TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient   •Identification of reprogramming-resistant genes and regions in porcine SCNT embryos•H3K9me3 and H3K27me3 are enriched in reprogramming-resistant genes and regions•KDM4A and GSK126 facilitates nuclear reprogrammingRemoving H3K9me3 and H3K27me3 by •TDG overexpression promotes DNA demethylation and improves reprogrammingTransient  KDM4A and GSK126 improves SCNT embryonic development, but does not reactivate TDG during embryonic genome activation. Meanwhile, transient TDG overexpression promotes DNA demethylation and also improves SCNT embryonic development in pigs.In this article, Miao and colleagues show that H3K9me3 and H3K27me3 are epigenetic barriers to porcine SCNT-mediated reprogramming. Removal of these modifications by  CD163 and pAPN in donor cells, cloned pigs exhibited resistance to porcine viruses such as PRRSV, TGEV, and PDCoV (XIST in donor cells (Pig cloning by somatic cell nuclear transfer (SCNT) holds great promise for agriculture and biomedicine and has been widely employed to produce genome-edited pigs for breed improvement , human dnd PDCoV . In addind PDCoV . Howevernd PDCoV  and knocor cells , the birKDM6A mRNA or KDM6B small interfering RNA reduced H3K27me3 enrichments in two-cell embryos and promoted blastocyst formation  was the first confirmed epigenetic barrier to prevent MZT. Overexpression with H3K9 demethylases in SCNT embryos, such as lysine-specific demethylase 4D (KDM4D) and KDM4B, was able to improve embryonic genome activation (EGA) and the blastocyst rate (∼90%) . Anotherormation . A previic cells . Howeveric cells . Before ic cells . Recentlic cells . All theIn the present study, we combined RNA sequencing (RNA-seq), ultra-low-input native chromatin immunoprecipitation sequencing (ULI-NChIP-seq), and whole-genome bisulfite sequencing (WGBS) to investigate histone modification and DNA methylation on the donor genome in pig SCNT embryos. More importantly, insufficient activation of thymine DNA glycosylase (TDG), a DNA demethylation regulator specifically expressed during the pig MZT, was defined as a novel barrier for nuclear reprogramming. Taken together, our study reveals a unique interplay between histone modification and DNA methylation in pig nuclear reprogramming and provides effective methods to overcome multiple epigenetic barriers in this process.in vivo-fertilized (IVO) embryos (four-cell stage) and SCNT embryos (eight-cell stage)  . Here, wl stage) . Both hil stage) A and S1BBy comparative analysis, 3,070 differentially expressed genes (DEGs)  were highly expressed in IVO4c compared with IVO2c, termed EGA-ON genes . Among tTo comprehensively dissect the difference of protein-coding and non-coding transcripts, we also performed a sliding window strategy as previously reported . CompareHaving ascertained reprogramming-resistant genes and regions in SCNT4c, we then asked whether their anomalous expression patterns are stabilized by epigenetic modifications from donor cells. Therefore, we performed ULI-NChIP-seq on PFF and SCNT4c with 200 cells, and generated the genome-wide maps of H3K4me3, H3K9me3 and H3K27me3 . StrikinWe first assessed the histone methylation on EGA-ON/OFF regions. As shown in By further assessing the histone methylation on PFF-ON/OFF regions, no difference in H3K4me3 was detected among these regions in PFF, and its enrichments were even lower on PFF-ON regions in SCNT4c E. MoreovXenopus SCNT embryos have been reported to exhibit H3K4me3 hypermethylation on their transcription start sites (TSSs)  . Howevers (TSSs) A–2D. Meas (TSSs) A–2D, conKDM4A and KDM6A individually or in combination (KDM6A injection showed no improvement on blastocyst formation (19.5%), and even impaired the beneficial effect of KDM4A (27.5%) when both mRNAs were injected (15.3%)  . Thus, wKDM4A-injected groups (26.8%), and this rate was even higher when combining these two methods (33.5%)  . Blastoc (33.5%) F and S3G (33.5%) F and 3G,KDM4A + GSK126 treatment, we performed RNA-seq of combined-treated SCNT4c (SCNT4c-KG). The hierarchical clustering and PCA analysis results revealed that the transcriptional patterns of SCNT4c-KG were close to IVO4c, but different from SCNT4c , human (HERVL and HSATII), and cattle (MLT1A0 and ERV1-2-I_BT) embryos . Moreovepression . In the pression . Strikinriptions A. In detriptions . In partriptions C and 5D.TDG expression could not be rescued by KDM4A + GSK126 treatment   E and 6F.FF genes  and atteFF genes .TDG expression , and PFF-ON gene promoters  were examined by bisulfite sequencing PCR , an ultra-low-input WGBS method, to generate their DNA methylation maps on a genome-wide scale. First, when compared with EGA-ON/PFF-OFF regions, DNA methylation levels were higher in EGA-OFF/PFF-ON regions in SCNT4c A. This cpression B. DNA depression C. Third,cing PCR , and thecing PCR  and S5. cing PCR D. Moreovcing PCR  and S4, In fertilized embryos, histone methylation and DNA methylation undergo a widespread decrease at the pre-implantation stage in mouse , human , cattle H3K9me3 and H3K27me3 have been verified as conserved reprogramming barriers among different species. In pig cloning, aberrant H3K9me3 and H3K27me3 have been observed at the one- to two-cell stage via immunofluorescence, while their detrimental effects to MZT remain unknown . By usinXenopus donor cells  5mC and 5hmC were comparably enriched in SCNT embryos when compared with PFF, while 5fC and 5caC were reduced in SCNT embryos  or a PicoPure RNA Isolation Kit (Applied Biosystems), respectively. Double-stranded cDNA was then synthesized by a SMARTer Pico PCR cDNA Synthesis Kit (Clontech) and an Advantage 2 PCR Kit (Clontech), and purified by AMPure XP beads (Beckman Coulter). After fragmentation with a Bioruptor Sonication System (Diagenode), cDNA libraries were generated by a VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme). Paired-end 150-bp sequencing was performed on an Illumina HiSeq X Ten platform.Illumina Casava (v1.8.2) was used for base calling. Low-quality reads were removed by Trimmomatic (v0.39). The filtered reads were mapped to the pig reference genome (Sscrofa11.1) with STAR (v2.5.3a) to obtain uniquely mapped reads. Transcripts were reconstructed by StringTie (v2.1.2) to define protein-coding novel transcripts by GffCompare (v0.10.2) and CPC2. Complete reference annotation consisted of novel and known transcripts. Gene expression was then quantified to FPKM using Cufflinks (v2.2.1). The expression levels of repetitive elements were assessed by bedtools (v2.29.2), and normalized by VST count. The RNA-seq signal intensities of repetitive elements were quantified and visualized by deeptools (v3.1.3). RepeatMasker annotation was downloaded from the UCSC Genome Browser. Pearson correlation calculation, hierarchical clustering analysis, and PCA were performed using cor, hclust, and princomp functions in R (v3.5.2), respectively. Mapping reads of genes and repeats were visualized using Integrative Genomics Viewer (IGV) software (v2.7.2).IVO4c/IVO2c > 3, average FPKM in IVO4c > 5; PFF-OFF genes in IVO4c, FCPFF/IVO4c > 3, average FPKM in PFF > 5. Both genes were classified into three groups based on the expression differences among IVO4c, PFF, and SCNT4c. We also defined FCSCNT4c-KG/SCNT4c > 2 and FCSCNT4c-TDG/SCNT4c > 2 as EGA-OFF genes reactivated in SCNT4c-KG/TDG, while FCSCNT4c/SCNT4c-KG > 2 and FCSCNT4c/SCNT4c-TDG > 2 were PFF-ON genes silenced in SCNT4c-KG/TDG. Adjusted p values of all DEGs were < 0.05. GO analysis was performed using clusterProfiler function in R.FC and adjusted p values among samples were analyzed by DESeq2 (v1.22.1), based on the reads count obtained by HTSeq (v0.9.1). DEGs in IVO4c/IVO2c > 5, average RPM in IVO4c > 5; PFF-OFF regions in IVO4c, FCPFF/IVO4c > 5, average RPM in PFF > 5. Both regions were classified into three groups based on the expression differences among IVO4c, PFF, and SCNT4c. We also defined FCSCNT4c-KG/SCNT4c > 2 and FCSCNT4c-TDG/SCNT4c > 2 as EGA-OFF regions reactivated in SCNT4c-KG/TDG, while FCSCNT4c/SCNT4c-KG > 2 and FCSCNT4c/SCNT4c-TDG > 2 were PFF-ON regions silenced in SCNT4c-KG/TDG. Fisher's exact test p values of all DERs were < 0.05.The expression levels of genomic regions among samples were assessed through a sliding window  made by bedtools, and normalized by RPM. Differentially expressed regions (DERs) in ULI-NChIP was performed as previously described . For eacIllumina Casava (v1.8.2) was used for base calling. Sequenced reads were trimmed for adapter sequence, low-quality, and low-complexity reads, then mapped to Sscrofa11.1 reference genome using Bowtie2 (v2.3.3.1). Multiple mapped reads, PCR duplicates, and reads with a map quality score of less than 30 were removed. The correlation coefficients of replicates, ChIP-seq intensity, and the occupying rates of exonic and repetitive sequences were quantified and visualized by deeptools (v3.1.3). Peak calling was performed by MACS2 (v2.2.5) with the parameters "macs2 callpeak –n Sample –g 2.2e9 -B -p 0.05" relative to input samples. Mapping reads of representative genomic regions were visualized using IGV software.A pool of PFF and four-cell SCNT embryos (50 cells) was harvested for PBAT-seq as previously reported . In brieIllumina Casava (v1.8.2) was used for base calling. Trimmed reads were then mapped to Sscrofa11.1 reference genome using bismark (v0.22.3). Multiple mapped reads, PCR duplicates, and reads with a map quality score of less than 30 were removed. The DNA methylation levels were quantified and visualized by bismark and deeptools (v3.1.3), respectively.th/75th percentiles, and the whiskers indicate the 2.5th/97.5th percentiles. Development rates and qPCR results are presented as the mean ± SD. p values were calculated using two-tailed Student's t test with SPSS Statistics 20 software (IBM). Differences are shown with asterisks indicating ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.All experiments were repeated at least three times. All middle lines in the box-whisker plots indicate the median, the edges indicate the 25GSE161527 and GSE139512.The Gene Expression Omnibus accession numbers for the data reported in this paper are GEO: Conceptualization, Y.-L.M., Z.-X.C., and X.L.; methodology, X.L., T.W., L.C., and J. Zhou; software, Z.-X.C., L.C., J. Zhang, and W.Z.; validation, X.L. and J. Zhou; formal analysis, X.L., T.W., L.C., and J. Zhou; investigation, T.W., Z. Li, G.B., S.Y., D.W., C.D., T.X., H.H., L.Y., Z. Liu, and X.Z.; resources, T.W., Z. Li, G.B., S.Y., D.W., C.D., T.X., H.H., L.Y., Z. Liu, and X.Z.; data curation, L.C. and J. Zhang; writing – original draft, X.L., T.W., and L.C.; writing – review and editing, Y.-L.M., X.L., T.W., and L.C.; visualization, X.L., L.C., J. Zhang, and W.Z.; supervision and project administration, Y.-L.M. and Z.-X.C.; funding acquisition, Y.-L.M.The authors declare no competing interests.


It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple ‘off switches’ for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.  MYC by Hurley et al. (G-quadruplexes (G4s) are DNA structures that can arise under physiological conditions from guanine-rich DNA sequences Figure  1–4). A. A1–4). y et al. . Importay et al. . This oby et al. . This gey et al. , supportin vitro models of transcription  caused DNA-damage at G4 sites in a transcription and replication dependent fashion . These studies have allowed for a far more direct investigation of the association between endogenous G4-formation and gene expression, revealing an almost inverted picture to the earlier investigations. Indeed, G4 ChIP-Seq performed in keratinocytes  and humaTo fully appreciate and investigate the role G4 formation may play in gene regulation, we need to consider the genomic context in which G4s exist. The 3D organization of genetic material in cells presents a high level of complexity, where the canonical double helix of DNA is wrapped around proteins in a macromolecular structure known as chromatin. Chromatin is made of nucleosomes: a repetitive unit consisting of histone proteins which wrap around a stretch of DNA (ca. 146 bp) contributing to the folding of DNA inside the nucleus Figure  22,23).,23.22,23REV1 were characterized by delayed or fully compromised DNA replication, particularly at G4-forming sequences .,32.10,32Despite the fact that there is now significant evidence suggesting that G4s may act as an active factor in shaping the structure of chromatin, for example by altering histone modifications and nucleosome placement, it is also conceivable that G4s form at open chromatin sites as a simple consequence of DNA accessibility and negative supercoiling that is also required for active transcription . Future In addition to nucleosome free DNA, a further structural requirement of transcription is the ability of duplex DNA to be unwound into single strands, one of which acts as the template strand for RNA polymerase, whilst the other is denoted the non-template strand. As polymerase transcribes the template strand, a three-stranded intermediate is formed comprising the two original DNA strands and the newly transcribed mRNA sequence, which make up a so-called R-loop Figure  34). In. In34). in vitro measures of transcription which may not encapsulate the complex interactions of G4s within cells. Thus, it would be worth further investigating if the presence of G4s in R-loops is similarly correlated with increased transcriptional output within a chromatin DNA context that is more representative of transcriptional processes in living cells.Such R-loops form significantly in actively transcribed genes and interestingly, genome-wide studies have also confirmed formation of G4-structures in transcriptionally active genes ,17,18. FIn contrast to this work suggesting G4s have a positive effect on transcription by stabilizing R-loop formation, it has been noted that over-stabilization of G4s can have deleterious effects on transcription. For instance, artificially enriching G4 content either by exposing cells to G4-ligands  or down-Although the enrichment of G4s in promoters of highly transcribed genes sprouted the notion that G4s may mediate transcriptional control , it is possible that this role expands beyond the local context and into additional long-range mechanisms of epigenetics. In fact, considering promoter sites directly proceeding genes constitute only a fraction of the human genome, research in this area may represent just the tip of the iceberg when it comes to investigating how G4s control gene expression. For example, a single gene may have its transcription regulated by multiple regulatory sites littered all throughout the genome—sites which are able to exert their regulatory powers over long-distances and are often dysregulated in disease states . In receOne of the most important long-range interactions utilized for epigenetic control is between transcriptional promoter and enhancer sites of DNA Figure  49). Wh. Wh49). To test this hypothesis, countless chromatin conformation capture (3C/Hi-C) experiments have been conducted, which allow for the cross-linking and downstream sequencing of DNA regions known as ‘topologically associated domains’ (TADs) that are connected not in sequence, but in space . The resin vitro with RNA polymerase, may in vivo be used to enhance rather than inhibit transcription and polymerase activity by enriching loop formation. Furthermore, these findings highlight even more how the simple strategy of considering individual G4 formation at specific gene promoters might significantly underestimate the potential roles of G4s in the regulation of gene expression.However, CTCF binding motifs are not the only sequences enriched at loop boundaries as G-quadruplex sequences are also abundant at the edges of loops . Moreovein vitro studies have demonstrated the high affinity of multiple regulatory proteins for G4 structures including, eukaryotic transcription factors SP1  and MED1 (Mediator 1) which are able to link nhancers . The liqnhancers . As suchin vitro for RNA repeat expansions, nucleobase homopolymers and some mRNAs by means of RNA–RNA interactions including base pairing, base stacking and other long-range promiscuous interactions  are enriched in promoter and enhancer regions of DNA. It is speculated that when the two guanine runs are brought into close proximity, they are able to hydrogen bond together and thus assemble into a ‘full-G4’, that may mediate promoter-enhancer interactions . SimilarThus, the formation of intermolecular or long-distance G4 structures in such regions may be a mechanism of DNA networking that stimulates LLPS and promotes enhancer activity Figure . This isAs such long-range and intermolecular interactions are favoured in crowded environments , it is pAn additional area in which epigenetic control appears at the G4 level can surprisingly be seen when considering how cells respond to environmental stresses. An example of this stress is demonstrated by guanine oxidation induced by reactive oxygen species (ROS) that arise from multiple exogenous and endogenous factors such as exposure to radiation and metabolism ,81. AmonVEGF and NTHL1 promoters .,91.87,91xidation ,89,94.Whilst mechanistically informative, a limitation of these studies is the reliance on plasmid systems with synthetically inserted G4-rich promoters which may not be representative of the role of G4s in a cellular and chromatin context. Despite this, similar results were observed when considering plasmids transfected into human glioblastoma cells where guanines were oxidized using the cytokine TNFa which naturally stimulates oxidative stress within cells, suggesting that these findings are consistent even when tested under more physiologically relevant conditions . FurtherKRAS promoter and may promote the transcription process  activity . Similar process .Altogether this work demonstrates that an apparently mutagenic event such as the oxidation of guanine bases by ROS, in a G4 context, could actually be considered as a way to epigenetically control gene expression. Such DNA damage coincides with relaxed helical tension and is particularly likely to occur in guanine-rich regions , thus prIn this review, we have explored a number of pathways by which quadruplex DNA may regulate gene transcription with an emphasis on how G4 formation may actually associate with transcriptional activation. This is in direct contrast to the initial and perhaps outdated perspective where G4s are labelled as direct inhibitors of polymerase activity and transcription. Despite this, the goal of this review is not to argue that G4s act solely as transcriptional activators, as there may be individual cases where G4s are repressive in nature , but to Early studies considered how individual G4s behave within plasmid systems or relied on ligands that artificially stabilize dynamic G4 structures and may additionally displace natural G4-binding partners. This work contributed to a narrative that depicts G4s as transcriptional roadblocks and might not reflect at all the endogenous role of these structures. Such a narrative is still often re-iterated in the current literature and reflects a simplistic view of G4 formation and transcriptional regulation that we strongly feel needs to be updated in light of recent studies. Going forward, it is important to consider the G4 not as an isolated entity within a specific genomic location, but as a structure that exists as part of an interconnected network of other biomolecules in living cells.Recent research conducted in this spirit has revealed multiple exciting mechanisms that may explain how G4s act as transcriptional regulators. This includes the role of G4s in mediating the placement of histone marks and in interacting with chromatin remodelling proteins, thus shaping the higher order structure of chromatin. Additionally, the notion that G4s can influence the formation and stability of transcriptional loops including R-loops and long-range enhancer-promoter loops has been demonstrated. Even relatively new mechanisms considering intermolecular G4s as triggers of liquid-liquid phase separation in combination with selective interactors (such as CSB) and DNA-damage induced transcriptional activation are being explored. Altogether these novel mechanisms strongly challenge the simple ‘on/off’ switch role that it is still often associated to G4 formation.By considering G4s as more than simple knot-like impediments for RNA polymerase to overcome, it will be possible to develop these new avenues of research which may be particularly important for investigations of transcriptional dysfunction in pathologies such as cancer and ageing. It follows that further research in this area may unravel a distinct layer of epigenetic regulation in which G4s are implicated as multi-faceted regulatory elements in a complex cellular environment.


Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, ‘nuclear morphogenesis’ may be a microscopic counterpart of the macroscopic process that shapes the animal body.An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in  Summary: The nuclear space becomes progressively heterogeneous during embryo development, forming distinct transcriptional sub-nuclear environments, paralleling the partitioning of the embryo body during morphogenesis. During embryogenesis, transcriptional regulation controls the expression of genes that pattern the animal body . This prDrosophila embryos at a late stage of development and that the developmental gene shavenbaby  resided in regions enriched for TFs when it is transcriptionally active , rhomboid (rho) and snail (sna) enhancers to measure their local chromatin environment during different stages of development and at different locations in the embryo. In summary, we found that the distributions of TFs and epigenetic marks became progressively heterogeneous as development proceeded. Thus, the nuclear space may undergo a transformation that parallels morphogenesis, where the animal body becomes increasingly heterogeneous and segmented. Additionally, even for the same gene, the nuclear environments at transcription sites depended on the location and timing of expression. Organizing the nucleus to form multiple reconfigurable regulatory environments in a single nucleus could permit genes to perform different tasks at different times and facilitate the complicated regulatory patterns during later stages of embryogenesis.However, the origins and spatial-temporal behaviors of these transcriptional microenvironments during development remain unclear. When does the nuclear microenvironment form? How and when do genes interact with these microenvironments? Here, we imaged the distributions of TFs and histone modifications in Hunchback (Hb) is a TF that is a gap gene in the early embryo . We obsey-axis) when the image has been shifted by a given distance compared with the original (x-axis) indicates the abundance of structures at that length scale . There was a monotonous increase in autocorrelation as the embryo ages, suggesting that Hb environments spanning several hundred nanometers became more prevalent as development progressed. Indeed, stimulated emission depletion (STED)  distributions across similar developmental stages with Airyscan and computed their autocorrelation . There was a similar upward trend, suggesting the formation of local TF environments in older embryos.To quantify the spatial distributions of Hb, we computed the spatial autocorrelation of Hb intensity G. The len (STED)  imaging n (STED) H, bottomn (STED) H, middlen (STED) H, top, tFig. S1F,G) and Engrailed , which are active within a narrower time frame during development, showed high autocorrelation when they are first expressed (stage 10) and when they reach maximum expression (stage 14) in nuclei on the ectoderm. At stage 16, when both are no longer expressed, their distributions became sparse with low autocorrelation. This two-state behavior for factors only expressed at later stages of development is unlike Hb and Kr, which showed a progressive increase in autocorrelation with intermediate states. This suggests that the general distributive properties of TFs may conform to the underlying nuclear architecture at the time of their expression.The TFs Ultrabithorax  is expressed  and imaged embryos at stages 5 and 10, in nuclei within 20 µm of the ectoderm to preserve optical resolution. We stained for H3K4me1 . However, using RNA fluorescence in situ hybridization (FISH) to detect transcription sites degraded the signal from histone modifications . To improve quantitative image analysis, we detected transcription sites without the denaturing step in RNA FISH to preserve the signal from histone modifications (Drosophila melanogaster line expressing a reporter mRNA containing MS2 stem loops driven by 18 kb of the cis-regulatory region of hb (hbBAC)   with a l (hbBAC) . This sp embryos . The mRNhbBAC transcription sites between stages 5 and 10 showed changes that are distinct from the global trend in To quantify the spatial distribution of histone marks around transcription sites, we computed the average radial intensity distribution centered on the transcription site F-J acco. We obseFig. S3A). We generated transgenic lines with MS2 reporter mRNA driven by enhancers from two genes active during this process: snail (sna) and rhomboid (rho). The constructs are named sna A2.2 W0.10 and rhoNEE, respectively. We observed that both were expressed in patterns corresponding to their endogenous genes : in an anterior-posterior band along the ventral side for sna A2.2 W0.10 . rhoNEE, on the other hand, showed no change in H3K4me1  and an increase in H3K4me3 only between early and mid cellularization . Thus, the regulatory environments around genes can change over relatively short time scales in a manner that depends on their cis-regulatory regions.To test whether we can resolve finer temporal changes in local histone environments, we focused on the 1 h cellularization process at end of the rapid nuclear division cycles at stage 5 , which wus genes , 2007  has a sharp ventral edge resulting from Snail repression and trails off gradually in the dorsal direction due to a decreasing gradient of the activator Dorsal  but differential engagement from enhancers, depending on whether the repressor Snail is present. Together, these results imply that histone environments around genes depend on the properties and states of cis-regulatory elements. These differences could refine expression patterns over time, as is the case for many developmental genes  of hunchback (hb) on a bacterial artificial chromosome (BAC) driving a yellow reporter mRNAs containing 24 MS2 stem loops generated by hb. The reporter constructs for rhoNEE and sna A2.2 W0.10 were derived from the plasmid pIB-hbP2-P2P-lacZ-αTub3 UTR (hbP2 enhancer between the restriction sites HindIII and AscI replaced with the rhoNEE or the sna A2.2 W0.10 sequence. The hbP2 promoter was retained. The plasmids were synthesized by Genscript. The constructs were injected into Bloomington Drosophila Stock Center line 27388 and integrated using RMCE by GenetiVision. Virgin flies from a transgenic D. melanogaster line containing MS2 coat protein (MCP) fused to EGFP driven by the nanos promoter  staining. The MCP-EGFP line does not contain a nuclear localization signal (NLS) to minimize MCP-EGFP forming aggregates in the nucleus in the absence of MS2 mRNAs.All Tub3 UTR  with theter from  were crorhoNEE, CTTGGGCAGGATGGAAAAATGGGAAAACATGCGGTGGGAAAAACACACATCGCGAAACATTTGGCGCAACTTGCGGAAGACAAGTGCGGCTGCAACAAAAAGTCGCGAAACGAAACTCTGGGAAGCGGAAAAAGGACACCTTGCTGTGCGGCGGGAAGCGCAAGTGGCGGGCGGAATTTCCTGATTCGCGATGCCATGAGGCACTCGCATATGTTGAGCACATGTTTTGGGGGAAATTCCCGGGCGACGGGCCAGGAATCAACGTCCTGTCCTGCGTGGGAAAAGCCCACGTCCTACCCACGCCCACTCGGTTACCTGAATTCGAGCT; and sna A2.2 W0.10, GTCGACCTAGTTCTGTTTTGTGACTCGGATTTACTATTTCGCATGGCTCCTCTTCGAACAATGTCAGTCGAGCTCTGTAGATCCCTGTGTTCCCTCTTCATTGTCAACTTGAACAAATGAGCCAGGGAACAAGGTGCAAAAATGGGACGGTCCTATTCTCAGCAAAAATTGACAAGAACAACAACAATGTCTATGGAAAATCGAACTTCATCCCAGCACCTGCAGAAATCCCGAGCGAGTCGGGGAAAAAGTATTTAACCCCCGAAAGGGTTTTCCCCAAAATAATGAAGTAATGAATGAAGCGGAAAACACTGGCCGCCAATCTACCTAATACTAATGAGCGGGCCAACCCGACCAGGAATTTTTGCAAGTCAGGTACTTCAACGGATATATGGGTTCGACAAGTGCGGATTTTCCCGCGACATCAATGAGGACTTGGCCGGGTTATCCGCGGTGCTCATCGGGCAATTCCGCGGCCGAGGACTTCATCGTAGTGATCATTAGGTAGATATGTGCATGGATGTGACATGGCGATCATTGCGCGGAATAACACACGTAATAACCGAGATATCCGGGATGACCCACCAGGTAGGATGTGAGGACATATAGAAAACCCCCAGCCAGTTTTTCCACTCGTCGTGGCTTGTTTTGCTTGAGTTTCGCTGACTGCGTAATTGGATAAGATGGGAAATTACTTTAAATCCTTCGCTGATCCACATCCGGACATTCGTCGAAGGAAAATCCATTGCAGGGAAATACGAAATGGAAATGCGGCTGGGTTATTGGCTCGACATTTCCCATCTTCCCTCACGCCATTGGTTGCAGGATCGCGGGGAATTGGAATTCCGCGCTGGAATTTTTTGTCACCTCTTGGGTTTATCAAAACTTTTGGGTTTGCTATGGATTTTTTCCAATTTTACCACCGCGCCTGGTTTTTTTTTTTTGACGACGCGGAAAATCGGACTTGGCTATGCGGGCTTGTCTGTTTTTCCGGGTACAAAGTCTGCATGTCAGCCTCCATGCGGGAGTGGGAGTTGGGAAAGTTTCCCATCGATAGTTGGAGGGGTGGCTTGAAAGTCTGGAGGTGCTAGCTGGGAAAGTTGTGTGTGCGCGATGAGGCAAGGAGTCAAAGATCAGGGGAGTTGGAAAGCGAGAATTGTGGGAATCGTCCAGGACTCAGCTGGATGCTGAGGGGCAGTATGATTTTTTTTACGTTATCAATCGAATTGATTTTAAGACAGCAGAACTTCACATACTAATAAGATGACCATGGGATTAGTTAAAATGTGTAACTCGTATTCGAATCGTCATTCTTTCACGGACCAATCGTGGGAACAGGAGATCTCTTCGATCCAAGCTCACAGGAGACTTGACACTCTTCGTCTATTCCTTGTCAAGTTTTTAATGACATCTCCTATGCCCTGAGCTATGTTTTCCTAGCTCTCATCGATCGCTGCCAATGAGCCACTGGAGATGATCCATAAGTCAGCGTAGAGTGCACCCCAGAGTTGACACTTGGTGTCTCGGAATTCGGCTCATTATCAGTGCTATTTTTGGAACACCTCTCTGCGAAGGTGTCATTTTTGTCAGTGCGTATCGCTCAGGTTCAACTCCCCACCAAAAACCGAATTTAGAGCATCGGCAGATGTACTTGAAGCACTCAATCTAAGTGAGGAAACCACCCCATGAACGAAGAGTACTAGGAGTCCTATTTGACTCGTGCTTAAAAATAGAAAATTACTTAGGGTGATCCATAGGTAGGGAGGCGATATTGTAACTTGCATTTCGGACCCGGACCTGCACGAGTTATTACGGGTGGGTTGTGAGCGTATCGGGAAATTGGAGAGCCACCAGATCTGTCATAACTTATACGGGGGATCCTTATTCCTGGGAGGGTGCGCCTGCGTCTGCTCTTCCGAGAGAGAGGTGGGAAATGGAGGAAGAGAGAGAGAGAGAGAGTGAGAGAGCAGGTAGAGGGAAGTGAGGGAAATACGCAATAAGGGTATGGGAAAAGTGCTGTTGTTGTTGCTAGGTAGCGACGCACACGTGCGAGTGTTTTTCTGTTTTGAAGAAGAACCACCACCAAATGG.The enhancer sequences were as follows: 1118w flies (for imaging TFs) and from crosses of the hbBAC, rhoNEE or sna A2.2 W0.10 reporter lines with the MCP-GFP line (for imaging transcription sites and histone modifications) were collected, fixed and IF stained using previously described protocols ;The secondary antibodies were as follows: Alexa488 donkey anti-mouse ; Alexa488 donkey anti-rabbit ; Alexa555 donkey anti-rabbit ; Alexa594 donkey anti-mouse ; and STAR RED goat anti-rabbit .in situ hybridization (FISH) and histone modifications using IF, we followed a previously described FISH protocol . One additional secondary antibody was used: Alexa488 donkey anti-sheep .To stain for transcription sites using RNA fluorescence protocol  and the x-y  and z direction  of stacks used for quantification. The laser power and gain were adjusted to maximize the signal to noise ratio within the dynamic range of the PMT or Airyscan detector for each sample preparation condition (i.e. different antibodies). Within a given sample preparation condition, laser power and gain were kept the same across embryos. The acquired Airyscan stacks were processed with Zen 2.3 SP1  in 3D mode to obtain super-resolved images.Confocal and Airyscan  imaging Drosophila embryos was carried out in ProLong Diamond without DAPI . Fixed embryos at the appropriate stage and orientation were imaged on a STEDYCON  in 2D STED mode on an Olympus BX53 microscope. Excitation lasers with wavelengths of 594 and 640 nm were used as appropriate for the specific fluorescent dyes. The wavelength of the STED laser was 775 nm. All samples were imaged using an Olympus UPlanSApo 100× Oil/1.4 objective. The resolutions used for the x-y and z directions were 25 nm and 250 nm, respectively. The laser power and gain were adjusted to maximize the signal-to-noise ratio within the dynamic range of the APD detector on the STEDYCON for each sample preparation condition. Within a given sample preparation condition, laser power and gain were kept the same across embryos.Mounting of fixed r = 0. Radially averaged auto-correlation functions were computed using an ImageJ macro . Airyscan images were used for the autocorrelation of Hb and Kr for their wider field of view compared with STED, in order to include a larger number of nuclei. Intensity distributions and autocorrelation functions were plotted using Matlab (MathWorks).Image processing was carried out using Fiji  with the


Drosophila Hox TF Ultrabithorax (Ubx) in the mesodermal and neuronal tissues using bioinformatics. Our investigations showed that Ubx preferentially interacts with multiple yet tissue-specific chromatin sites in putative regulatory regions of genes in both tissues. Importantly, we found the classical Hox/Ubx DNA binding motif to be enriched only among the neuronal Ubx chromatin interactions, whereas a novel Ubx-like motif with rather low predicted Hox affinities was identified among the regions bound by Ubx in the mesoderm. Finally, our analysis revealed that tissues-specific Ubx chromatin sites are also different with regards to the distribution of active and repressive histone marks. Based on our data, we propose that the tissue-related differences in Ubx binding behavior could be a result of the emergence of the mesoderm as a new germ layer in triploblastic animals, which might have required the Hox TFs to relax their binding specificity.Transcription factor (TF) networks define the precise development of multicellular organisms. While many studies focused on TFs expressed in specific cell types to elucidate their contribution to cell specification and differentiation, it is less understood how broadly expressed TFs perform their precise functions in the different cellular contexts. To uncover differences that could explain tissue-specific functions of such TFs, we analyzed here genomic chromatin interactions of the broadly expressed  Drosophila Hox TFs provide an ideal model.The development of multicellular organisms is coordinated by distinct sets of transcription factors (TFs) that work in networks to initiate cell fate decision, specification, cell localization and tissue differentiation ,2,3. TFsHox genes are evolutionary conserved and specify the segment identity along the anterior-posterior axis of the developing embryo [g embryo ,9,10,11.g embryo ,13,14. Fg embryo , or fromg embryo ,17. All g embryo . DespiteTo contribute to the field and tackle this question in a global fashion, we used the Hox TF Ultrabithorax (Ubx) and bioinformatic approaches to analyze the binding behavior and preferences of Ubx in two tissues, the mesoderm and the neuronal system. These investigations showed that Ubx binds preferentially multiple chromatin sites within single genes, and interacts with the same genes in different tissues but mostly via tissue-specific chromatin sites. In order to identify the origin of these differences, we investigated the chromatin landscape and the Ubx binding motifs within these regions, which revealed that Ubx interacts primarily with non-canonical Hox/Ubx motifs in the mesoderm, while it preferentially uses the classical Hox/Ubx motif for chromatin interactions in the neuronal system. Intriguingly, these novel binding motifs were predicted to have lower binding affinities for Ubx than the classical Hox/Ubx motif. Finally, we also found that the distribution of histone marks at Ubx chromatin sites is different in the mesoderm and the neuronal systemIn sum, our analysis of Ubx binding behavior in two tissues identified important differences with regards to underlying sequence patterns, binding affinities and binding site distributions, which we hypothesize to be a consequence of mesoderm development and evolution.Hox proteins are active in most cell types within a segment. On example is the Hox protein Ubx, which is expressed in the ectoderm and nervous system from para-segment 5 to 12 corresponding to the segments T3 to A6, while Ubx expression in the mesoderm is found in A1 to A6 A–C. Intrmidline (mid) locus: Ubx interacts with the mid promoter in both tissues, while the remaining interactions are distinct (We aimed at closing this gap by using the Hox protein Ultrabithorax (Ubx) and its chromatin interactions in two distinct tissues, the mesoderm and the neuronal system, as a model. To this end, we re-investigated existing Ubx ChIP-Seq profiles retrieved from neuronal and mesodermal cells at two developmental time points: embryonic stages 10–13 (4–9 h after egg lay (AEL), early time point, specification) and embryonic stages 14–17  . This medistinct F.Drosophila embryos.Taken together, the differential binding analysis revealed that Ubx interacts with different chromatin sites in the mesoderm and the nervous system of We next wanted to characterize the differential binding behavior of Ubx in more detail. To reduce the complexity of the data, we focused on the stages 10–13 for the subsequent analyses. We first asked how many of the Ubx chromatin interactions occur in the same or different genes in both tissues. To this end, we associated Ubx chromatin peaks to genes, which revealed that 35% of genes were bound by Ubx in both tissues, which will be referred to as “common genes” in the rest of the manuscript (3552 overlapping/common genes). The remaining genes were specifically bound only in the mesoderm  or the neuronal system  . These dteyrha-meyrha (tey), an E3 ubiquitin ligase encoding gene that is multiply bound by Ubx peaks in the mesoderm and neuronal tissue (snail (sna), a transcription factor encoding gene which is bound only once in both tissue contexts  and multiple peaks (multiPeaks) using the WEADE tool . We founl tissue F, and ancontexts G. Intrigcontexts E.In sum, this analysis showed that Ubx interacts at the same time with many genes in the mesoderm as well as the neuronal system, and does so preferentially via multiple and tissue-specific chromatin sites.So far, our analysis has focused on the “common genes” category. In a next step, we wanted to elucidate whether Ubx binding to the same genes in different tissues is of different quality. To this end, we focused on those genes bound by Ubx multiple times in enhancer regions  in both tissues, and searched for over-represented DNA motifs. To increase specificity of this search, we sub-divided the Ubx peaks in these regions into three categories: (1) Ubx peaks within common genes that are only present in enhancer regions in the mesoderm, meso_ONLYpeaks, (2) Ubx peaks within the common genes that are only present in enhancer regions in the neuronal system, neuro_ONLYpeaks, and (3) Ubx peaks within the common genes that are present in both tissues within a 2 kb region, which we considered to be due to their close distance part of the same regulatory control element and thus bound by Ubx in both tissues, 2kb_common. Interestingly, this categorization of peaks uncovered that the majority of Ubx chromatin peaks (around 75%), despite being present in both tissues in the same genes, are specific to one of the two tissues, and only a smaller proportion was present at the same location in both tissues B.We next performed motif enrichment analysis in all three categories using MEME ,23. We fAfter having identified novel sequence patterns within Ubx-bound chromatin regions, we asked whether the known and novel Hox/Ubx motifs are different with respect to predicted affinity or location in the gene loci. Our analysis of peak distribution in regulatory regions revealed that the classical Hox/Ubx motif, which was the one found over-represented among the neuron-specific Ubx chromatin interactions, was located preferentially in intronic regions. In contrast, Ubx binding in the mesoderm is different, as most of the novel, STREME-identified Hox/Ubx binding sequences were more or less evenly distributed in intronic or intergenic  regions D. Using Taken together, the analysis indicated that Ubx primarily binds to the classical Hox motif in the neuronal system, while it interacts with novel, low affinity sequences in the mesoderm.In the next step, we examined whether the genes commonly bound by Ubx in the mesoderm and neuronal system are characterized by other features than differences in the underlying Hox/Ubx sequence motifs. Regulatory regions and their activity are defined by the chromatin status. Thus, we analyzed the distribution of two histone modifications, H3K27ac and H3K27me3, at tissue-specific Ubx peaks located in enhancer regions of gene loci in the two different tissues. Both histone marks are key modifications of the nucleosome component Histone H3, with H3K27me3, represents a mark associated with repressive chromatin ,26, whilTaken together, these results showed that the chromatin sites bound by Ubx specifically in one of the two tissues behave differently with regards to two histone marks. Our finding that mesoderm-specific Ubx sites marked by H3K27ac carry the same histone mark in the nervous system was surprising, in particular as the associated genes primarily correlated with epidermal/neuronal tissue development.The evolutionary conserved Hox proteins perform very specific functions in vivo; however, in vitro, they bind rather generic and frequently occurring DNA sequences. This raises two important questions related to Hox specificity: first, how do different Hox proteins control segment-specific features, and second, how does a single Hox protein regulate the development of different cell and tissue types present in one segment in a specific manner. The first question is related to all Hox proteins and has been extensively studied in the last decades ,30,31. HDrosophila mesoderm and neuronal system. We found that Ubx interacts with highly specific and non-overlapping chromatin sites even when interacting with the same gene locus. Most genes were bound by Ubx at multiple sites located in putative regulatory regions , while single Ubx binding events were less frequent. Analysis of the Ubx sites identified the classical Hox/Ubx binding motif to be enriched in regions bound by Ubx in the neuronal system, but not the mesoderm. In this tissue, a variety of sequences with similarities to the classical Hox motif were found, which we assume to represent divergent Ubx binding motifs specifically related to the mesoderm. Finally, tissue-specific Ubx sites were not only different in the underlying sequence patterns but also in the distribution of active and repressive histone marks. In sum, these results revealed for the first time that Ubx binding in different tissues is highly specific with regards to the location, sequence patterns and underlying chromatin marks.To close this gap, we investigated the binding behavior of the Hox protein Ubx in the Drosophila mesoderm emerges from the neuro-ectoderm and is determined when the predominantly active histone marks  set at gene regulatory regions during the activation of the zygotic gene program are complemented or replaced by repressive histone marks (H3K27me3) [One puzzling result was our finding that regions marked as “active” and specifically bound by Ubx in the mesoderm were also in the “active” chromatin configuration in the nervous system, in particular as the genes associated with these regions were predicted to function primarily in the nervous system. This distribution of chromatin marks at tissue-specific Ubx sites indicates that the Hox control of nervous system is more restricted and fixed, as active marks at Ubx sites are related to and exclusive for the tissue, which is not the case in the mesoderm. How could such divergent and more “relaxed” binding behavior of the Hox TF Ubx in the mesoderm be explained? Several aspects related to mesoderm development might be helpful to formulate a hypothesis. First of all, the mesoderm is one of the three germ layers present in Bilateria, while a “true” mesoderm, which arises as a result of gastrulation, is absent in Cnidaria, a group of dipoblastic, radially symmetric organisms ,33. Thus3K27me3) ,40,41. TBased on these assumptions, we hypothesize that the emergence of a new tissue, the mesoderm, might have required the Hox TFs, which define different body parts and thus need to be active in all tissues, to “relax” their molecular properties, such as their binding behavior and preferences, to enable this major transition from diploblastic to triploblastic organisms, and this change in binding preferences seems to be co-adapted for Hox tissue specificity.1118w fly line served as a wildtype and embryos were prepared for staining and stained as described in [The ribed in , and theribed in ). The emribed in .The data used in the manuscript was deposit on NCBI Gene Expression Omnibus: GSE121752, and annotated against genome dm6. The data sets contained Ubx ChIP-Seq peaks and histone mark disruptions of H3K27ac and H3K27me3 from the mesoderm and neuronal system, as well as of embryonic stages 10–13 and 14–17. Bioinformatics analysis was performed as described in  analysis


Schistosoma japonicum could inhibit the expression of COL1A1 in activated HSCs. In addition, studies have found that Ets proto-oncogene 1 (Ets-1) suppresses the production of ECM by down-regulating matrix related genes such as COL1A1 induced by transforming growth factor β, and ultimately inhibits liver fibrosis. In this study, the major aim was to investigate the effect and mechanism of Ets-1 on inhibiting COL1A1 gene promoter activity in HSCs by recombinant Schistosoma japonicum protein P40 (rSjP40). We observed the rSjP40 inhibited the expression of COL1A1 by inhibiting the activity of the COL1A1 promoter, and the core region of rSjP40 acting on COL1A1 promoter was located at -1,722/-1,592. In addition, we also demonstrated that rSjP40 could promote the expression of Ets-1, and Ets-1 has a negative regulation effect on the COL1A1 promoter in human LX-2 cells. These data suggest that rSjP40 might inhibit the activity of COL1A1 promoter and inhibit the activation of HSCs by increasing the expression of transcription factor Ets-1, which will provide a new experimental basis for the prevention and treatment of liver fibrosis.Liver fibrosis is a severe disease characterized by excessive deposition of extracellular matrix (ECM) components in the liver. Activated hepatic stellate cells (HSCs) are a major source of ECM and a key regulator of liver fibrosis. Collagen type I alpha I (COL1A1) is one of the main components of ECM and is a major component in fibrotic tissues. Previously, we demonstrated that soluble egg antigen from  Liver fibrosis is a common pathological change of chronic liver disease, which usually occurs after chronic liver injury. It is known that the main processes of liver fibrosis are the activation of hepatic stellate cells (HSCs) and subsequently the excessive deposition of extracellular matrix (ECM) components . After tSchistosoma japonicum protein P40 (SjP40) is one of the main components in the SEA, which belongs to the heat shock protein family of schistosomiasis -induced hepatocyte apoptosis and accelerates liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH) mice , a member of the Ets transcription factor family, has been reported to be involved in fibrosis progression by regulating fibroblast activation and proliferation, and regulating the synthesis and degradation of ECM. Studies have found that up-regulation of Ets-1 expression enhances transforming growth factor SH) mice . Howeverfibrosis . HoweverIn this study, we observed that whether the transcription factor Ets-1 was participated in the inhibition of COL1A1 in human HSC cell lines LX-2 cells treated with rSjP40.COL1A1 promoter sequences constructed as previously described  supplemented with 10% FBS . Cells were maintained in a humidified incubator containing 5% COCell lysates were prepared on ice for extracting proteins using RIPA buffer containing protease inhibitor (1 mM) and phosphatase inhibitors (1 mM). An equal amount of each lysate (50 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After the proteins were transferred onto polyvinylidene difluoride  membranes, the membranes were blocked in TBST containing 5% nonfat milk for 1 h and then incubated with the indicated primary antibodies diluted with 5% nonfat milk overnight at 4 C. The primary antibodies used for the western blot were as follows: mouse antibody against COL1A1  (1:200 dilution), and rabbit antibody against Ets-1  (1:500 dilution) or GAPDH  . After being washed for five times with TBST for 10 min, the membranes were incubated with the indicated secondary antibodies (horseradish peroxidase (HRP)-conjugated anti-mouse IgG  and HRP-conjugated anti-rabbit IgG  ) for 1 h at room temperature. Then the protein bands were visualized with an ECL reagents  using Image Lab software . GAPDH was viewed as an internal control. Finally, Image J  software was used to quantify the intensity of the protein bands.COL1A1 promoter and the pRL-TK reporter plasmids were cotransfected into LX-2 cells according to the manufacturer’s instructions of FuGENE . After transfection for 18 h, LX-2 cells were treated with rSjP40 or not. The cells were then harvested after 48 h of stimulation. Dual-luciferase reporter assay was performed and the firefly and renilla luciferase activities were detected.The indicated plasmids of 4 cells per well. When cultured to 70–90% confluency, 4 μL of lipofectamine 2000 reagent  was diluted with 100 μL of DMEM and incubated at room temperature for 5 min. Next, 2 μL of siRNA of Ets-1 or negative control siRNA were combined with lipofectamine 2000/medium mixture and allowed to complex by incubation for 20 min at room temperature. The mixed solution was added to 1 ml of cell in the twelve -well plate. After transfection for 4–5 h, the culture medium was replaced with the fresh medium and the cells were incubated in the presence or absence of 20 μg/ml rSjP40 for another 48 h.LX-2 cells were seeded in a twelve-well plate at a density of 5×10COL1A1 promoter.ChIP assay was carried out by SimpleChip Kit . Immunoprecipitation was performed with anti-Ets-1 antibody at 4°C overnight and normal IgG provided in SimpleChip Kit was used as a negative control. Precipitated DNA was analyzed by PCR using the primers as: 5′-CAATGGAATCTTGGATGG-3’ (sense); 5′- TGAGAAACTCTGTAGGGC-3’ (antisense), which were designed based on the Ets-1-binding sites on the p-value < 0.05 was considered significant.All experiments were analyzed by the Student’s t test. All values included in the figures represent mean ± SD. A COL1A1 promoter is located upstream of transcription initiation and plays a key role in regulating COL1A1 transcription. In this study, we further investigated the effect of rSjP40 on the transcriptional activity of COL1A1 expression. We constructed the COL1A1 promoter plasmid pGL3-COL1A1 . The fluorescence reporter plasmid containing COL1A1 promoter was transfected into LX-2 and detected by dual luciferase reporter assay. Dual luciferase reporter gene assay showed that significantly higher activity was observed in pGL3-COL1A1 than pGL3-basic (p < 0.05, COL1A1 promoter activity in a concentration-dependent manner (COL1A1 promoter.In our previous studies, we have confirmed that rSjP40 could inhibit COL1A1 expression in LX-2 cells . COL1A1 t manner . Hence, COL1A1. We used bioinformatics to analyze COL1A1 promoter sequences and predict possible binding sites for common transcription factors. Truncated mutation of transcription factor binding site was carried out and five luciferase reporter gene plasmids were constructed for experimental study (COL1A1 promoter (COL1A1 at the core region of -1,722/-1,592.Further, we attempted to seek the core region at which rSjP40 could inhibit the promoter activity of al study : pGL3-COal study . Then thpromoter  were trapromoter . Howeverpromoter . Comparepromoter , we confCOL1A1 promoter activity, we used JASPAR and PROMO database to predict the possible transcription factor binding sites in the main active region of COL1A1 promoter -1,722/-1,592. The predicted transcription factor was Ets-1, which was located at -1,679/-1,673 and shared similar loci in both databases (COL1A1 promoter at the region of -1,679/-1,673. To perform the quality control experiment of ChIP kit, anti-Histone H3 antibody was used as the positive control and the template was then harvested to perform PCR using RPL30 primers provided in the ChIP kit.To further explore whether transcription factors are involved in rSjP40 inhibiting atabases . TherefoCOL1A1 promoter by regulating the transcription factor Ets-1, we mutated the base "T" at the ETS-1 binding site -1,677 on the COL1A1 promoter into "A" and labeled it as pGL3-COL1A1 mut. The effect of rSjP40 on COL1A1 promoter was detected by dual luciferin reporter gene. We found that rSjP40 could inhibit the luciferase activity of COL1A1 promoter (pGL3-COL1A1, COL1A1 promoter (pGL3-COL1A1 mut, COL1A1 promoter. Then we further explored whether rSjP40 affects COL1A1 expression by regulating Ets-1. Successful knockdown of Ets-1 in LX-2 cells were confirmed by western blot (COL1A1 promoter activity and COL1A1 expression in LX-2 cells through ETS-1 dependent mechanism.Previous studies have shown that Ets-1 is expressed in HSCs and regulates the transcription of ECM genes . We furtern blot . And knoern blot . These rLiver fibrosis is a typical response to chronic liver disease and is characterized by large and excessive extracellular matrix in the liver. Currently, liver fibrosis is considered to be an evolutionarily conserved wound healing response to tissue injury, primarily driven by inflammatory and immune-mediated mechanisms . Liver fSchistosoma japonicum and Schistosoma mansoni, which are inflammatory diseases that cause fibrosis and portal hypertension (cercaria in the deposited eggs, which destroys the normal tissues of the host through chronic granulomatous inflammation mediated by T lymphocytes (Schistosoma mansoni, and they have high homology in amino acid sequence. SjP40 is often deposited in tissues in clusters to form larger granulomas. It has been reported that SjP40 and its antibody can be detected from the host as early as 21 days after the infection of Schistosoma japonicum, and is considered as a potential candidate antigen for the early diagnosis of schistosomiasis (Schistosoma japonicum egg protein rSjP40. Our results indicate that rSjP40 can inhibit the expression of COL1A1 in LX-2 cells, which is consistent with our previous results, demonstrating that rSjP40 can inhibit liver fibrosis by inhibiting the synthesis of COL1A1 in HSCs (COL1A1 promoter. The results of this study proved that rSjP40 can inhibit the activity of COL1A1 promoter. At the same time, it was confirmed for the first time that the core region of rSjP40 acting on COL1A1 promoter was located at -1,722/-1,592.Schistosomiasis is mainly caused by rtension . These pphocytes . Howeverphocytes . The maisomiasis . Then wh in HSCs . In thisCOL1A1 promoter activity by regulating Ets-1. CHIP experiments showed that Ets-1 could directly bind to the COL1A1 promoter at the site-1,679/-1,673. Then we mutated the base "T" at -1,677 of the Ets-1 binding site on the COL1A1 promoter into "A", and the activity of the COL1A1 promoter significantly increased, indicating that Ets-1 has a negative regulation effect on the COL1A1 promoter in human LX-2 cells. In addition, rSjP40 could not inhibit the activity of the mutated COL1A1 promoter at the Ets-1 binding site, further confirming the role of rSjP40 by affecting Ets-1. Finally, knockdown of Ets-1 in LX-2 cells reversed rSjP40-induced down-regulation of COL1A1 expression.As one of the most widely studied transcription factors in the Ets family, Ets-1 is involved in cell proliferation and apoptosis, angiogenesis, and tumor progression . StudiesCOL1A1 promoter by increasing the expression of transcription factor Ets-1, down-regulating the expression of COL1A1, thereby inhibiting the activation of HSCs and inhibiting liver fibrosis. The results of this study will provide a new experimental basis for the prevention and treatment of liver fibrosis.In conclusion, this study indicates that rSjP40 inhibits the activity of


Pleurotus ostreatus. Culture media composition was tested to choose the nitrogen source. Yeast extract (YE) was selected as a better nitrogen source than ammonium sulfate. Then, the effect of glucose and YE concentrations on biomass production and LA as response variables was evaluated using central composite experimental designs with and without copper. The results showed that the best culture medium composition was glucose 45 gL−1 and YE 15 gL−1, simultaneously optimizing these two response variables. The fungal transcriptome was obtained in this medium with or without copper, and the differentially expressed genes were found. The main upregulated transcripts included three laccase genes  regulated by copper, whereas the principal downregulated transcripts included a copper transporter (ctr1) and a regulator of nitrogen metabolism (nmr1). These results suggest that Ctr1, which facilitates the entry of copper into the cell, is regulated by nutrient-sufficiency conditions. Once inside, copper induces transcription of laccase genes. This finding could explain why a 10–20-fold increase in LA occurs with copper compared to cultures without copper when using the optimal concentration of YE as nitrogen sources.This research aimed to establish the relationship between carbon–nitrogen nutritional factors and copper sulfate on laccase activity (LA) by  Fungal laccases are glycosylated, multi-copper oxidases that catalyze the oxidation of hydroxyl functional groups on various substrates and the molecular oxygen reduction to water ,2,3. As Pleurotus ostreatus is a white-rot fungus biotechnological model for studying and producing fungal laccases [P. ostreatus cultures is affected by complex and not fully understood gene expression regulatory mechanisms at multiple levels.laccases . This fulaccases , its genlaccases , and it laccases . HoweverP. ostreatus monokaryotic (mk) strains, mkPC15 and mkPC9 [lacc1, lacc4, lacc6, lacc7, lacc9, lacc10, and lacc11), two on chromosome XI (lacc5 and lacc12), and one on chromosomes IV, VII, and VIII  [Twelve laccase genes have been identified in nd mkPC9  distribucis-acting elements promoter upstream of the transcriptional start [cis-acting promoter elements differ for each laccase gene on P. ostreatus. They can be divided into three groups: (i) response to nutrient-sufficient conditions (carbon and nitrogen sufficient) including catabolic responsive elements (CREs) and nitrogen binding site (NIT); (ii) response to inducers, such as metal responsive (MRE), xenobiotic responsive (XRE), and antioxidant responsive elements; (iii) response to stress, including heat shock sequence (HSE), and stress responsive elements (STREs) [Laccase gene expression regulation is controlled by al start . In addi (STREs) ,14. The  (STREs) ,16. In alacc2 gene, and small subunits produced by translating two different genes [Other laccase regulation mechanisms occurring at the post-transcriptional and post-translational levels could explain changes in the molecular weight of laccase isoenzymes or the heterogeneity observed in the electrophoretic migration of their isoforms . Most isnt genes ,18. Concnt genes . N-glycont genes ,21. On tnt genes . On the nt genes .Copper is a transcription inducer for some laccase genes and a cofactor in their catalytic center. This metal is present in the environment, and it is taken up by high-affinity transmembranal copper transporters of the CTR family .Because of all these regulatory levels, a wide range of maximum laccase activities have been reported for submerged fermentation processes aimed at the industrial production of enzymes or enzymatic cocktails to be used in the treatment of lignocellulose wastes in industrial processes.P. ostreatus senses carbon and nitrogen sources and concentrations through different mechanisms and pathways [4+) or L-glutamine as nitrogen sources [pathways  that difpathways . Catabolpathways , turns opathways . CCR is pathways . On the  sources ,31. In f sources . Both ca sources ,34,35.P. ostreatus is currently scarce, this work aimed to consider the interaction between the availability of nutritional factors  and copper sulfate at 1 mM to determine how the medium composition affects laccase induction by copper. The results to be presented indicate that carbon and nitrogen availability influence the laccase activity only when copper is present in a nutrient-sufficient condition where the transcription of nmr1 and copper transporters are upregulated.As systematic research on the interactions between nutritional factors on the laccase activity of 4∙5H2O (>98.0%); ammonium sulfate, (NH4)2SO4 (99%); calcium chloride, CaCl2 (99.9%); magnesium sulfate heptahydrate, MgSO4∙7H2O (99%); potassium chloride, KCl (99%); potassium phosphate monobasic, K2HPO4 (99%); thiamine hydrochloride (99%) sodium citrate dihydrate HOC(COONa)(CH2COONa)2∙2H2O (>99%), and sodium hydroxide anhydrous pellets, NaOH (>98%) were purchased from Merck . The citric acid (99.5%), malt extract agar, and yeast extract were supplied by Scharlau . 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonate), ABTS (>98.0%), 3,5-dinitrosalicylic acid, and DNS (>98.0%) were manufactured from Sigma–Aldrich, St. Louis, MO, USA. D(+)-Glucose anhydrous PA-ACS and sodium acetate anhydrous (99.9%) were obtained from PanReac AppliChem . Acrylamide and bis-acrylamide solution , 0.5 M Tris-HCl, pH 6.8, 1.5 M Tris-HCl, pH 8.8 solutions were provided for Bio-Rad, Alcobendas, Spain.Copper (II) sulfate pentahydrate, CuSOPleurotus ostreatus strain ANDES-F515, provided by the Laboratory of Mycology and Phytopathology of the Universidad de Los Andes-LAMFU and deposited in the ANDES Natural History Museum (MHN ANDES), was isolated in the Bosque de la Merced, Santa Bárbara village, Bojacá, Cundinamarca, Colombia. The dikaryotic mycelium was maintained and conserved on malt extract agar at 4 °C, with periodic replication of the growth zone for eight days of incubation at 25 °C in the dark.The −1 of (NH4)2SO4 were added to a minimum salt medium (MSM) composed of 0.5 gL−1 K2HPO4, 0.25 gL−1 MgSO4∙7H2O, 0.1 gL−1 CaCl2, 0.5 gL−1 KCl, 0.5 gL−1 thiamine, 2.1 gL−1 citric acid, 2.94 gL−1 sodium citrate, and 6.5 final pH adjusted with 0.1 M NaOH. For the experiments made using an organic nitrogen source, yeast extract 5 or 10 gL−1, and glucose were the only medium components. Glucose  was added to achieve different C:N ratios in both experimental conditions.The submerged fermentation (SmF) cultures were performed in 250 mL flasks containing 100 mL of culture medium. For the experiments made using an inorganic nitrogen source 1.0 or 10.0 gLThe elemental compositional analysis of yeast extract (YE) used in this study was performed and revealed a C:N ratio estimated at 4:1.−1) and YE  according to the results from the experimental design shown in 4∙5H2O (0.25 gL−1).Only glucose and YE were used in the culture media to evaluate models of surface response optimization using a central composite experimental design. The composition of the media were pairwise combinations of glucose . A laccase activity unit was defined as the enzyme required to oxidize 1 μmol ABTS per minute at 25 °C. Reducing sugars were measured using the 3,5-dinitrosalicylic acid (DNS) reagent by DNS method at 540 nm [Laccase activity was determined with ABTS ) as the substrate . The asst 540 nm  using glt 540 nm .v/v) acrylamide/bis-acrylamide and were adjusted with 25% (v/v) of buffer solution at 0.5 M Tris-HCl, pH 6.8, and at 1.5 M Tris-HCl, pH 8.8, respectively. The running buffer was 0.025 M Tris–0.192 M Glycine pH 8.3. Samples were mixed with native sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue. Native-PAGE was running in the Mini-PROTEAN Tetra Vertical Electrophoresis Cell  at 100 V. The isozymes were revealed through 30 min gel staining with ABTS 2 mM.Non-denaturing and non-reducing electrophoresis conditions (native-PAGE) were used to visualize the isoenzymes present in samples from the liquid phase of the culture medium . The staSamples with tiny pellets produced in the culture media up to day 7 were taken with a plastic Pasteur pipette (graduated from 1 mL). The samples were placed in a 1 mL Eppendorf tube and then carefully took a single pellet  to be pl−1 glucose and 15 gL−1 YE, with or without 1 mM CuSO4, were harvested on day 12 of culture for RNA isolation. The mycelium was collected by filtration, immediately frozen in liquid nitrogen, and ground to a fine powder in a mortar. Then, 100 ng of the powder were transferred to a 1.5 mL microcentrifuge tube for total RNA extraction using a Fungal RNA EZNA Kit  according to the manufacturer guidelines. Finally, the integrity and quantity of RNA were validated using Bioanalyzer (version 2100) and Qubit 2.0 fluorometer.The fungal biomass produced in the cultures containing 45 gLSar1, gapdh1, actin1, and pep were used as reference genes for normalization (Reverse transcription (RT) was performed using 800 ng per sample of the total RNA to obtain cDNA in a 20 µL volume using an iScript cDNA synthesis kit . The complete reaction mix was incubated according to manufacturing instructions in a thermal cycler . RT products were diluted 1:20 and kept at −20 °C until real-time qPCRs were performed in a CFX96 real-time system  using SYBR green dye to detect the product amplification . Each relization .https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/ (accessed on 21 July 2020)). The sequence available at www.genome.jgi.doe.gov/PleosPC15_2/PleosPC15_2.home.html (accessed on 7 December 2021) was used as a P. ostreatus genome reference to align the resulting reads STAR v2.3.16 [www.sandberg.cmb.ki.se/media/data/rnaseq/rpkmforgenes.py (accessed on 30 July 2020)) to calculate values of reads per kilobase of transcript per million mapped reads (RPKM).Illumina compatible libraries were prepared to be sequenced using the Illumina Nova Seq 6000 system from an mRNA isolate originating from RNA total. Following sequencing, RNA-seq data were filtered for assurance quality using FastQC and trimmed with BBDuk to remove adapters and low-quality reads  < 0.05 as the cut-off for statistical significance were used. A DEG with log2 Fold Change ≥ −2 was established as an upregulated gene, and log2 Fold Change ≤ −2 was established as a downregulated gene.Differentially expressed gene (DEG) analyses were performed using the EdgeR Bioconductor package and a dispersion parameter of 0.1. These analyses determined the transcriptional changes in the two culture conditions by comparing the gene expression values based on transcript reads. The gene expression values with LogGene annotations were based on the Joint Genome Institute (JGI) automated annotation to transcript identifications of the mkPC15 v2.0 reference genome. JGI automated annotation uses the following databases: Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), InterPro (IPR), Eukaryotic Orthologous Groups (KOG) and Enzyme Commission numbers (EC number), Transporter Classification Database (TCDB), Carbohydrate-Active EnZymes (CAZymes) and MEROPS database (proteolytic enzymes) . In the ® version 18 software was used to construct the statistical design, evaluate statistical significance, obtain the regression models, and find the simultaneous local optimum of one or more response variables.MinitabA principal component analysis (PCA) was performed to explore the correlations between nutritional variables, glucose, ammonium sulfate, and yeast extract with the production of fungal biomass and laccase activity with and without copper sulfate. It was conducted using the data shown in y) and the terms (β y ε) of Equation (1) or Equation (2) regression model that allows for the calculation of an optimal point.The central composite experimental design allows estimating the curvature of a response surface for a chosen response variable  and 14:1 C:N in their composition.The effect of nitrogen source and carbon–nitrogen ratio (C:N) were evaluated in submerged fermentation (SmF) cultures of The principal components analysis (PCA) results are shown in To explore if nutrient-sufficient or nutrient-limited culture conditions induced morphological changes in the mycelium, we studied the distribution of clamp connections in apical hyphae of the fungal pellets. P. ostreatus. Few vacuolated hyphae were observed in the cultures with nitrogen-sufficient conditions  results are shown in p-value < 0.05) the changes in biomass production as a function of glucose and YE concentrations with R2 0.8231 and 0.8035 for biomass production with and without copper sulfate, respectively. On the other hand, the laccase activity induced by copper sulfate was explained by a quadratic model  with glucose and YE concentration as varying factors (R2 0.9560); however, in the absence of a laccase inducer  in the culture, the media composition was statistically insignificant  and insufficient (R2 0.4039) to explain this response variable.The CCD model fit to a quadratic model explained  for cultures with and without copper. Optimum biomass production in the culture medium with copper was experimentally slightly better, and the model successfully predicted this result.According to the CCD models, the contour plots a,c showeIn the case of maximum laccase activity b,d, the P. ostreatus in the presence and absence of 1 mM copper sulfate. For this characterization, biomass production, glucose consumption, total protein, and laccase activity were measured at different culture times until day 21 (maxµ) were 0.52 and 0.69 d−1 for the copper-free and copper sulfate-containing media. Although maxµ was 32% higher in the presence of copper, the total biomass produced at the end of the culture time (21 d) was similar in both systems  (The GY4515 composition medium was selected to characterize the growth of l day 21 . The funl day 21 a showed ctively) a.The glucose consumption profile was similar in both systems. Glucose concentration decreased with the culture time to a final glucose concentration equivalent to 20% of the initial concentration b. At theThe most significant difference between both cultures was the laccase activity profile. While the highest laccase activity values in the medium without copper sulfate were detected before day 12, the highest activity values occurred beyond this day in the media with the laccase inducer. At the end of the culture, the laccase activity decreased in the media without copper, while it continued increasing when the inducer was present. A minimum 10-fold higher laccase activity was obtained by adding 1 mM copper sulfate to the culture media.P. ostreatus transcriptome in the GY4515 culture medium with o without copper sulfate are shown in lacc10, lacc6, and lacc2 appeared among the top 10 overexpressed transcripts in the cultures supplemented with copper. Additionally, the transcripts of the small subunit of laccase POXA3a, two cupredoxin, a gene similar to the pox2 gene, and a transcript coding for a putative copper-binding protein were also among the more overexpressed in these conditions. Conversely, in the absence of copper sulfate, the transcripts of three suspected copper transporters, a lipid transporter, the regulator of nitrogen metabolism Nmr, and five genes with unknown function were among the 10 most overexpressed.The top 10 more differentially expressed genes (up- and downregulated) revealed by the analysis of the P. ostreatus genome was carried out to determine the genes responsible for the activity in the GY4515 medium with and without copper sulfate. In lacc2 gene transcript was the only one significantly detected, while in the presence of this laccase inducer, the lacc2, lacc6, and lacc10 gene transcripts were the most highly upregulated. To a lesser extent, the transcripts of lacc3 and lacc5 genes were also overexpressed in the presence of copper sulfate. The lacc8 gene could not be amplified.The relative quantification of the transcripts of the 12 laccase genes annotated in the −1 glucose, and 15 gL−1 YE (GY0515), whereas the nitrogen-limited medium was 45 gL−1 glucose and YE, 4.0 gL−1 (GY4504). As copper-induced laccase activity depended on both glucose and YE concentration, the effects of copper in nutrient-limited conditions were tested. For this, two cultures in nutrient-limited conditions, carbon- and nitrogen-limited conditions, were chosen: the carbon-limited medium was 5.0 gL−1 , carbon-limited (GY0515), and nitrogen-limited (GY4504). The micrographs of the clamp connections in terminal fungal hyphae at seven days of culture are shown in P. ostreatus cultivated on natural substrates is currently scarce; therefore, this work aimed to consider the interaction between two nutritional factors  and copper sulfate at 1 mM to determine how the medium composition affects laccase induction by copper in submerged fermentation. Increased fungal biomass production is usually a sign of sufficient nutrient conditions when carbon and nitrogen sources are assimilable in the culture media in the presence of sufficient concentration of other growth factors. Our study chose two nitrogen sources  to establish which one best increased fungal biomass production and laccase activity in Smf. This approach allowed comparing the performance of nutrient-sufficient, only carbon-sufficient and nitrogen-sufficient conditions, assessing laccase activity in the culture media in the absence or presence of laccase-inducing copper sulfate.Systematic research on the interactions between nutritional factors on laccase activity production by YE was a more assimilable nitrogen source than ammonium sulfate to stimulate biomass and laccase activity in the evaluated culture media conditions; therefore, in subsequent experimentation, glucose and YE concentrations were varied using a central composite experimental design, which applies in the surface response methodology, to optimize the composition of the culture medium for optimal laccase activity. It was found that glucose and YE concentrations did not sufficiently explain the laccase activity in cultures without a copper inducer; however, when copper sulfate was in the culture media, both glucose and YE concentrations influenced this enzymatic activity causing an increase of around 20-fold. Furthermore, from this analysis, it was possible to infer that the organic nitrogen-sufficient conditions incremented laccase production.P. ostreatus and other white-rot fungi has been frequently reported to be affected by nitrogen sources [Laccase activity in  sources . Therefo sources , the pos sources . On the  sources  and SSF  sources .P. ostreatus growth with and without copper was laccase activity by the copper-induction of three laccase genes: lacc2, lacc6, and lacc10. At the transcriptional level, fungal growth without copper overexpressed copper transporter of high affinity (ctr1) and the transcriptional factor, nitrogen metabolic repression (nmr), a signal of nitrogen-sufficient condition. Nitrogen-sufficient conditions could mainly regulate the crt1 expression because, in nitrogen-limited conditions, laccase inducement by copper sulfate was lower than in carbon-limited conditions.The profiles of fungal growth, laccase activity, laccase gene family expression, and genome-wide gene transcription were studied using high-resolution techniques, such as real-time qPCR and RNA-seq, in the culture medium composition with sufficient carbon and nitrogen. The time course of laccase production during the culture was different in the presence or absence of copper, suggesting that the regulatory mechanisms of laccase activity production are different in both systems. Comparisons showed that the main change in lacc2, lacc6, and lacc10. Furthermore, the P. ostreatus transcriptome from the GY4515 culture medium with copper sulfate revealed that the lacc2, lacc6, and lacc10 genes responded to the addition of this laccase inducer under nutrient-sufficient conditions.The transcriptome data also suggest that, in the GY4515 medium, the yeast extract and the concentration used could induce the metabolic repression of nitrogen. This result was corroborated by the overexpression of a transcript with an NMR-like domain. These results suggest repression by nitrogen could be related to the regulation of copper transporters that, once present in the culture medium, facilitate the entry of this metal into the fungal cell and induce the transcription of genes C. albicans cultivated in nutrient-rich media, which showed fewer vacuolated compartments compared to the hyphae grown in low nitrogen media. Compared to that reported for this pleomorphic fungus, our results are similar [P. ostreatus hyphae grown in nutrient-rich media had fewer vacuolated compartments similar to those reported for C. albicans. Our study showed more vacuolated compartments grown in media with low nitrogen content in the culture media with higher C:N ratios. In another study, P. ostreatus was cultured in Potato-Dextrose-YE (PDY). The pellets from the culture media showed a young mycelium barely vacuolated (at the 5th culture day); however, when glucose concentration decreased for the culture time, the old mycelium was vacuolated (at the 12th culture day) [P. ostreatus in SmF seem to signal nutrient-limitation in this fungus that could affect laccase activity.We explored the fungal microscopic-morphological changes in terminal hyphae, identified by the distal clamp connections, in culture media using ammonium sulfate or yeast extract. We observed interesting microscopic-morphological changes according to the sources of nitrogen and the C:N ratios. A large pattern of vacuoles was found using yeast extract. This behavior has also been observed in hyphae of  similar . The P. ure day) . TherefoAnother sign of nutrient-sufficient conditions for carbon and nitrogen was tested, a culture medium whose composition increased biomass production using only glucose and YE in the culture medium. At the same time, the effect of glucose and YE concentrations on laccase activity in the absence and presence of 1 mM copper sulfate was also evaluated. The GY4515 medium was the composition closest to the maximum biomass production predicted by the statistical model. Surprisingly, the effect of the nutrient conditions in laccase activity was strongly dependent on copper sulfate in the culture medium.−1 resulted in a more than five-fold increase in laccase activity. An additional increase of up to 40 gL−1 in glucose concentration did not improve the laccase activity. On the contrary, lower activities were obtained [Cerrena sp., high carbon and nitrogen concentrations in the fermentation medium were beneficial for laccase production. In contrast, regardless of carbon concentration, low nitrogen concentrations drastically reduced laccase production [P. ostreatus nutrient-sufficient conditions when the fungus grew in a culture medium with a lot of glucose 102.68 gL−1 and yeast extract 43.65 gL−1 concentrations, where the lignocellulosic biomass pretreatment and laccase activity improved the pretreatment system [Without copper sulfate, the glucose and YE concentrations were insufficient to explain the changes in laccase activity in the different culture media evaluated in this study. However, increasing the glucose concentration from 5 to 2 gLobtained . In repooduction . Howevert system .P. ostreatus in SmF [P. ostreatus and other fungi, the design of experiments (DOE) of response surface methodology was also used; however, copper sulfate and nutritional factors were tested at various concentrations [P. ostreatus growth in GY4515 was characterized with and without copper, the main difference was laccase activity. Furthermore, the local transcriptional analysis of the laccase gene family showed that lacc2, lacc6, and lacc10 were overexpressed in the culture with copper.With copper sulfate, the changes in laccase activity in the different culture media were statistically explained by the glucose and YE concentrations evaluated using a CCD. Here we only tested a fixed concentration of copper sulfate (1 mM) because this was the concentration that had the highest increase in laccase activity when different copper sulfate concentrations (0.5 to 5 mM) were tested in s in SmF . In othetrations ,60,61,62The Zymogram results indicate that the isoenzymes induced by copper are affected by different nutrient conditions. In nitrogen-sufficient conditions that are also carbon-limited, the expression of isoenzymes around 70 KDa (Presumably lacc6) was negatively affected, while the isoenzymes Lacc2 and Lacc10 (around 40 KDa) were positively affected. All isoenzymes induced by copper were negatively affected by the inverse nutrient conditions . These results show an important effect of nitrogen-limited laccase activity induced by copper, suggesting that processes associated with organic nitrogen metabolisms influence copper uptake. In contrast, in nitrogen-sufficient that are simultaneously, the carbon-limited conditions, only some specific laccase isoenzymes induced by copper are affected. In these carbon-limited conditions, laccase activity is still influenced by copper because there is probably copper uptake.lacc2, lacc6, lacc10, pox2, and PoxA3a among the top 10 overexpressed transcripts. This last transcript does not code for a laccase enzyme but for a protein that can form heterodimers with Lacc2 (PoxA3) [Copper-induced laccase isoforms enzymes were previously identified in SmF . The mos (PoxA3) ,64. We dnmr1-like transcript listed in the top ten transcripts in culture without copper. In these nutrient-sufficient conditions, three ctr1 copper transporters were also found. This copper transporter has already been shown to negatively regulate CTR1 transcription in the dikaryon strain dkN001 and its monokaryon parents mkPC9 and mkPC15 of P. ostreatus [It is noteworthy to observe how these isoforms changed in the nutrient-limited conditions in the culture medium. In carbon-limited conditions, the two bands at 70 KDa disappeared, while in nitrogen-limited conditions, two bands at 70 KDa and two bands at 40 KDa were drastically affected. These results may support the idea that nutrient-sufficient conditions confer the fungus more susceptibility to laccase inducers. A marker for nutrient-sufficient conditions was found on the streatus .P. ostreatus in initial nitrogen-limited conditions medium produce hyphal with vacuolated morphology. These hyphal could have a lower expression level of copper transporters such as ctr1 than hyphal obtained in the nitrogen- sufficient condition. Consequently, copper uptake mechanisms are less prevalent in nitrogen-limited than in nitrogen-sufficient conditions, affecting laccase genes regulation by transcriptional mechanisms in the cis-acting elements promoter sequences as metal responsive elements (MRE) [Finally, using different nitrogen sources and C:N ratios allowed us to observe that nitrogen-limited conditions had a stronger relationship with the vacuolated patterns. Moreover, nitrogen-limited conditions affected laccase induction by copper. All these results again suggest a likely relationship between nitrogen metabolisms and copper uptake. In this relationship, ts (MRE) .P. ostreatus cultures favored the expression of a transporter family of genes with a high affinity to copper: the CTRs, which were able to uptake copper from the environment. This effect was evidenced by an increase in copper-dependent laccase activity by 20-fold, the presence of four laccase isoforms that were detectable in zymogram and by an increase in three laccase transcripts lacc2, lacc6, and lacc10 determined by real-time qPCR and RNAseq.This study describes a systematic analysis of the composition of the culture medium based on two nutritional factors: glucose as a carbon source and ammonium sulfate or yeast extract as nitrogen sources. This analysis determined what type of nitrogen source and what combination of glucose and yeast extract concentrations favored the production of fungal biomass and laccase activity induced by copper. The nutrient-sufficient conditions for both carbon and nitrogen in