role of FOS in intramuscular preadipocyte differentiation was determined by loss and gain of function using siRNAs and an overexpression vector. In addition, the potential target genes of FOS were predicted and compared with FOS, they exhibited contrasting expression patterns at various stages of lipogenesis in intramuscular adipocytes. Taken together, our study results indicate that FOS negatively regulates goat intramuscular adipogenesis and provides new information about the role of FOS in adipogenesis.Capra hircus) (N 3) were purchased from Sichuan Tiandi Goat Biological Engineering Co., Ltd. . Goat intramuscular preadipocytes were isolated and cultured in accordance with previously described methods [The animal experimentation study received approval from the Laboratory Animal Ethics Committee at Southwest Minzu University and the Animal Disease Control Center in Sichuan Province, China. Jianzhou Daer goats  siRNA were synthesized by Gene Pharma . The sequences of the NC and si-RNAs were as follows:Briefly, the coding region of the goat siRNA1: (5′-GUUCCUUCUAUGCAGATTUCUGCUGCAUAGAAGGAACTT-3′);siRNA2: (5′-GAGAUUGCCAAUCGCUGATTUCAGCAGAUUGGCAAUCUCTT-3′);NC: (5′-UUCUCCGAACGUGUCACGUT TACGUGACACGUUCGGAGAATT-3′).Goat FOS overexpression vector and siRNA transfections were performed using TurboFect . Intramuscular preadipocytes were transfected with the transfection reagent  at a 70% confluence. Then, the medium was changed to a medium containing 50 µmol/L of oleic acid, and differentiation was induced for 2 days at 16 h post transfection.The cell culture method was the same as that described above. First, goat intramuscular adipocytes were fixed with 4% formaldehyde for 20 min at room temperature. The Oil Red O staining was performed according to the method described by Xiong et al. . Next, OBodipy and DAPI stainings were performed according to the method described by Chen et al. . Bodipy UXT gene was used as an internal control. The qPCR primer sequences are listed in Total RNA was isolated from harvested intramuscular adipocytes, using RNAiso Plus . Briefly, goat intramuscular adipocytes were collected and resuspended using RNAiso Plus. Chloroform was added, and the suspension was shaken vigorously and then allowed to stand, followed by centrifugation. The supernatant was then aspirated and transferred to a new tube. Isopropyl alcohol was added to precipitate the RNA, and centrifugation was performed. The liquid above the sediment was discarded, and the precipitate was washed with DEPC ethanol and then centrifuged repeatedly. The supernatant was discarded, and the RNA precipitate was allowed to air-dry. Finally, an appropriate amount of DEPC water was added to dissolve the RNA. The A260/A280 values of the RNA were determined as in the range 1.8–2.0 using a UV spectrophotometer, which met the test requirements. cDNA was reverse-transcribed using a reverse-transcriptase kit  and the quantitative PCR spiking system described by Chen et al. . The UXTIntramuscular adipocytes were collected after 2 days of differentiation induction and were lysed according to the instructions of the triglyceride determination kit , and the triglyceride content was determined according to the procedures described in the manufacturer’s instructions.The total protein from the goat intramuscular adipocytes was extracted using RIPA cell lysate on ice. According to the instructions of the BCA protein assay kit , the protein concentration of each sample was determined and denatured at 100 °C. Western blotting analyses were performed as previously described in the literature ,18. In bt-test was utilized to analyze comparisons between two groups. The one-way ANOVA test was utilized to analyze comparisons between multiple groups, and Duncan’s method was used to demonstrate the significance of the data, where “*” means a significant difference (p < 0.05) and “**” means an extremely significant difference (p < 0.01).All the data were analyzed for significance using SPSS 26.0 software and expressed as mean ± SEM. The qPCR images were generated using GraphPad Prism 8.0 software. Student’s two-tailed Previous research has found that in COS-1 cells, 95% of the FOS proteins are localized in the nucleus, and 5% are in the cytoplasm . To invep < 0.01) in the expression of FOS compared to that of the NC    A. In add < 0.01) B,C. Howe < 0.01) D. PPARγ  < 0.01) E. In sumNext, the knockdown of the FOS was achieved using siRNA-mediated techniques in the intramuscular adipocytes of goats. The results showed that the interference efficiency of siRNA1 was 48% and that of siRNA2 was 40% A compareAP2, PPARγ, C/EBPα, and SREBP1 were significantly increased in the knockdown groups  (The expression of lipogenic-related genes was examined after knocking down FOS in adipocytes. The mRNA levels of n groups A. Surpri < 0.05) B,C, but  < 0.05) D. Meanwh < 0.05) E. These https://jaspar.elixir.no/ 27 September 2023), and the promoter sequences, including PPARγ, C/EBPβ, and C/EBPα, were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/ 27 September 2023). As shown in As FOS was found to be a negative regulator of goat preadipocyte differentiation and to repress the expression of adipogenic genes, we hypothesized that FOS may inhibit lipid deposition by targeting downstream genes. According to the relevant literature, FOS can bind to PPARγ and C/EBP promoter sequences and regulate their expressions. Consequently, the expression changes in PPARγ, C/EBPβ, and C/EBPα were examined in adipocytes induced to differentiate from day 0 to day 5. The results showed that the expressions of PPARγ, C/EBPβ, and C/EBPα were opposite to that of FOS in adipocytes induced to differentiate A–C. ThenPPARγ, which is a major transcription factor in adipogenesis and has two isozymes, PPARγ1 and PPARγ2 [LPL is highly expressed in adipose tissue, muscle, and liver [LPL was upregulated in FOS-overexpressing cells and was downregulated in the knockdown cells. We hypothesized that the main reason for the inhibition of lipid accumulation by the overexpression of FOS was a decrease in triglyceride synthesis, which was also supported by a decrease in the mRNA level of LPL in siRNA-treated cells. What is surprising is that the lipid metabolism-related genes, FASN, ACC, HSL, and ATGL, were significantly downregulated in overexpression and knockdown cells. FASN can produce long-chain fatty acids in the presence of malonyl coenzyme A, whereas ACC is the rate-limiting enzyme for the biosynthesis of long-chain fatty acids, and both are key enzymes in lipogenesis [Intramuscular fat deposition is precisely regulated by numerous key genes and transcription factors. Therefore, it is important to reveal the transcription factors that influence adipogenesis to enhance intramuscular fat deposition. Herein, we presented evidence that FOS serves as a negative regulator of intramuscular adipocyte differentiation. Our research provides important theoretical support for refining the molecular network of the key transcription factors regulating intramuscular fat deposition in goats. Previous studies have reported that FOS is encoded by the proto-oncogene c-fos, a nuclear-like protein with an alkaline zipper. In COS-1 cells, 95% of the FOS proteins are localized in the nucleus, and 5% are in the cytoplasm . In thisd PPARγ2 . Previoud PPARγ2 . The oved PPARγ2 . In addid PPARγ2 . The C/Ed PPARγ2 . It has d PPARγ2 . C/EBPβ d PPARγ2 . Taken tnd liver , and catnd liver . Overexpnd liver . Our resogenesis ,33. ATGLogenesis . HSL is ogenesis . The extogenesis . When thogenesis . The speogenesis . These pPPARγ, C/EBPβ, and C/EBPα promoters possess multiple binding sites for FOS. Moreover, it has been reported that PPARγ, C/EBPα, and C/EBPβ exhibit promoter-binding activities with FOS [c-Fos inhibits 3T3-L1 cell differentiation by affecting the PPARγ2 promoter activity [Bioinformatics analysis has demonstrated that with FOS ,39,40,41activity . We hypoactivity ,43. Perhactivity . It has activity . In addiactivity ,46. As mPPARγ, C/EBPβ, and C/EBPα, along with alterations in the expressions of critical lipid-metabolism genes, such as FASN, ACC, and LPL. These results not only underscore the potential of FOS as a novel target for improving the quality of goat meat but also provide essential data for further investigation into the intricate molecular mechanisms governing the FOS regulatory network in IMF deposition.In conclusion, our findings highlight that FOS serves as an inhibitory factor in the differentiation of intramuscular adipocytes in goats. This inhibition is associated with notable changes in the levels of key adipogenic transcription factors, including


Estrogen receptor beta  plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.Greb1 but not at the ERβ-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.We here performed ERβ ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the ERβ cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1  and showed that ERβ and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERβ and LRH-1 co-binding at the ERβ-repressed gene By characterizing the genome-wide endogenous ERβ chromatin binding, gene regulations, and extensive crosstalk between ERβ and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.The online version contains supplementary material available at 10.1186/s12915-023-01773-1. The highly regulated function of the ovary is essential for female fertility and endocrine homeostasis. The ovary is the source of oocytes and the major provider of the female steroid sex hormones estrogen and progesterone. Ovarian dysfunction is relatively common, with incidences of primary ovarian insufficiency and early menopause estimated at 3.7% and 12.2%, respectively , and lifESR1) and β , or the membrane G protein-coupled estrogen receptor 1 (GPER1). ERα is expressed in several female organs, including theca cells of the ovary, as well as in the hypothalamus, and the pituitary gland [Lhcgr, Cyp11a1, Cyp19a1, Runx2, and Ptgs2), confirming the significance of ERβ during folliculogenesis and ovulation [Estrogen, along with progesterone, regulates female fertility. The production of estrogen is a cooperative interaction between theca and granulosa cells of the ovary. Theca cells produce androstenedione that diffuses to the neighboring granulosa cells where it is aromatized by aromatase (CYP19A1) to estrone (E1). E1 is then converted to 17β-estradiol (E2) by hydroxysteroid 17β dehydrogenase 1 (HSD17B1). Aromatase in the granulosa cells also converts the testosterone produced by the theca cells into E2. The effect of E1 and E2 is mediated through binding to the nuclear receptors estrogen receptor α  and abundant motif (present in 45.9% of the bound sequences whereas the background frequency is 3.6%), as would be expected  as the ERE. The NR5A motif can be bound by two nuclear receptors: the steroidogenic factor 1 (SF-1/Nr5a1) and the liver homolog 1 (LRH-1/Nr5a2). Both are expressed in the mouse ovary and essential for ovulation [We next determined which transcription factor binding sequence motifs were significantly enriched in the ERβ-bound DNA. Using HOMER, we identified that the estrogen response element (ERE) was the most enriched  and ERβKO (n = 4) mice. We identified 803 differentially expressed genes (DEGs) following the loss of ERβ, with a relatively uniform distribution between up- and downregulated genes  was the most significantly downregulated gene following loss of ERβ  in the absence of ERβ , oxidation-reduction , and ion transport   , fatty acid metabolism , TGFβ receptor signaling , and male gonad development   was upregulated. We further noted upregulation of plexin C1 (Plxnc1) in ERβKO ovaries  and angiogenesis  were enriched among both up- and downregulated genes. When all DEGs (regardless of direction) were analyzed for biological process enrichment, lipid metabolism, glucose homeostasis, response to hypoxia, response to stimulus, and multicellular organism development were among the enriched functions  in humaRβKO n =  mice. WeInhbahigh and Kctd14high), two types of luteal cells (regular and large), theca cells, two types of cumulus cells (Nupr1high and Ube2chigh), ovarian surface epithelial cells, macrophages (Lyz2high), and stromal cells. Following the loss of ERβ, a significant decrease in luteal cells, along with an increase of theca and Nupr1high cumulus cell populations were indicated , lipid metabolism , positive regulation of angiogenesis , cell differentiation , and multicellular organism development ,  was among the most enriched functions of the target genes , Cpm, Epb41l2, Mylk3, Mdfic, Zfp219, Epb41l1, Pik3cd, Skil, and Fosl2. Notably, the majority of the direct targets (158 out of 245 or 64%) had an ERβ binding site in an intron, most often  in intron 1. This included Greb1 (4 binding sites in intron 1), Angpt4, Pak3 and Bcl2 (each 2 binding sites in intron 1), and Dhh and Pdgfrb (each 1 binding site in intron 1). Thus, a primary mechanism whereby ERβ regulates genes appears to involve its binding to intron 1.In an effort to identify the direct ERβ transcriptional target genes in the ovary, we integrated our RNA-seq and ChIP-seq data. This revealed that as much as a third  of the ERβKO ovarian DEGs were located nearest (their TSS) to one or multiple ERβ-binding chromatin sites  had two intronic ERβ-binding sites. However, neither LRH-1 nor SF-1 transcripts were significantly regulated in the ERβKO ovary. The remaining nuclear receptor genes that were located by ERβ-binding sites , as well as several nuclear receptor coregulators , were also not detected as significantly regulated in the knockout ovaries. This lack of transcriptional regulation is in line with the result generated by Binder and colleagues using isolated granulosa cells from WT and ERβKO ovaries, where only Pgr was differentially expressed following ERβ knockout after ovulatory stimuli [Since nuclear receptors may crosstalk at several levels , we next explored our datasets to identify potential nuclear receptor regulation by ERβ. We found that the TSS of multiple nuclear receptor genes were located closest to several ovarian ERβ-binding sites and could thus be potential direct targets  in the ERβKO ovary but at least 2-fold upregulated in the WT ovary, and with high confidence (FDR < 0.0001) across the individual samples. The expression of 14 genes fulfilled these strict requirements  were bound at the promoter also in humans. These three direct targets with conserved promoter binding were either upregulated (Skil and Epb41l2) or repressed (Inha) by ERβ in the mouse ovary . Thus, although there are major species differences between human and murine fertility, the chromatin binding of ERβ on key targets is relatively conserved. This indicates that key ovarian genes  and transcription factors  are of particular importance for ERβ-mediated female fertility also in humans.To explore a potential relation to human health, we compared the genes located by ERβ-bound sites in the mouse ovary (endogenous ERβ) with those previously assessed in human cell lines (only ERβ ChIP-seq data from exogenous ERβ in other cell types than ovary were available), and performed with the same validated antibody , 47. Thip = 1e−459, Fig. Two species-conserved ERβ-bound genes of particular importance, LRH-1 and SF-1, are nuclear receptors that bind the DNA motif NR5A) that we found enriched at a considerable fraction  between genes co-bound by ERβ and LRH-1 and those bound by only one receptor. To condense the list, all biological pathways were run through REVIGO , if ERβ might recruit LRH-1 (only ERE motifs), if both may bind simultaneously, or if they compete. In an effort to explore this, we first investigated to which extent the de facto dual-bound sequences (approx. 200 nt) harbored both NR5A and ERE motifs. We found that the largest group  harbored an NR5A but not an ERE motif. The second largest group  held both an ERE and an NR5A motif  of chromatin events where both motifs are present: LRH-1 and ERβ either bind adjacent to each other or compete for binding. To explore the latter question, we first investigated the distance between the dual ERE and NR5A motifs . We found that in nearly all cases (92%), the two motifs were within 100 bp of each other -seq data performed in the granulosa cells under the same settings as the LRH-1 ChIP-seq . We iden2FC < -0.9, FDR < 0.05). This generated a list of 28 genes . These genes are also, according to public databases, all expressed in granulosa cells in the mouse. Out of these 28 genes, 9 genes are bound by both receptors near each other at sites that harbor both an ERE and NR5A motifs  in at least one location. Among these genes, we find those encoding key developmental proteins such as germ cell-specific gene 1-like  and Fibrillin 2 . Further, Fkbp5, known to be upregulated by several hormone receptors, has multiple ERβ and LRH-1 binding sites  is expressed both in mouse and human granulosa cells and encodes a co-chaperone that interacts with several hormone receptors  and determines their responses [Cyp11a1, and downregulated Greb1) are also of interest  in breast cancer MCF-7 cells [To explore which gene regulations this dual binding may be critical for, we scrutinized the genes with dual de facto binding of ERβ and LRH-1 (ChIP-seq), that were most strongly upregulated by ovarian ERβ  was included for comparison, as well as a gene desert where neither is expected to bind (negative control). Primers were designed to cover the selected areas with amplicons of approximately 100 bp, and qPCR on a 4th ERβ mouse ovary ChIP experiment (average DNA size of 100–200 bp after sonication) confirmed ERβ binding at the identified sites and not at the gene desert . Further, while not enriched compared to Cry2, amplification products were detected for the Fkbp5 (and Cry2) sites, but not at the Cyp11a1 site. This may indicate that only one of the receptors, ERβ or LRH-1, binds at one time and that they may compete for binding at these sites. However, we cannot exclude that co-binding may occur to a lesser degree at some of these sites (including Fkbp5 and Cry2).To experimentally test whether ERβ and LRH-1 bind to the same regulatory DNA fragments simultaneously, we performed a ChIP-reChIP experiment. We explored DNA regions of target genes indicated to be bound by both receptors , Gata4 , and Runx2 (no ERβ-binding site) are regulated upon loss of ERβ in granulosa cells [Gata4, and several previously identified key ERβ-regulated genes were not found to be affected in our study  and did not exhibit any ERβ-binding sites). A difference is, of course, that our analysis included the whole ovary, and our data is impacted by differences in cell composition.Our transcriptomic analysis of ERβ in WT and ERβKO ovary supports a role for ERβ in ovarian lipid metabolism, as has previously been indicated in isolated granulosa cells during ovulation , and we sa cells , 30. HowNupr1high (nuclear protein 1) cumulus cells. Nupr1 has been linked to inflammatory response and has been shown to be induced in the cumulus-oocyte complex during the ovulatory process [Nupr1 was also recently identified in murine atretic granulosa cells [Nupr1 could explain the overall phenotype of a reduced number of cumulus cells. Lastly, we also observed a trend of increased Lyz2high macrophage cells in the ovaries of the ERβKO mice. This increase in macrophages aligns with our previous findings from colon tissue, where ERβ is anti-inflammatory and reduces macrophage infiltration [It is well known that FSH promotes the development of ovarian follicles, and that LH regulates the preovulatory maturation of oocytes, ovulation, and formation of the corpus luteum. These surges also activate pro-inflammatory genes through cAMP signaling in the ovary and the secretion of proteolytic enzymes by the follicle. These enzymes degrade the follicular tissue at the site, resulting in oocyte release , 61. ERβ process . Howeversa cells . As one sa cells , an incrltration , 65. OurHsd17b1, Hsd17b3, Cyp11a1). ERβ also bound chromatin by the aromatase gene. Although its expression was not altered upon ERβ loss, we note that this regulation is expected to occur specifically at diestrus and may thus only be detected at this specific time point. Overall, since we did not compare ovaries of different estrous stages, we may have missed regulations that occur at specific stages.A spike in estrogen level normally occurs just before ovulation, and this provides positive feedback to the hypothalamus and pituitary, which in response will induce the LH and FSH surges. ERβKO females exhibit a reduced level of E2 at diestrus , 28. ThiFshr) and LH (Lhcgr) are both expressed in granulosa cells. They are G protein-coupled receptors and signal through cAMP-dependent and cAMP-independent mechanisms which result in activated protein kinases and downstream activation of transcription factors and other proteins. In accordance, most of the LH and FSH surge genes affected by ERβ deletion  did not exhibit an ERβ-binding chromatin site and can thus be concluded to be downregulated as a consequence of the reduced surge in knockout animals rather than being direct targets of ERβ. Meanwhile, both the FSH and LH receptor exhibited ERβ binding sites , indicative of being direct transcriptional targets of ERβ. Fshr was significantly upregulated in the knockout, whereas Lhcgr was not regulated. Thus, Fshr appears to be repressed by ERβ in the ovary. However, we cannot exclude that this regulation is a feedback mechanism to compensate for the reduced surges.In accordance with ERβKO mice exhibiting a reduced size of the LH surge, we could confirm the deregulation of numerous ovarian LH and FSH surge genes. The receptors for FSH  but was completely absent (0.3 FPKM) in the ERβKO ovary. This could simply be a consequence of the ovulatory phenotype , but our finding that ERβ binds intergenic chromatin located downstream (29 kb from TSS) of the gene itself, indicates a potential direct regulation. Bhmt expression has also been linked to the transcription factor Cebpa (C/EBPα). Cebpa mediates LH-activated ERK1/2-dependent granulosa cell differentiation and is essential for ovulation and luteinization. Cebpa knockout females are subfertile, similar to ERβKO females [Cebpa gene was also bound by ERβ (two intergenic chromatin sites: 24 kb upstream and 6 kb downstream of TSS) and downregulated following ERβ knockout, ERβ may regulate Bhmt both directly and indirectly (via Cebpa).One of the strongest impacted genes upon ERβ deletion was  females . As the Lrp1, Lrp4, Lrp5, Lrp8, Lrp11) all held ERβ bindings sites in intron 1. ERβ was furthermore essential for the expression of two of these (Lrp8 and Lrp11). Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis and Lrp8 has been associated with reproductive traits in ducks (where it is exclusively located in granulosa cells) and has been suggested to regulate follicular growth [C. elegans have found that first introns are more conserved in length, are bound by more transcription factors, and that the transcription factors that bind first introns are largely different from those binding promoters [On a mechanistic note, we observed that nearly half of the ERβ chromatin binding sites were located in introns. As an example, multiple low-density lipoprotein receptor-related proteins genes . FrCtnna2, Lrp8, Slc38a3, Cabp1, Tspan11). This may suggest that a subset of granulosa cells genes require the cooperative activity of both ERβ and LRH-1 in the transcriptional complex for their expression. This connection between ERβ and LRH-1 is likely intrinsic to granulosa cells of the ovary, where both these nuclear receptors are highly expressed.Importantly, we identified the NR5A (LRH-1 and SF-1) motif as a DNA-binding element associated with ERβ in the ovary. We show that this motif was nearly as enriched among ERβ bound sequences as its own ERE motif. While it may be theoretically possible that the NR5A motif, which is an extension of a half-ERE site, can be bound by ERβ, this is not supported since the NR5A motif was not found to be enriched in ERβ ChIP-seq of other cells that do not express LRH-1 (exogenous ERβ expressed in colon or breast cancer cell lines ). AccordOur finding that the larger group of de facto dual-bound sequences contained only the NR5A motif and no ERE may support the hypothesis that LRH-1 binds first, and then recruits ERβ to the chromatin. This is in line with reports of LRH-1 acting as a pioneer factor in mice where it can bind nucleosomal DNA in vitro and promote chromatin accessibility during zygotic genome activation in two-cell embryos . It is fGreb1 chromatin area that harbors dual NR5A and ERE motifs. This confirms their nearby interaction at the chromatin level. This particular gene is known to be upregulated by ERβ when introduced into other cell types , but was indicated by our data to be downregulated by ERβ in the ovary. Whether this is a cell type or species difference, or whether the presence of LRH-1 impacts the direction of regulation, needs further studies. Furthermore, the same experimentation showed that at certain other sites we could not detect any bound DNA (Cyp11a1) or no clear enrichment (Fkbp5). Our experimentation using transactivation assays showed that they can inhibit each other’s transcriptional activity at their classical response element. It is thus possible that ERβ and LRH-1 bind simultaneously at some sites, but one at a time at other sites, perhaps competing for binding. Whether one receptor’s transactivation (at its respective response element) is inhibited by the other nuclear receptor, is because they can bind each other’s motif , or because of protein-protein interaction, remains to be investigated. Further, as sites far apart may directly interact with each other, investigating the related 3D chromatin architecture, using for example Hi-C sequencing, as well as exploring chromatin accessibility  to show which sites lose transcriptional factor accessibility following ERβ deletion, can help to further elucidate their precise mechanism of action. As LRH-1 is essential for fertility, and its deletion leads to the formation of large preovulatory follicles with failure to ovulate, similar to the ERβKO phenotype [Upon testing the hypothesis that they bind simultaneously, our ChIP-reChIP experiments demonstrated that both ERβ and LRH-1 could indeed bind co-operatively within the henotype , our datOverall, the strengths of our study include the usage of a highly validated antibody, the unbiased approach of ChIP-seq and RNA-seq, the analysis of the endogenous ERβ activity within the tissue environment, and the comparison with knockout animals. To be noted, we also compared our data with ERβ ChIP-seq in ERβKO animals, and a direct comparison (WT ChIP vs ERβKO ChIP) confirmed over 70% of sites (WT ChIP vs input). We consider this a high fraction, considering the caveats of using ChIP on non-existent proteins. The accuracy of the cistrome is evidenced by the significant enrichments of the ERE and well-known associated motifs AP-1 and GATA. Moreover, a recent study of several ovarian transcription factors included ChIP-seq of ERβ. Although  this was performed with a non-validated antibody , without replicates nor corresponding input, it did nevertheless identify 3976 of our 4875 binding sites . This suSome weaknesses of the study should be highlighted. Since we analyzed non-synchronized ovaries in RNA-seq, the gene expression data is diluted by cells not expressing ERβ. This means that low-abundant ERβ-regulated genes are likely to be missed. Also, genes that are regulated at specific estrous stages will most likely not be detected. Thus, more genes are likely regulated than what is demonstrated in this study. This may explain some discrepancies between this study and the literature. Consequently, while we gained in studying the complete impact on the in vivo ovarian context, further studies are needed to comprehensively define the full spectrum of regulated genes, including at specific phases of the estrous cycle. However, it should be noted that a complete overlap between identified ChIP-binding sites and regulated genes is not expected. The identified overlap here is similar to what has been previously demonstrated for ERα in homogenous cell lines , 69, 73.We here provide wide-ranging insights into the endogenous ERβ gene regulatory landscape in the ovary and identify its ovarian target genes at the genome-wide level. Our work reveals mechanistic insights including that ERβ and LRH-1 have a shared cistrome and that they can bind simultaneously at some sites, and not at other sites, and that they can inhibit each other’s transcriptional activity. Altogether, we provide a foundation that enables a better understanding of ovarian physiology and female fertility. This data can support research for an effective treatment to overcome ineffective follicle development and oocyte maturation.N = 80, age 4–20 weeks). ERβKO animals were generated by heterozygous breeding of ERβ+/− mice generated from ERβflox/+ mice  crossed with transgenic Rosa26-Cre deleter mice (Taconic) [Ovaries from mice with (WT) and without expression of ERβ (ERβKO) was used for this study (Taconic) . Deletion = 14) and ERβKO animals (n = 8) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. The sections underwent heat-mediated antigen retrieval in citrate buffer  followed by permeabilization  and blocking of endogenous peroxidase activity . Slides were further blocked for unspecific binding of secondary antibodies  and unspecific avidin/biotin binding (DAKO). Incubation with primary anti-ERβ antibody  in 0.1% IGEPAL in PBS was performed overnight at 4 °C. Negative controls without primary antibody were used for each slide. The sections were incubated with appropriate biotin-conjugated anti-IgG secondary antibody  for 1 h at room temperature (RT). Following incubation with avidin-biotin complex  for 45 min at RT, the staining was developed with Liquid DAB+  Substrate Chromogen System (DAKO). The slides were counterstained with Mayer’s Hematoxylin and dehydrated in ethanol and xylene. Images were captured using a BX53 light microscope and CAM-SC50 camera . The ERβ staining was assessed using a combinative semiquantitative scoring of staining intensity and area.Sections of formalin-fixed paraffin-embedded mouse ovary from WT (n = 2) was fixed in 4% formaldehyde (24 h), stored in 70% ethanol, embedded in paraffin, and sectioned. The sections were used for RNA in situ hybridization with mouse Esr2 probe (316121) using RNAScope 2.5HD Assay – Brown according to manufacturer’s protocol (RNAScope ACDBio). Mouse Ubs (310771) primer and Dapb were used as a positive and negative control, respectively, in mouse liver.WT ovary (n = 5) or ERβKO (n = 5) mice was homogenized with a tissue lyser . Total RNA was isolated with QIAzol and purified using the miRNeasy Mini Kit  according to the standard protocol, and on-column DNAse treatment was applied. Quantitative and qualitative analyses were performed with NanoDrop 1000 spectrophotometer and Agilent Tapestation 2200 , respectively.Frozen ovarian tissue from WT (Actb and Gapdh). All primer sequences are provided in Additional file One microgram RNA was reverse transcribed using the iScript cDNA synthesis kit (Biorad) according to standard protocol. Ten nanograms of cDNA were used to perform qPCR in a 10 μl volume, using iTaq universal SYBR Green supermix (Biorad), as recommended by the supplier. Non-template negative controls and melting curve analysis were used to ensure specific amplification. Samples were run in duplicates using the CFX96 Touch System (Biorad) and the relative gene expression was calculated as the mean per group using the ΔΔCt method, normalized to the geometric mean of two reference genes  and ERβKO mice  were homogenized using a tissue lyser . Total protein was extracted in RIPA buffer  with 1× protease and phosphatase inhibitor, incubated for 30 min on ice, vortexed for 60 s, and centrifuged at 13,000 rpm for 15 min at 4 °C. Protein concentration was measured by BioRad Protein Assay Kit (Bio-Rad) and a UV spectrophotometer. On a 10% SDS-polyacrylamide gel , 20 µg of total protein and ladder were loaded and electrophoresed. Using Transblot Turbo Transfer Kit and system the separated proteins were transferred to a PVDF membrane for 10 min . The membrane was incubated in 5% milk for 1 h at RT before incubation with primary antibodies against ERβ  and GAPDH  in 5% milk overnight. Membranes were washed four times with 0.1% TBST for 10 min, and then incubated with anti-mouse IgG HRP-conjugated secondary antibody . Clarity western ECL substrate  was used to visualize the protein and the images were recorded using an imaging device (ThermoFisher).Frozen ovaries from WT  and ERβKO ovaries  were collected from 4–16-week-old mice (N = 42), washed with PBS, cut into small pieces, and cross-linked with formaldehyde . Formaldehyde was removed by PBS washes and glycine (0.125 M) was added to stop further cross-linking. The samples were stored at -80 °C. Cells were separated using a Dounce homogenizer on ice, passed through a 100 µM cell strainer to remove connective tissue, washed with ice-cold PBS, and cell pellets were collected by centrifugation at 4500 rpm. At 4 °C, with ice-cold reagents and incubations on shaking, cell pellets were lysed (10 min) in LB1  and centrifuged . The pellets were then resuspended (5 min) in LB2 , centrifuged, and dissolved in LB3 buffer  and sonicated to generate 200–500 bp chromatin fragments. Chromatin samples were centrifuged at 13,000 rpm (5 min), and supernatants were transferred into low-binding DNA tubes and incubated overnight with ERβ antibody (PPZ0506) or IgG  as a control. After overnight incubation, 30 µl of protein G Dynabeads were added  and incubated (4 h). Beads were washed with TSE1 , followed by TSE2 , LiCl buffer  and TE buffer (10 mM Tris-HCl and 1 mM EDTA) for 10 min in each buffer. Samples were eluted with a NaHCO3 (0.75%) buffer containing SDS (1%) and proteinase K. Cross-linking was reversed overnight at 65 °C, followed by treatment with RNase A  and DNA purification using QIAquick PCR purification columns . The resulting DNA was used to prepare ChIP-seq libraries as using the NEB Next Ultra II DNA Library Prep Kit as previously described [Fresh WT ovaries  or ERβKO (n = 4) mice was used for library preparation (Illumina TruSeq Stranded mRNA) and sequenced  at Sweden’s National Genomics Infrastructure (NGI) facility. The sequencing generated at least 13 million paired-end reads (2 × 150 bp) per sample, which were mapped to the mouse genome (GRCm38) using STAR. Gene counts and FPKM values were generated with FeatureCounts and StringTie. DESeq2, with raw counts as input, was used to calculate differentially expressed genes (DEG). The FDR was estimated by the Benjamini-Hochberg procedure. The differential expression of genes was considered significant if FDR < 0.05 and log2FC >|0.4|. Only genes with an FPKM average > 1 in at least one group were used for downstream analysis. The data is available at GEO  > 8) from frozen ovaries from WT . AnalysE196650,  was usedCIBERSORTx  was usedN = 14, using 7 mice/14 ovaries per replicate). First ERβ was ChIP:ed onto protein G Dynabeads as performed to the ChIP-seq (above). Following the washing steps , the ERβ-bound chromatin complexes were eluted with 0.1 M citrate buffer (pH 3) into low-binding DNA tubes. This was incubated overnight with LRH-1 antibody . An additional negative control without LRH-1 antibody was included. Next, following incubation with protein G Dynabeads and washing , the samples were eluted per the ERβ ChIP protocol. The resulting DNA was used in qPCR to amplify sites predicted to be bound by both ERβ and LRH-1  or only ERβ (Cry2). ChIP-reChIP results were depicted both as fraction of input as well as the fraction of ERβ ChIP. Primers of a DNA desert were used as negative control. Primer sequences are provided in Table SN = 4, 8 ovaries pooled) was repeated as confirmation of ERβ binding (as above for ChIP-seq). The result is normalized to input and illustrated as fold change over negative control.ChIP-reChIP was performed in duplicates, using ovaries from WT mice  in Opti-MEM. After 5 h the medium was replaced by fresh complete medium without antibiotics. On day 3, 500 ng of either ERE-TATA-LUC [SW480 cells (authenticated with SNP profiling and tested for mycoplasma) were used for the transactivation luciferase assay. The cells, which do not express ERβ nor LRH-1, were cultured in DMEM-high glucose (Merck) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (Invitrogen), and 2 mM L-Glutamine at 37 °C with 5% COTATA-LUC  or NR5A-TATA-LUC  reporterp-value < 0.05 was considered statistically significant .GraphPad Prism was used for statistical analysis . The results are expressed as mean ± SEM. For the luciferase reporter assay, a two-way analysis of variance (ANOVA) was used for multiple comparisons between the different conditions followed by Fisher’s LSD test. A Additional file 1: Fig. S1. Integrative genomic viewer showing the transcripts of Esr2 detected in the ovarian RNA-seq of WT mice (n = 5). The red box marks the insert region of isoform 1 (ERβ_ins) in the ovary of WT mice. Fig. S2. Additional ChIP-seq results and comparisons. Venn diagrams illustrating (A) detected ERβ-binding sites when normalizing ERβ ChIP-seq triplicates of WT ovaries against ERβ ChIP-seq of ERβKO ovaries, and (B) comparison of results when normalizing against input versus normalizing against ChIP-seq of ERβKO ovaries. (C) Enriched motifs among ERβ chromatin-binding sites in promoter (−1 kb to +100) and enhancer regions (−50 kb to +2 kb), respectively. (D) Venn diagram representing the overlap of our ERβ ChIP-seq and whole ovary RNA-seq of WT and ERβKO mice, compared with microarray of isolated granulosa cells from WT and ERβKO ovaries before and after ovulatory signal [Fig. S3. Additional replicates of ERβ-LRH-1 ChIP-reChIP and the luciferase reporter assay. (A) Replicate 2 of ERβ-LRH-1 ChIP-reChIP normalized against ERβ-ChIP, and (B) replicate 1 and 2 normalized against input. Additional replicates of (C) ERE-TATA and (D) NR5A-RE luciferase reporter assay in SW480 cells.y signal , with ERAdditional file 2: Table S1. Statistics of the ERβ ChIP-seq.Additional file 3: Table S2. All ChIP ERβ-binding sites.Additional file 4: Table S3. All differentially expressed genes between WT and ERβKO ovaries, and the enriched biological functions.Additional file 5: Table S4. ERβ-binding sites by nuclear receptors.Additional file 6: Table S5. Conserved ERβ-binding sites between mouse and human.Additional file 7: Table S6. Common ERβ and LRH-1 binding sites.Additional file 8: Table S7. Primer sequences.Additional file 9. Images of original uncropped blots of ERβ protein detection.Additional file 10. Supporting data values for figures where n < 6.


During the evolution of deuterostome, whole genome duplication (WGD)-enriched lineage diversifications and regulation mechanisms occurs. However, only limited research exists on foxl2 duplication in teleost or other vertebrate species. In this study, two foxl2 paralogs, foxl2 and foxl2l, were identified in the transcriptome of spotted knifejaw (Oplegnathus punctatus), which had varying expressions in the gonads. The foxl2 was expressed higher in the ovary, while foxl2l was expressed higher in the testis. Phylogenetic reconstruction, synteny analysis, and the molecular evolution test confirmed that foxl2 and foxl2l likely originated from the first two WGD. The expression patterns test using qRT-PCR and ISH as well as motif scan analysis revealed evidence of potentially functional divergence between the foxl2 and foxl2l paralogs in spotted knifejaw. Our results indicate that foxl2 and foxl2l may originate from the first two WGD, be active in transcription, and have undergone functional divergence. These results shed new light on the evolutionary trajectories of foxl2 and foxl2l and highlights the need for further detailed functional analysis of these two duplicated paralogs.As a member of the forkhead box L gene family,  From yeruit fly ,3,4. Altruit fly . Coupledruit fly ,7,8,9.foxl2 plays important roles in sex differentiation and gametogenesis [foxl2 represses male cues such as sox9 in the ovary by regulating estrogen signaling and prevents the trans-differentiation of granulosa cells into Sertoli-like cells and theca cells into Leydig-like cells. It regulates steroidogenesis during gonad development as a repressor of steroidogenic enzymes such as StAR, cyp11a1, and cyp17a1 [foxl2 plays important roles in goat and bivalve mollusks [foxl2 in female mouse leads to sterility [foxl2 also results in female to male sex reversal [foxl2 is a female-biased gene in chicken [foxl2 is a conserved female reproductive factor. Additional foxl2 genes have been widely identified in many teleost species and were originally named foxl3 or foxl2b, including the European sea bass (Dicentrarchus labrax), rice field eel , medaka (Oryzias latipes), and zebrafish (Denio rerio) [foxl3 was predominantly expressed in the testis [foxl3 was restricted to the germ cells within the gonads, not the somatic cells in medaka. Adult female medaka with disrupted foxl3 were observed to produce functional sperm in an expanded germinal epithelium, revealing the significance of foxl3 as a germ cell–intrinsic factor in influencing the sperm–egg fate decision [rec8 and fbox47 could serve as the downstream targets of foxl3 to regulate germline sex determination [As a member of the FOXL gene subfamily, ogenesis . It has  cyp17a1 ,13. As amollusks ,15. The terility . In XX N chicken , turtle  chicken , and fro chicken . All theo rerio) ,22,23,24e testis ,25,26. Mmination .foxl2 paralog is initially attributed to the teleost-specific whole genome duplication (WGD). With the findings of the additional foxl2 duplicates outside of the teleost lineage, it has been suggested that these two paralogs are originated from an ancestral gene at the base of the vertebrate by the first two rounds of the WGD [foxl2 and foxl2l, which were different from previous studies. To avoid confusion and make it easier for readers to search the related data directly in NCBI, we chose the names, foxl2 and foxl2l, according to NCBI in this study.The additional  the WGD . In the foxl2 was under significant purifying selection pressure [foxl2 and foxl2l paralogs.The WGD have increased the complexity and genome size of vertebrates. Duplicated genes not only promote diversification and evolutionary innovation ,29, but pressure . This mifoxl2 paralogs, foxl2 and foxl2l, were isolated in the spotted knifejaw transcriptome. To confirm the origin and evolutionary destiny of the duplicated foxl2 and foxl2l paralogs, we performed phylogenetic reconstruction, chromosomal synteny analyses, and positive selection pressure tests in vertebrates. The results of qRT-PCR, in situ hybridization, and the motif scan analyses indicated the potential function diversification of these two paralogs in spotted knifejaw. This study enhances our understanding of the WGD and functional diversification in evolution history. It also provides a basis for studying the evolutions and functions of duplicated foxl2 paralogs.In this study, two Six healthy two-year-old spotted knifejaws, consisting of three females and three males, were randomly selected from Laizhou MingBo Aquatic Co., Ltd., Yantai, China and temporarily cultured in an institute aquarium tank at 25 °C with continuous aeration. The individuals were anesthetized with MS-222 and euthanatize by severing the spinal cord. The specimens, including the brain, kidney, heart, spleen, liver, ovary, and testis, were collected and snap-frozen in liquid nitrogen, then stored at −80 °C for subsequent RNA extraction.Oreochromis niloticus), medaka (O. latipes), and puffer fish (Takifugu rubripes) were retrieved from the NCBI online database and used as queries. To identify the FOXL gene family members of spotted knifejaw, a local BLAST with an E-value of 1 × 10−5 against the spotted knifejaw gonad transcriptome was used [The FOXL gene family members of Nile tilapia  were aligned via Clustal X to test the similarities and differences between these two duplicates.The deduced foxl2 and foxl2l [foxl2 and foxl2l sequences of the selected species  were used in this analysis.MEME suite was used to distinguish the possible functional divergence between d foxl2l . The foxIn accordance with the manufacturer’s protocol, the TRIzol reagent  was used to extract total RNA from the specimens. To remove the genomic DNA and proteins, the DNase I  and RNAclean RNA kit  was applied. RNA quality and quantity were measured via agarose gel electrophoresis and the NanoPhotometer Pearl . Following the manufacturer’s instructions, the M-MLV kit (TaKaRa) was applied for cDNA synthesizing.foxl2 and foxl2l genes were designed via an online tool IDT (http://www.idtdna.com/Primerquest/Home/Index (accessed on 13 March 2023)) in 3′ untranslated regions .Specific primers for spotted knifejaw  regions . A pre-efoxl2/2l-ISH-Fw/Rv-specific primers  from a local fish farm for in situ hybridization (ISH). The testis and ovary samples were collected and fixed in 4% PFA overnight at 4 °C, dehydrated in gradients of increasing methanol, and then stored in 100% methanol at −20 °C. Then, the testis and ovary samples were embedded in paraffin after clearance via xylene. ISH was performed on the paraffin sections of the gonad specimens. ISH was performed using DIG-labeled RNA sense and antisense probes which were synthesized via the DIG RNA Labeling Kit (SP6/T7)  using the  primers .foxl2 and foxl2l genes. The screening criteria is according to the manual of PAML v4.7 [Eleven teleost species were selected to verify the differences in selective pressure between the AML v4.7 . Two Bayfoxl2 paralogs, foxl2 and foxl2l, were identified in the gonad transcriptome of spotted knifejaw via tblastx with an E-value of 1 × 10−5. According to the differential expression analysis results of the gonad transcriptome, foxl2 was expressed higher in the ovary than the testis, while foxl2l was expressed higher in the testis than the ovary  family, rbp2 and rbp7 of the RBP (retinol binding protein) family, nmnat3 and nmnat1 of the NMNAT (nicotinamide nucleotide adenylytransferase) family, clstn2 and clstn1 of the CLSTN  family, and hes6 and hes4 of the HES (hairy and enhancer of split) family. These likely paralogous genes between the foxl2- and foxl2l- containing scaffolds might be originated from their corresponding ancestral gene during the first two WGD and simultaneously with the duplication of foxl2 and foxl2l. Hence, it could be speculated that foxl2 and foxl2l near these genes might also arise from the first two WGD and are differentiated in function.A genome-wide search of the FOXL family genes found only two paralogs of foxl2 and foxl2l, multiple amino acid alignments of deduced protein sequences of these two paralogs were constructed with the selected teleost species . As shown in foxl2 genes shared high identities among different species and foxl2l showed similar results. However, the comparison between foxl2 and foxl2l genes revealed a clear difference in their protein sequences. Only the FH domain was conserved in both foxl2 and foxl2l, whereas the C-terminal region differed significantly.In order to detect the similarity between foxl2 or foxl2l was highly conserved among the selected species. But, similar to the results retrieved from the protein sequence alignment  in the PAML package were applied to examine the differences of selection pressure between d foxl2l . The phyfoxb1, two foxc1, two foxf2, and two foxk2. Overall, FOX gene family members have expanded via the WGD in evolution history. In this study, an expansion was also identified in the FOXL gene subfamily. Four FOXL genes, foxl1, foxl2, foxl2l, and foxl3, could be found in almost all vertebrate studied in this research.As an ancient class of DNA-binding transcription factors, FOX gene family members occupied various functions in cell proliferation, organ differentiation, embryonic development, and immunoregulation ,7,8,9. FGenerally, new genes can emerge via a variety of evolutionary mechanisms, such as exon shuffling, retroposition, duplication, mobile elements, gene fission and fusion, lateral gene transfer, and de novo origination. Among these mechanisms, duplications consisted of a single gene duplication, segmental duplication, and the WGD plays significant roles in evolutionary innovation ,41,42.foxl2 paralogs, foxl2 and foxl2l, were firstly identified and named in the spotted knifejaw genome. In vertebrates, transcription factors, ribosomal proteins, cyclins, and kinases are more frequently duplicated during the WGD process [foxl2 and foxl2l clustered into one clade, while foxl1 and foxl3 clustered into another clade, indicating that the duplicated transcription factor, foxl2l, may originated from the WGD, but not from the other evolutionary events. The additional paralogue foxl2l was initially thought to be resulted from the teleost-specific WGD, but the additional foxl2l was also found out of the teleost lineages such as amphibians, reptiles, aves, and mammals, indicating that foxl2l might have been duplicated from the ancestral foxl2 gene earlier than 3R. The synteny analysis results also supported this speculation. As shown in foxl2 and foxl2l flanking fragments were not found. Many corresponding genes in the same gene family could be located around foxl2 and foxl2l, such as pik3cb and pik3cd, rbp2 and rbp7, nmnat3 and nmnat1, and clstn2 and clstn1. The results were consistent with the speculation that foxl2 and foxl2l did not originate from the teleost-specific WGD but from the earlier first two WGD. According to the research and analysis of the correlation between foxl2l and per3 in European sea bass, the same conclusion can be obtained [In this study, two  process ,44. As sobtained .foxl2 and foxl2l had undergone functional diversification in vertebrates in accordance with the analyses in this study.After duplication, new gene pairs will undergo rapid changes in the sequences and structures, resulting in different evolution fates . Generalfoxl2 and foxl2l, which indicated primary functions in DNA binding. However, the amino acid sequences of these two paralogs were not conserved in the other parts, suggesting that functional diversification may have occurred between foxl2 and foxl2l. Based on the comparation between M0 and M3, the foxl2 and foxl2l paralogs were under selective pressure. However, no models of the positive selection were statistically significant for these two paralogs, suggesting that they likely underwent purifying selection during the course of evolution. Additionally, the MEME motif scan results showed that only three motifs  were conserved between these two paralogs while other parts of the protein sequences differed significantly, which verified the speculation that functional diversification has occurred between foxl2 and foxl2l.As shown in foxl2 and foxl2l were performed via qRT-PCR. It showed that foxl2 was expressed higher than foxl2l in the heart, liver, brain, and ovary, whereas it is contrary in the spleen, kidney, and testis. Meanwhile, the ISH results were consistent with the expression profiles, showing that foxl2 was expressed higher than foxl2l in the ovary and lower in the testis. All results indicated a functional diversification between the foxl2 and foxl2l paralogs. Previous studies have also shown different expression levels of foxl2 and foxl2l in the gonads. In rainbow trout, foxl2l is expressed in the differentiating female gonad during the first oocyte meiosis but remains undetectable in males [foxl2l genes of Atlantic salmon and European sea bass is predominantly expressed in the testis rather than the ovary in the adult stage, which is different from the profiles in rainbow trout. Even foxl2l is also expressed in the ovary, where the levels are much lower than foxl2. It can be indicated that foxl2l participates not only in the onset of oocyte meiosis, but also in regulating testis development [foxl2 and foxl2l are consistent with our results obtained from spotted knifejaw. It indicates that foxl2 is mainly expressed in the ovary, while foxl2l is mainly expressed in the testis [foxl2 was detected in the gonads of sex-changed wrasse and half-smooth tongue sole [foxl2 was also highly up-regulated in the sex differentiation process of Chinese tongue sole [foxl2 in sex change and sex differentiation. What function foxl2l plays in this process is unknown for the time being, and further studies are needed to investigate it.In the current study, the expression profiles of in males . Howeverelopment ,25. In te testis . Sex difgue sole ,49. In ague sole . All thefoxl2 and foxl2l shared conserved DNA-binding characteristics in the FH domain, whereas functional divergence had already occurred after their origination via the WGD. foxl2 is predominantly expressed in the ovary and foxl2l in male germ cells. Foxl2 coupled with foxl2l would have complementary roles in gonadal development and gametogenesis.Considering the aforementioned results, we speculate that foxl2 and foxl2l paralogs in spotted knifejaw. We first characterized the foxl2 and foxl2l paralogs in spotted knifejaw and speculated a potential origin via the first two WGD. Our findings suggest a probable functional diversification of these two duplicated paralogs in vertebrate evolution history. This study provided adequate information and new sights into the functional divergence of foxl2 and foxl2l and can be treated as fundamental groundwork for following studies. Further analysis of the exact functions of foxl2 and foxl2l remains to be addressed in suitable vertebrate species.In this study, we investigated the origin and function of the


Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies. Primary9/L . Childre9/L . Older a9/L ,6. ITP is considered an immune-mediated disease and platelet autoantibodies (PAA) are thought to be important actors . Testinggagitalics is not necessary, but it must be consistent in the whole papper), polymerase (pol), and envelope (env), flanked between two regulatory long terminal repeats (LTRs) [Human endogenous retroviruses (HERVs) originate from ancestral infections of somatic cells with subsequent integration into germ line of primates millions of years ago. During evolution, the continuous mutations have blocked the production of infectious virions. However, HERVs maintain their retroviral structure with three principal genes: group associated antigens (s (LTRs) . Most HEs (LTRs) ,16, reprs (LTRs)  and syncs (LTRs) , are esss (LTRs) ,20. Posts (LTRs) ,22,23,24s (LTRs) ,26. Theis (LTRs) ,26,27,28s (LTRs) ,30,31,32s (LTRs) ,34; theis (LTRs) , reminisHERV expression is modulated by environmental factors via epigenetic mechanisms. Tripartite motif containing 28 (TRIM28), also called KAP1 or TIF1-β, is a nuclear co-repressor of Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs) ,36. SET Despite the wealth of data supporting the role of HERVs, TRIM28, and SETDB1 in eliciting and/or maintaining autoimmune diseases, no studies explored their expressions in patients affected by ITP. Therefore, the aims of this study were to assess and compare the transcriptional levels of pol genes of HERV-H, -K, and -W, the three retroviral families most widely studied ,21; env All children and adolescents affected by CITP . This kit provides treatment with DNase during the first step of RNA extraction. To exclude contamination with residual genomic DNA, total RNA extracts were directly amplified without reverse transcription. RNA concentration and purity were spectroscopy determined  at absorbance of 260 and 280 nm. RNAs were stored at −80 °C until use.2 25 mM, 1 μL of RNase inhibitor 20 U/l, 2 μL ImpromII (Promega), 2 μL mix dNTPs 100 mM (Promega), 0.4 μL random hexamers 250 μM (Promega), and dd-water in a final volume of 20 μL. The reaction mix was carried out in a GeneAmp PCR system, 9700 Thermal Cycle  under the following conditions: 5 min at 25 °C, 60 min at 42 °C, and 15 min at 70 °C for the inactivation of enzymes; the cDNAs were stored at −20 °C until use.A total of 400 ng of RNA was reverse-transcribed with 2 μL of buffer 10× 4.8 μL of MgCl2, 200 nmol of specific probes, and 500 nmol of specific primers.Transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of SYN1, SYN2, and HWERV-W; and TRIM28 and SETDB1 were achieved as previously described in detail ,24,33,34−ΔΔCt method [All amplifications were performed in a 96-well plate at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and at 60 °C for 1 min. Each sample was run in triplicate. Relative quantification of target gene transcripts was performed using the 2t method . GAPDH wt method ,24,33,34p < 0.05 was taken to be statistically significant.One-way ANOVA test was used to compare the age between patients with CITP and the two subgroups (B1 and B2) of HC. Mann–Whitney test was used to compare the transcripts of pol genes of every HERV family; env genes of SYN1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 between children with CITP and control children. Spearman correlation test was performed to estimate the correlations concerning transcription levels of each HERV sequence and TRIM28 or SETDB1 in every group of children. Statistical analyses were performed using Prism software, version 7 . In all analyses, p = 0.1998). As reported in p = 0.4111); Group B2: median 21.8, IQR 21.5–22.4 (p = 0.1432).The transcription levels of housekeeping gene GAPDH were similar between patients with CITP and HC: Medians and IQR 25%–75%: Group A: median 22.3, IQR 21.8–22.7; Group B1: median 22.1, IQR 21.6–22.5 . When expressions of HERV-env genes from patients on eltrombopag alone were compared to those from HC, only SYN1 showed significantly higher values . Medians and IQR 25%–75% of SETDB1: on eltrombopag: median 1.64, IQR 1.27–1.80; no treatment: median 1.80, IQR 1.46–2.17 (p = 0.3397). The median transcriptional levels of TRIM28 and SETDB1 were lower in the 11 patients on eltrombopag treatment alone as compared to the 18 patients with no treatment, but they did not reach statistically significant differences, with borderline r TRIM28 . MediansCurrent results highlight for the first time that children and adolescents with CITP exhibit higher transcriptional levels of pol genes of HERV-H, -K, and -W and env genes of SYN1 and SYN2 in whole blood as compared to age-matched healthy subjects. Some clues that endogenous retroviruses could be involved in inducing alterations in platelet count can be found in the literature. Retrovirus-like particles were detected in lysates of purified platelets ,44. ReveThe cause and the clinical relevance of the enhanced expression of HERV sequences in CITP children remain elusive. Several experimental studies demonstrate that defective transcription of TRIM28 and SETDB1 may lead to increased HERV activation, via DNA hypomethylation and limited chromatin condensation ,39. The Viral infections often precede the onset of primary ITP by a few weeks. The virus-driven stimulation of the immune system is thought to be highly implicated in inducing targeted injuries ,55. SeveThe number of HERVs with high mRNA levels in CITP patients suggests that their upregulation is a wide and complex phenomenon. As regards the single HERV genes, pol genes of HERV-H, HERV-K, and HERV-W contribute to the production of reverse polymerase, that catalyzes the mRNA retro-transcription into the DNA. This may result in new mutations or dysregulated transcription of cellular genes. It must be underlined that the primers and probes we used revealed preserved sequences of many pol genes from different HERV families, which, however, did not allow outlining of the effects of single HERV loci. SYN1 and SYN2 share syncytial and immunomodulatory properties. They target T-cell activation and significantly suppress T-cell functions ,20. SYN1Eltrombopag is a nonpeptide thrombopoietin receptor agonist used for treatment of CITP as a highly effective and well-tolerated therapy for achieving hemostatic platelet counts ,70. A thEpigenetic processes encompass chromatin-based mechanisms in the regulation of gene expression with no structural DNA changes. Environmental factors can cause epigenetic alterations, such as those induced by TRIM28 and SETDB1. TRIM28, through binding to lysine residues of target proteins, causes their phosphorylation and proteasome-driven degradation. TRIM28 recruits SETDB1 for SUMOylation, a post-translational event involved in several cell functions, such as protein degradation, RNA splicing, and transcriptional inhibition ,72. TRIMBetter understanding of the physiopathology of ITP and the failure of tolerance checkpoints responsible for autoantibody formation may open the way to new diagnostic and prognostic approaches and innovative therapeutic strategies. For instance, early detection and longitudinal analysis of the variables here taken into consideration might predict acute or chronic evolution of ITP and allow a patient-tailored therapy. A number of anti-HERV therapeutic strategies might be adopted, such as specific anti-RNAs, monoclonal antibodies, cytotoxic T lymphocytes against HERV antigens, and antiretroviral therapies ,78,79. H