The proteomic expression analysis of human colorectal cancer showed the upregulation of SRP9 with hypoxic adaptation of the tumor microenvironment of heterogeneous primary human tumor tissues , suggestDrosophila (ELAV)-like 1, Hu Antigen R (HuR) to TP53 mRNA, an interaction that led to the promotion of p53 translation, which reveals the competitive mechanism of 7SL1 and HuR for binding to TP53 3’-UTR (via the HuR-ETS1-cyclin dependent kinase inhibitor 1A (p21)/cyclin dependent kinase inhibitor 1B (p27) axis  axis . In panc27) axis Figure 1A study on forkhead box P3 (FOXP3), a transcription factor that is crucial for the development and inhibitory function of regulatory T-cells (Treg), indicated that overexpression of FOXP3 resulted in the repression of the transcription of 7SL1, whereas the knockdown of FOXP3 showed the upregulation of 7SL1 RNA transcription . The mecvia the interaction with transcriptional factor complex IIIC (TFIIIC) as well as SUZ12 polycomb repressive complex 2 subunit (SUZ12) (Although a role of the polycomb repressive complex 2 (PRC2) component, working on the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated epigenetic control of RNA polymerase II (Pol II) transcribed coding gene transcription, has been well-established, a recent study on EZH2-mediated epigenetic regulation of RNA polymerase III (Pol III) transcription indicated that EZH2 is involved in the repression of Pol III transcription of tRNA(Tyr), 5S rRNA, and 7SL1 RNA genes  (SUZ12) , suggestIn a single-cell sequence analysis –6, it waRecently, it was demonstrated that the immune stimulatory property can be utilized for the development of CAR-T cell therapy . The vecDespite advances in surgery, standard radiation therapy, and chemotherapy, immunotherapy was added as a fourth treatment to cancer, as the discovery of immune modulation through immune check points and exploration of new combinations of cancer multimodality therapies led to overcoming long-term resistance and tumor recurrence . Cancer The RAPP pathway study revealed the immune stimulatory property of endogenous non-coding RNA, 7SL1, which is a component of SRPs. A previous study suggests several important implications. First, given that the co-deploy peptide antigen with 7SL1 can exert an efficient anti-tumor effect , this apHS, KS, and HI conceptualized the study objectives and obtained the funding, wrote the manuscript. TH, SK, YD, HE, and TS outlined content of the manuscript. TS suggested which disease information should be addressed in the manuscript. All authors have read and approved the final manuscript version for publication.This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology , and Japan Agency for Medical Research and Development (AMED) . Partial support was received from Princess Takamatsu Cancer Research Fund and Mitsubishi Foundation to HI.Authors are thankful to every lab member.Partial institutional endowments were received from Taiho Pharmaceutical Co., Ltd. , Hirotsu Bio Science Inc. ; Kinshu-kai Medical Corporation ; Kyowa-kai Medical Corporation ; IDEA Consultants Inc. ; Unitech Co. Ltd. .The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction – an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in  Cellular DNA exists in a wide range of topological states in which the double helix is over or under twisted from its relaxed contour of ∼10.5 bp per helical turn. This upset in twist imparts torsional strain on the DNA duplex that causes it to wrap around itself, a phenomenon known as supercoiling. Supercoiling is a fundamental and necessary characteristic of chromosomes in all cells, with major roles in DNA compaction and strand opening operations. By reducing or increasing the free energy requirement of duplex melting at transcription start sites, supercoiling strongly modulates gene expression, particularly in bacteria, in which initial loading of RNA polymerase (RNAP) is not aided by ATP hydrolysis . DNA supE. coli are wrapped around each other forming long precatenanes , which, according to Liu and Wang's  twin-domatenanes . Such st Topo IV , the pre Topo IV . The res Topo IV  possiblyli cells , likely li cells ,14, and li cells  and permli cells ,17.E. coli chromosome has a supercoiling density of σ = −0.06, or ∼6 fewer helical turns per 100 than would be present if it were relaxed. This negative state is thought to be maintained by selective relaxation of positive supercoils at replication and transcription twin-domains , all known topoisomerases function as ‘relaxases’, reducing supercoiled DNA towards the relaxed state . Cells m-domains . In bact-domains . A signi-domains  and bact-domains . These p-domains , thus in-domains ; however-domains ,28.E. coli range from one barrier every 10 kb , with binding being most strong to underwound regions . PsoraleE. coli genome. Our method also revealed an unexpected bilateral symmetric pattern of supercoiling over the chromosome that is centered on a skewed origin—terminus replication axis. The interplay between transcription and replication and their effects on genome supercoiling are discussed.To address these issues, we have developed a highly sensitive psoralen-based supercoiling assay (Psora-seq) that is fast, reliable, and uses commercially available materials. Using this method, we show that transcription from highly expressed ribosomal genes disrupts supercoiling over a 50 kb region—substantially farther than previously measured. Models of genome supercoiling, generated by extrapolating the supercoiling dynamics observed at ribosomal genes to all other genes, showed good agreement with the supercoiling profile from Psora-seq, suggesting that transcription is a major determinant of supercoiling in the E. coli strain MG1655 was used for all experiments except for the invA inversion strain, which has a 18.4 kb kanamycin marked inversion including the rrnA operon  was added to 70 μg/ml, which resulted in DNA fragment sizes ∼0.1–1 kb  365 nm UV light in a UVP CL-3000L crosslinker. Genomic DNA was prepared by the CTAB method  were washed according to the manufacturer's instructions, then half the DNA (200 μl) is added to the beads and rotated for 1 hour at room temperature in the dark, followed by 16 h rotation at 4°C in the dark. Unbound DNA was removed by washing beads according to the manufacturer's instructions, then resuspended in 100 μl 95% deionized formamide  and heated to 95°C for 5 min to release bound DNA. The DNA/bead mixture was pipetted repeatedly for 30 s, then placed on a magnetic stand for 30 s before collecting the eluate. The remaining 200 μl of sheared DNA (input sample) was prepared by heating in formamide as above. Affinity purified and input samples were precipitated and resuspended in 10 μl TE each. DNA was quantified by Qubit Fluorometer (ThermoFisher) and diluted to 0.2 ng/μl in TE for sequencing.E. coli fragment . A standard curve was generated using KAPA DNA Standards for Illumina platforms (cat# 07960409001) and library fragment size was estimated by agarose gel electrophoresis. Pooled libraries were adjusted to a final concentration of 6-20 pM before denaturing and sequencing. Paired-end sequencing was performed on either an Illumina MiSeq sequencer, or an Illumina NextSeq sequencer, with a 150-cycle MiSeq V3 reagent kit (Illumina) using the re-sequencing workflow. Sequencing reads were aligned to an MG1655 reference genome (accession NC_000913) using Illumina integrated BaseSpace sequencing data analysis software. Aligned reads (0.5–3 million per sample) were extracted, sorted into 1-kb bins, and exported to Excel format using two custom Matlab scripts, sequencingcompile.m and sequencingcompile2.m (https://github.com/DavidBatesLab/Matlab-scripts.git). The 35 bins containing ribosomal genes, which cannot be accurately mapped to the genome due to high repetitive sequence, were replaced with average values from the surrounding 6 kb and relative reads per bin were normalized to genome average. Psora-seq enrichment was calculated by dividing normalized pulldown values by normalized input values then log2 transformed.Sequencing libraries were created using the Nextera XT DNA Sample Preparation Kit according to manufacturer protocol except that sequencing libraries were manually normalized by quantifying by qPCR, adjusting each library to a final concentration of 2 nM prior to pooling. PCR amplification was performed on a 7900HT Real-Time PCR System (Applied BioSystems) using the KAPA SYBR FAST qPCR kit and primers specific to the adapter sequences on either side of the E. coli ribosomal operons was co-oriented by the direction of rrn transcription and aligned to the midpoint of each operon. To quantify the relative change in supercoiling generated by an operon, psoralen profiles were normalized so that the mean log2 binding value over the 80-kb range was zero. Aligned and normalized data from all 7 operons and from 6 independent samples were then averaged together to create a consensus ribosomal operon psoralen profile , then summed these predicted twin-domains to create a composite twin-domain map. Individual twin-domains were patterned after ribosomal operon twin-domains scaled by the relative rate of transcription. Transcription rates were obtained from two data sets, RNAP binding by ChIP-chip  and mRNA2 transformed, and converted to standard (z) score. For ChIP-chip data, microarray coordinates were converted to MG1655 NC_000913.3, then sorted into 1-kb bins and converted to z-scores as above. Psora-seq data (log2 reads IP/input) were also converted to z-scores.Genomic protein binding information was obtained from previous ChIP-seq studies ,45,46. Mwww.real-statistics.com). Statistical tests between population means were first confirmed to have equal variance by F test, then analyzed using two-tailed Student's t test. Linear regressions were carried out using the least squares method. The LINEST Excel function was used for regression analysis of ribosomal gene twin-domains.Graphing and statistical analyses were performed in Microsoft Excel with the Real Statistics Resource Pack for Macintosh  when the axis of symmetry was skewed counterclockwise of the natural ori-ter axis by 14 degrees  symmetry between supercoiling of the left and right chromosome arms Figure . Symmetrs Figure . Althougs Figure ,53 and gdif locus within the terminus region  and range (magnitude) of twin-domains generated at ribosomal operons, psoralen binding at all 7 operons was averaged and twin-domain dimensions were analyzed by regression Figure . The resrrnA ribosomal operon  exhibited a reversed twin-domain at the rrnA locus compared to wild-type  between transcription strength (RNAP binding x operon length) and twin-domain amplitude among all genes  and top 1% expressed genes (red), respectively. Because twin-domain amplitudes did not scale proportionally with transcription among weakly transcribed genes, we expect weaker twin-domain signatures are masked by nearby transcription or other modulators of supercoiling. Supporting this conclusion, ribosomal operon twin-domain amplitude was reduced by about 40% during late-exponential phase, which has ∼10-fold lower rate of rRNA transcription initiation compared to mid-exponential phase, and twin-domains were completely absent during stationary phase  for which we obtained transcription data. Measuring twin-domain amplitude as the change in psoralen binding across the operon . Treatment of cell cultures with rifampicin one hour before psoralen crosslinking resulted in correlations of r ≤ 0.01 to both transcription models  but relatively modestly correlated to the mRNA model , which does not include expression data for ribosomal genes. High correlation between RNAP model and Psora-seq data was present at all 7 ribosomal operons  exceed the highest expressed genes in eukaryotes by a factor of ∼2–4  half being more negative and the lower (terminus) half being more positive Figure . SupercoA Figure , possiblA Figure . In contA Figure , or HU, A Figure ,25. The A Figure  concludice versa ,70. HowegidA and mioC .All Psora-seq data generated in this study are available on the NCBI Sequence Read Archive (BioProject PRJNA805241). Custom Matlab scripts that were used to compile and sort sequencing data are available on the GitHub repository (gkac244_Supplemental_FileClick here for additional data file.


Thousands of long noncoding RNAs (lncRNAs) are actively transcribed in mammalian genomes. This class of RNAs has important regulatory functions in a broad range of cellular processes and diseases. Numerous lncRNAs have been demonstrated to mediate gene regulation through RNA-based mechanisms. Simultaneously, non-functional lncRNA transcripts derived from the activity of lncRNA loci have been identified, which underpin the notion that a considerable fraction of lncRNA loci exert regulatory functions through mechanisms associated with the production or the activity of lncRNA loci beyond the synthesized transcripts. We particularly distinguish two main RNA-independent components associated with regulatory effects; the act of transcription and the activity of DNA regulatory elements. We describe the experimental approaches to distinguish and understand the functional mechanisms derived from lncRNA loci. These scenarios reveal emerging mechanisms important to understanding the lncRNA implications in genome biology. The development of high-throughput sequencing technologies has shed light on transcriptome knowledge leading to the identification of thousands of transcriptionally active genomic regions that do not derive from protein-coding genes but produce long noncoding RNAs (lncRNAs) ,2,3. In Typical regulatory DNA elements such as promoters and enhancers drive lncRNA transcription or are embedded into lncRNA loci ,10. SurpIn this review, we discuss the underlying mechanistic principles associated with the activity of lncRNA loci beyond the RNA-dependent function. In particular, we discuss recent insights into the regulation originated from the activity of transcription and the regulatory elements of hosted lncRNA loci. We also provide an updated overview of the available methods for interrogating the mechanistic scenarios derived from these observations to distinguish the functional components of given lncRNA loci.cis-acting mechanism) [Cis-acting lncRNAs regulate gene expression at the local level by remaining tethered to their transcription site to modulate gene regulation through chromatin looping, modulate the binding of transcription factors, and the establishment of histone modifications to their target regions [In general, lncRNAs are coexpressed with adjacent or neighboring protein-coding genes ,15. Althchanism) ,17. Cis- regions ,19,20. AGene regulation may be influenced by neighboring transcription, where the act of transcription of a given locus per se can regulate nearby genes . This neS. pombe, the phosphate-responsive permease gene (tgp1+) is regulated by the transcription factor Pho7 in response to phosphate stimulus. The lncRNA nc-tqp1 is transcribed upstream of tgp1+ upon phosphate starvation. The activity of noncoding transcription originated from nc-tqp1 inducing tgp1+ silencing by an increase in nucleosome occupation over the promoter region, which physically prevents Pho7 binding [nc-tqp1, noncoding transcription originated from serine-induced lncRNA SRG1 silences SER3 by increasing nucleosome density over SER3 promoter in response to serine stimulus. The increased nucleosome compactness interferes with the binding of transcription factors on the SER3 promoter [nc-tqp1 and SRG1 are upstream lncRNAs whose transcription act interferes with the binding of transcription factors by modulating nucleosome displacement over promoter regions.Noncoding RNA transcription can be originated not only from intragenic but also from intergenic genomic regions . This ob binding . Similarpromoter . Thus, nIn addition to the gene silencing induced by the physical displacement of nucleosomes, lncRNA transcription may influence the chromatin state through modifying the profile and deposition of histone marks, binding of TF, and the activity of RNAPII. During the progression of transcription, the RNAPII associates with chromatin remodeling complexes, transcription factors, and histone chaperones, contributing to the chromatin state dynamics during transcription . There aTcra locus is involved in V(D)J recombination during thymocyte development [In mammals, the elopment . This loelopment . IntriguD. melanogaster, the Polycomb Response Elements (PRE) silence genes by targeting Polycomb Group (PcG) complexes, which are necessary for homeotic gene regulation [Fab-7 PRE in the bithorax locus preventing the repressive effect by the PcG complex [LYZ) is transcribed under lipopolysaccharide (LPS) stimulation [LYZ by the enhancer. Upon LPS stimulation, the upstream enhancer becomes active and transcribed into the lncRNA LINoCR leading to CTCF eviction from the enhancer and subsequent LYZ activation [Uph) is transcribed upstream to Hand2. The Uph transcription is necessary to maintain the binding of GATA-4 and the activation of two overlapped enhancer elements-driven Hand2 gene expression [In gulation . Noncodi complex . Thus, lmulation . The instivation . Oppositpression . In suchThymoD is transcribed from an upstream intergenic region from the Bcl11b gene, which harbors an enhancer element. The act of transcription repositions the enhancer element from the nuclear periphery to the nuclear interior, triggering the enhancer activation. Moreover, ThymoD transcription facilitates CTCF/Cohesin complex occupancy over the enhancer and the Bcl11b promoter, which juxtaposes enhancer–promoter communication into a loop structure [Although architectural proteins such as CTCF and cohesin complex mediates chromatin structuration through facilitating a loop formation between the enhancers and promoters , lncRNA tructure   embedded into lncRNA loci can exert cis-regulation separately from the lncRNA transcript or the act of transcription ,52. PromIn parallel, examining available high throughput sequencing data such as chromatin accessibility (DNase I and ATAC-seq), histone modifications , and TF binding from ChIP-seq can help to clarify the context in which a lncRNA locus can exert its function. Moreover, chromosome conformation capture  data can inspect to elucidate the possible interacting pattern of the putative regulatory element with potential target genes .The process of transcription by itself can drive local gene expression regardless of the synthesized transcript as described above. To unravel whether the transcription process coming from a locus of interest exerts cis-regulation, transcription termination is the frequently used method. Inserting polyA-terminator sequences immediately from the lncRNA TSS allows premature transcription termination ,14. UsinCollectively, all the experimental approaches described before should be applied to unraveling the complex molecular mechanism of a lncRNA locus, which in turn led us to identify if the candidate lncRNA locus is, in fact, a functional RNA transcript. However, it could be experimentally complex, especially in investigating the dynamic regulation of lncRNA loci during cell differentiation systems or organism development. Finally, these elegant and carefully experimental approaches can lead us to dissection of all the possible regulatory scenarios coming from lncRNA loci. Examples of lncRNA locus where one or more mechanistic principles may act together to regulate gene expression in a particular context have been reported ,49,59.Our knowledge about gene regulation has evolved with the discovery of the lncRNA genes. Even though evidence demonstrates that some lncRNA transcripts are byproducts of transcription or transcriptional noise, others are functional RNA molecules involved in gene regulation. Even more, the DNA sequences where lncRNA emerges and the process of transcription can influence local gene expression. This underlines the importance of applying genetic tools to study the RNA and DNA as regulatory modalities emerging from lncRNA loci, in which several possible modes of action can arise from disentangling the molecular basis of lncRNA loci. The examples described here highlight the need to apply rigorous experimental approaches to identify functional lncRNA transcripts as well as putative regulatory DNA elements associated with gene regulation. These emerging mechanisms of action by themselves can exert gene regulation with relevant biological functions such as cell differentiation, imprinting, development, and diseases. Fortunately, emerging exciting genome editing tools like CRISPR-Cas9 coupled together with information about the epigenetic landscape have enabled distinguish the lncRNA mechanisms of action. Although these efforts, it remains to clarify the molecular mechanisms and the functional roles of lncRNA which are complex loci given the existence of processes associated with their production and the activity of regulatory DNA elements that originated it. Future research will allow characterizing on genome-wide scale potential non-functional lncRNA transcripts as well as new putative functional regulatory mechanisms associated with lncRNA loci. It will be also interesting to unveil if these regulatory mechanisms observed in lncRNA loci also take place in other noncoding loci such as miRNAs, piRNAs, or cirRNAs and their contribution to cell biology and disorders.


Caenorhabditis elegans serves as a powerful tool for understanding mechanisms of drug action with its short life cycle, genetic amenability and conserved cellular processes. Further investigation is required particularly in in vivo systems to gain broader understanding of ketamine’s actions. In this study, we aimed to decipher ketamine-mediated alterations using C. elegans as a model. We show that ketamine specifically induces apical extracellular matrix modifications (aECM) in the vulva and the cuticle. Ketamine treatment phenocopies neuronal migration and vulval invagination defects of chondroitin mutants despite wild-type like chondroitin staining pattern. Normal vulval expansion and defective vulval eversion phenotypes of ketamine-treated animals are suggestive of alterations in the network of aECM factors which do not impinge on chondroitin. Ketamine ameliorates impaired movement of a group of roller mutants characterised with collagen defects in the cuticle and RNA-seq identifies that 30% of the cuticular collagens are upregulated in response to ketamine. Ketamine alters aECM, neuronal migration and collagen expression in C. elegans. We propose C. elegans as a putative animal model to investigate ketamine-mediated ECM modifications.Ketamine is a widely used anesthetic agent since 1960s and has recently been exploited for its rapid antidepressant action at subanesthetic doses. It has been demonstrated that ketamine induces alterations in extracellular matrix (ECM) in rodent models which in part plays a role in its anti-depressant action. The nematode  N-methyl-d-aspartate (NMDA) receptor blockade is the underlying mechanism for anesthetic action of ketamine, however how ketamine exerts its antidepressant effect is complex and still not clear. Increased synaptogenesis after low-dose ketamine treatment via modulation of extracellular matrix (ECM) has been proposed as one of the mechanisms of ketamine’s antidepressant action which is sustained long after the drug is metabolised5. It has been previously demonstrated that ketamine induces epithelial phenotypic changes (EPC) and alterations in ECM in animal and cell line models10. The nematode Caenorhabditis elegans serves as a powerful tool for understanding mechanisms of drug action with its short life cycle, genetic amenability and conserved cellular processes. C. elegans has a simple anatomy and fully characterised, invariant cell lineage. A wild-type animal has 959 somatic cells which are traceable from zygote to adult stage. The transparent body of C. elegans consists of wide variety of epithelial tubes despite only 959 somatic cells. The tubular structures such as cuticle and the vulva are lined with extracellular matrices composed of collagens, lipids, chondroitin and heparan glycosaminoglycans (GAG) some of which are shared with the mammalian ECM components11.Ketamine is a widely used anesthetic agent since 1960s and has recently been exploited for its rapid antidepressant actionC. elegans is vulva which is generated via orchestration of mainly EGFR/Ras/MAPK and Notch signalling. The egg-laying apparatus vulva in the hermaphrodite C. elegans is comprised of 22 cells which are derived from epithelial precursor cells through three rounds of cell divisions within 5 h12. More than hundred mutants isolated through mutagenesis screens were identified on the basis of vulval lineage defects, and were broadly classified as being vulvaless (vul) and multivulva (muv)15. Vulval morphogenesis can also be disrupted without a cell lineage defect. One such vulval phenotype arises due to defective epithelial invagination causing a misshaped vulva with a squashed vulval lumen appearance termed as Sqv. Disruption of the chondroitin biosynthesis has been reported to account for formation of Sqv phenotype on the basis of sqv mutants carrying mutations in genes which encode components of chondroitin biosynthesis.One of the widely investigated tissue in C. elegans as a model. We show that ketamine specifically induces apical extracellular matrix modifications (aECM) in the vulva and the cuticle. Ketamine treatment phenocopies neuronal migration and vulval invagination defects of chondroitin mutants despite wild-type like chondroitin staining pattern. Normal vulval expansion and defective vulval eversion phenotypes of ketamine-treated animals are suggestive of alterations in the network of aECM factors which do not impinge on chondroitin. Ketamine ameliorates impaired movement of a group of roller mutants characterised with collagen defects in the cuticle and RNA-seq identifies that 30% of the cuticular collagens are upregulated in response to ketamine. Our findings identify BLIMP-1/BLMP-1 as a putative novel molecular target for ECM modulatory effects of ketamine. We propose that ketamine modulates ECM components in part via orchestration of Wnt and TGF-beta signalling pathways and regulatory network of transcription factor BLMP-1.In this study, we aimed to decipher ketamine-mediated modifications using Caenorhabditis elegans has a rapid life-cycle. After hatching, it becomes an egg-laying adult within 2.5 days at 25 °C. We have monitored control and ketamine-treated animals for 48–72 h. At the 54-h mark, all animals in the control plate were adults whereas only a small fraction of ketamine-treated animals could reach adulthood . At this stage, ketamine-treated plate contained 4% ± 0.6 of L2 stage, 15% ± 2.6 of L3, 27% ± 1.6 of L4 and 36% ± 1.2 of young adult animals. Given that ketamine works as an anesthetic, we tested whether developmental delay might be due to slow pharyngeal pumping rate. The pharynx of the worm works as a neuromuscular pump where proper pharyngeal movements are required for a normal feeding. Feeding quality can be measured as a read-out of pharyngeal pumping rate per minute (ppm). Feeding rate of ketamine-treated and no-treatment control animals were measured in young adults . Although the difference was subtle it reached significance levels. In order to explore whether abnormalities caused by ketamine extends to reproduction process, the brood size was determined for control and ketamine treated animals. The average brood number in ketamine-treated animals decreased by 63% as compared to control animals . . Fig. b. Wild-tmen Fig. b,d. The ied Fig. e,f. Therzmp-1::GFP and cdh-3::CFP; ceh-2::YFP reporter markers  displayed correct expression of zmp-1::GFP reporter similar to control animals (n = 63). VulB, VulC and VulF were visualised using cdh-3::CFP; ceh-2::YFP reporter in L4-stage animals where ketamine-treated animals (n = 102) showed no obvious expression defects as compared with the controls (n = 84). Figure unc-68 and nmr-1 were tested for formation of Sqv phenotype21 , nmr-1(ak4) and nmr-1(ak4); unc-68(kh30) mutant animals show highly penetrant Sqv phenotype clearly identified under a dissecting microscope. For unc-68 mutants . HSNs are bilaterally symmetric serotonergic motor neurons which control egg laying in the hermaphrodite animals. HSNs are born in the tail during embryogenesis and migrate anteriorly towards the middle of the embryo .Disruption of vulval morphogenesis without a cell lineage defect in ketamine-treated animals is similar to phenotypes displayed by ryo Fig. c24,25. A26. HSN axon then enters the ventral nerve cord and grow anteriorly towards the head. Having confirmed that HSNs display normal migration at early larval stage in ketamine-treated condition, the migration and axonal guidance of HSNs were further analysed at adult stage where axonal growth of HSNs is completed. Using a different reporter that is tph-1::GFP marker, adult worms were treated with ketamine and their HSN migration and axonal guidance was examined. As shown in Fig. t-test, paired, mean ± sem, p = 0.0055; **p < 0.01).Each HSN neuron sends a single axon which stereotypically extend towards ventral position and around the vulvasqv-5 (squashed vulva-5) mutants and uridine 5′-diphosphate–sugar transporter sqv-7 mutants; we embarked on assessment of chondroitin abnormalities in ketamine-treated animals23. To do this, an antibody specific for Chondroitin-0-sulphate was used which detects chondroitin stubs digested with chondroitinase ABC (CSase ABC) enzyme. Embryos of the ketamine-treated adults were examined for their chondroitin proteoglycan (CPG) layer morphology. The eggshell of the C. elegans is composed of three outer layers with CPG layer being defined only a decade ago .Given that ketamine-treated worms display Sqv phenotype that is characteristic of animals carrying mutations in the genes encoding for chondroitin synthesis pathway components such as the chondroitin synthase ago Fig. b27. The V-5 Fig. b. In ordPCR Fig. c. The exrol”  mutants depicted in Fig. rol-6 mutants’ movement defect was ameliorated with 25% rollers as compared with 100% in untreated rol-6 mutants  . For rol-1, rol-8 and sqt-1 mutants, control and ketamine-treated animals both displayed 100% roller phenotype. The video recordings showing amelioration of Rol phenotype for rol-6 and rol-9 mutants are shown in Supplementary Videos Ketamine-treated animals display Sqv phenotype characteristic of chondroitin mutants yet have wild-type like chondroitin levels and distribution. Sqv phenotype arises due to disruption of vulval lumen formation which is coordinated via apical extracellular matrix components including chondroitin proteoglycans, ZP domain proteins and other glycoproteins. The eggshell is also formed through aECM components and ketamine-treated animals did not show an obvious eggshell rupture as evidenced with the staining of CPG layer against chondroitin stubs. It was curious whether ketamine-treatment modulates cuticle formation which is another tubular structure formed of aECM in the worm. The epidermal cuticle which maintains the body shape is formed of collagen and non-collagen components. Cuticle collagen gene mutants can affect the body shape in various ways one which is twisted body axes termed as a “Roller” phenotype, shortly “ol” Fig. a. rol-6 mutant is shown in Supplementary Fig. rol-6 mutants, the cuticle structure of the rol-9 mutants is ameliorated via ketamine exposure.The cuticle and the internal organs of the cle Fig. a and witcle Fig. b, body wnts Fig. c, the lod rol-9sc48 mutantrol-6, rol-4 and sqt-1 genes are involved in similar biological processes related to collagen function yet only rol-6 mutants show amelioration of Rol phenotype. rol-8 and rol-9 are non-cuticular genes where rol-8 is a collagen and rol-9 is molting protease inhibitor29. rol-8 mutant does not respond to ketamine treatment however rol-9 mutants despite their non-collagen classification responds to ketamine-treatment. One interesting fact about these six mutants analysed for their response to ketamine in terms of rolling movement is that rol-6 and rol-9 which are responsive to ketamine are right-handed rollers whereas rol-4, rol-8, rol-1 and sqt-1 are left-handed rollers. rol-1 was not detected in the differentially regulated gene list. There are 173 predicted cuticular collagens in C. elegans 55 of which are upregulated as shown in Table 30.In order to identify the underlying mechanism of ketamine-induced modifications, we performed RNA-seq experiments which showed that ketamine treatment in wild-type animals upregulated 473 genes and downregulated 254 genes  and sqv-5(k175) yet these mutants display an abnormally protruding vulva at adult stage similar to our observation in the case of ketamine-treated animals22. Closer look into the different types of vulval cells have shown that the Sqv phenotype in ketamine-treated animals does not result from defects in cell differentiation. To date, Sqv phenotype has been described for worms defective in the GAG biosynthesis and modification41. One exception is lin-35; spr-1 double mutant animals which carry mutations in the mammalian orthologues of Retinoblastoma (Rb) and CoREST, respectively42. lin-35; spr-1 double mutants display a more severe Sqv phenotype with an abnormally compressed vulval lumen at the L4 larval stage. Despite their Sqv phenotype, lin-35; spr-1 animals have wild-type like chondroitin levels and distribution similar to ketamine-treated animals. The initial inflation of the vulval lumen at early L4 stage is dependent on chondroitins. Vulval lumen matrix have recently been shown to contain dynamic apical ECM components filling the vulva lumen during morphogenesis19. After the early luminal inflation by chondroitins, the vulval lumen is shaped by the transient localisation of these apical ECM components including ZP proteins, the extracellular leucine-rich repeat only proteins and lipocalin. aECM assembly in the vulval lumen mediates the correct vulval eversion independent of chondroitin. This observation is in line with our ketamine results where initial vulval inflation is normal and vulval eversion is disrupted which is suggestive of alterations in the network of aECM factors which do not impinge on chondroitin. This is also in parallel with the wild-type like immunostaining observed in ketamine-treated animals. However, it is possible that mild modifications in the chondroitin biosynthesis and distribution which were not detectable in our study, is sufficient to induce Sqv phenotype.We have found that the vulval epithelium cells of the ketamine-treated animals fail to fully migrate dorsally after detachment from the cuticle leading to defective invagination thus Sqv phenotype. At L4 stage, ketamine-treated animals show no gross vulval lumen abnormality. By the adult stage, ketamine-treated animals display an abnormally protruding vulva. When the animals are filled with eggs, ketamine’s effect on vulval epithelium becomes more visible. Some of the 43. Low-dose ketamine administration ameliorates symptoms of MD which is sustained long after the drug is metabolised. One of the proposed mechanism of anti-depressant action for ketamine is the altered perineuronal net (PNN) composition as evidenced from recent studies conducted on rats5. PNNs are composed of ECM molecules including chondroitin, linker proteins and hyaluronic acid. They surround specific types of neurons in the brain. ECM constitutes about one fifth of the brain’s volume and is involved in development and plasticity. The brain ECM has considerable importance for the physiology of synaptogenesis with evidence supporting their involvement in the etiology of psychiatric disorders such as major depression, schizophrenia, bipolar disorders, and autism45. ECM components hold the potential to be candidate therapeutic targets for neuropsychiatric diseases. In addition to its therapeutic use in the clinic to treat depression, ketamine has been used to generate schizophrenia model in rats by its virtue of ECM modification10. Therefore, ketamine administration can result in adverse side effects including schizophrenia-like phenotypes and dissociation limiting its therapeutic use. As opposed to findings in the rodent models on remodelling of PNN mainly via alterations in the chondroitin, we have found that ketamine-induced aECM alterations resulting in vulval invagination defect is independent of chondroitin. The vulval epithelial phenotype upon ketamine-treatment may not include chondroitin however, it is possible that chondroitin localised in the neural ECM is modified which was not investigated in this study.Repeated subanesthetic dose of ketamine infusion therapy is currently being used in the clinic to treat major depression (MD)Caenorhabditis elegans shares similar set of signalling molecules as its mammalian counterparts and has been creatively used as a model organism to decipher molecular mechanisms of antipsychotic drugs46. One strategy to investigate novel targets of antipsychotic drugs in C. elegans was to exploit the developmental delay induced by these drugs which was in part due to decreased pharyngeal pumping rate47. Similar to these drugs, ketamine-treatment induced developmental delay and inhibition of pharyngeal pumping. Animals carrying mutations in known ketamine receptors in the worm, that is RyR and NMDA receptors, namely unc-68 and nmr-1 showed delayed development (data not shown) and Sqv phenotype implicating a mechanism of action for ketamine independent of its anesthetic action. There are very few studies conducted on ketamine’s action in C. elegans and high doses of ketamine is administered in these studies, up to 30 mM as opposed to 2.5 mM in our study20. To our knowledge, this is the first study to show ECM modulatory role for ketamine in the worm. The observations presented in our paper is in the long-lasting category as opposed to ketamine’s transient anesthetic action. Ketamine plays NMDA-independent roles in several cellular processes and mechanism underlying the long-lasting effects of ketamine is not well understood49. In a glioma cell line model where NMDA receptor was knocked down, ketamine was still able to exert antidepressant effects50. Given that nmr-1; unc-68 double mutants display Sqv phenotype similar to single mutants, ketamine’s long-lasting effect is likely to be independent of its NMDA blockade. Through which receptors ketamine functions to modify ECM in the worm requires further investigation. Studies performed on rodent models and cell lines have shown that the predominant site of action for ketamine is neurons due to expression pattern of ketamine receptors. However, recent studies demonstrated extraneuronal sites for ketamine51. Our findings suggest that ketamine is likely to act nonautonomously to induce vulval and cuticular changes.sqv-5 ad sqv-7 display HSN migration defects along with their characteristic Sqv phenotype. Genes involved in correct HSN migration at adult stage include epidermal microRNA mir-79 and hst-2 which function in maintenance of heparan sulfate homeostasis52. However, these mutants do not display Sqv phenotype. Ketamine-treated animals show HSN migration defect at adulthood but not at the beginning of development (L1). Modifications of the extracellular matrix later in development may account for this difference. Ketamine may act nonautonomously to induce HSN migration defects. Amelioration of roller phenotype in ketamine-treated rol-6 and rol-9 mutants suggests that ketamine modulates multiple components of aECM. Collagen defect is the underlying cause for the movement of worms in a rolling fashion. Consistent with our phenotypic observations, RNA-seq experiments identified a group of ketamine-responsive cuticular collagens corresponding to 30% of the entire cuticular collagen genes in C.elegans. Upregulated gene list included all the roller mutants assessed for their movement defect except rol-1. Upregulation of majority of the cuticular collagens suggests a complex network for ketamine’s mechanism of action. We observed that the roller mutants which do not respond to ketamine are all left-handed rollers, namely rol-4, rol-1, rol-8 and sqt-1. The right-handed rollers rol-6 and rol-9 show enhancement in their movement suggesting ketamine modulates specific class of collagen complexes. The handedness of the roller phenotype stems from specific mutations which has an effect on the processing and trimerization of pro-collagens53. Cuticle development is a complex process. There is an extensive interaction network within the cuticular collagens which is not well understood. Mutations that induce left and right body-handedness has been described however the underlying mechanism remains obscure. Further research with the use of ketamine may shed light on possible mechanism for handed rolling behaviour. The collagen-related defects can be dose-dependent which might account for the rol-4 mutants’ subtle movement improvement which did not reach significant levels54.Chondroitin biosynthesis mutants 34. Previous reports in mammalian systems have demonstrated a modulatory role for ketamine in Wnt and TGF-beta pathways38. Our enrichment analysis has shown that BLIMP-1/BLMP-1 might be a putative molecular target for ECM modulatory effects of ketamine by the virtue of overlapping collagens with altered expression. BLIMP-1/BLMP-1 functions both as a transcriptional repressor and a transcriptional activator and was reported to positively regulate bed-3 which showed increased expression levels in the ketamine-treated animals suggesting ketamine may upregulate collagen and bed-3 expression via BLIMP-1/BLMP-1-mediated gene regulation39. One study conducted on a cell line model inferred that ketamine might inhibit Blimp1 expression55. Our findings suggest that BLIMP-1/BLMP-1 might be a putative novel molecular target for ECM modulatory effects of ketamine. Given that ketamine-regulated factors hold the potential to be therapeutic targets for treatment of treatment-resistant depression, using C. elegans as a model to decipher ketamine-mediated alterations may provide insights into its’ roles in the mammalian systems.Enrichment analysis of differentially regulated ketamine-responsive genes identified collagens some of which have been reported to be regulated via Wnt and TGF-beta pathwaysC. elegans can be extended to the mammalian systems. In conclusion, we propose C. elegans as a putative animal model to study ketamine-mediated ECM alterations.A limitation of this study include the types of tissues examined for ketamine-induced modifications. Although we demonstrated a neuronal migration defect, further studies focused on neural ECM are required. Ketamine may not act on the chondroitin in the context of vulva, however which aECM molecules are altered by ketamine-treatment remains to be deciphered. Further evidence is required whether these findings in otIs33[kal-1::GFP] IV; for assessment of HSN migration and branching defects at adult stage zdIs13[tph-1::GFP] IV; for detection of vulval abnormalities syIs49[zmp-1::GFP + (pMH86) dpy-20( +)] IV, syIs51[cdh-3::CFP + unc-199( +)] V; syIs55 [ceh-2::YFP + unc-119( +)] X, ncIs13[ajm-1::GFP] and ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20( +)] I; to test whether sqv phenotype can be induced in mutants with defective NMDA and RyR receptors nmr-1(ak4) II and unc-68(e540) V; to evaluate roller phenotype amelioration rol-1(e91) II, rol-4(sc8) V, rol-6(e187) II, rol-8(sc8) II, rol-9(sc148) V and sqt-1(sc13) II.Nematode strains were cultured on Nematode Growth Media (NGM) seeded with OP50 bacteria and maintained according to standard techniques. Experiments were performed at 25 °C unless otherwise specified. The animals used for ketamine treatment are wild-type Bristol N2. Strains carrying following transgenes or alleles were used: For visualisation of HSN neuron at L1 stage C. elegans is common when exposed to various drugs including anesthetics. 2.5 mM concentration allowed to carry out experiments without severe developmental delay and with observable phenotypes. Pharyngeal pumping rate was determined on young adult animals for three 20-s periods. One pump is defined as a single cycle of contraction and relaxation of the terminal bulb muscles. At least 10 animals were used for per three independent measurements.Synchronised L1 wild-type animals were grown on OP50-seeded NGM plates containing 2.5 mM ketamine and in M9 buffer as vehicle for control condition. Ketamine-treated and control animals were grown at 25 °C and all experiments were carried out at this temperature. Ketamine concentrations of 1 mM, 2.5 mM, 5 mM, 10 mM were used where higher concentrations induced high toxicity and severe developmental delay (data not shown). The 2.5 mM concentration was selected on the basis of strength of the phenotypes. Developmental delay in Ketamine-treated and control animals were transferred to individual bacteria-seeded NGM plates with a single L4 stage animal per plate and grown at 25 °C. Animals were transferred to new plates every 24 h until they stopped egg-laying. The number of progeny on each plate was determined with three independent experiments using 10 hermaphrodites per condition.57. Ketamine-treated and control embryos were washed with M9 buffer and embryo pellets were fixated with 4% PFA overnight at 4 °C after four rounds of freeze-crack step in liquid nitrogen. Fixative was removed by washing in PBS containing 0.25% Triton-X (PBST). Samples were treated with 50 mU/ μl chondroitinase ABC (CSase ABC) enzyme prepared in 0.04 M Tris Acetate, pH 8. As per negative control, samples were incubated in 0.04 M Tris buffer without the CSase ABC. The incubation period for with and without CSase ABC treatment was 3 h at 37 °C. Enzyme was heat inactivated at 95 °C for 5 min. For blocking, samples were treated with 10% donkey serum for one hour at room temperature. Primary antibody incubation was performed using anti-chondroitin antibody  at 4 °C overnight. For the secondary antibody, donkey anti-mouse Alexa 488 (Life Technologies) was used with a dilution of 1:250. VectaShield mounting medium with 49.6-diamidino-2-phenylindole (DAPI) was used to mount embryos on the microscope slides.The immunofluorescence was performed as described previously with some modifications58. Control and ketamine-treated animals were synchronized as described above. Worms were collected at the stage of adult day 1 and washed in ice-cold M9 buffer twice. Supernatant was removed and TRIzol reagent was added to the worm pellet with a ratio of 1:2. Animals were frozen in liquid nitrogen with three cycles of freeze–thaw. RNA extraction was carried out using Direct-zol RNA kit (Zymo Research) as per manufacturer’s instructions. Briefly, worm pellet lysed in TRIzol was spun for 1 min at 12.000 g. Supernatant was mixed with absolute ethanol (1:2) and loaded onto spin columns. After washing steps, in-column DNase I treatment was performed. cDNA was synthesized from 4–10 μg total RNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Real-time PCR was conducted using Roche LightCycler 480 II. sqv-7 and sqv-5 transcript levels were analyzed using ∆∆Ct method with ubc-2 as a normalization control.Transcript levels were measured as previously reportedC. elegans reference genome (WBcel235) using Hisat2 v2.0.5. The mapped reads were assembled by StringTie (v1.3.3b) . FPKM of each gene was calculated using featureCounts v1.5.0-p3. Differential expression analysis was performed using the DESeq2R package (1.20.0). Genes with at least a two-fold difference at an FDR < 0.05 were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes were implemented by the WormBase Enrichment Suite based on the WS262 WormBase release. Annotation and visualization of the gene set enrichment data was performed using WormCat 2.0.Synchronised L1 wild-type animals were distributed over NGM plates containing 2.5 mM ketamine or in M9 buffer as vehicle and grown at 25 °C. RNA was isolated from whole worms at the stage of adult day 1 as described above. RNA Integrity Number (RIN) quality score was assessed using a Bioanalyzer 2100. mRNA libraries were constructed using using the Illumina Tru-Seq Stranded mRNA Library Construction Kit. 150-bp paired-end reads were generated on an Illumina HiSeq 2500 machine with 70–90 million reads per sample. FASTQ output files were aligned to the For image acquisition Leica DMI1 inverted microscope was used. Sqv phenotype was scored under the dissecting microscope (Leica M165 FC).Statistical analysis was performed using GraphPad Prism Version 9.3.1. For all analysis α-level is 0.05.Supplementary Legends.Supplementary Figure 1.Supplementary Table 1.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Sperm routinary fitness evaluation is not sufficient to predict bull reproductive capacity as they present differences in fertility up to 40%. Among the defects which compromise spermatozoa functionality, new approaches consider the study of sperm chromatin, which is the core structure containing paternal genetic information. Sperm chromatin needs to be compacted to maintain the integrity of DNA, which occurs by binding nucleoproteins with high affinity to DNA. In the last stages of sperm maturation, chromatin is hyper-compacted by basic proteins called protamines in a process named protamination. In this review, we summarized intrinsic and extrinsic factors that are suggested to influence protamination in bull spermatozoa, considering old and new evidence from human and murine spermatozoa. Also, the current approaches to evaluate bull protamination and its relationship with fertility were described. Nevertheless, the physiological mechanisms of protamination are still poorly understood. The use of assisted reproduction techniques (ARTs) in cattle production is expanding as it promotes genetic gains by breeding dams with sires of high genetic potential . Howeverin vivo  throughout sperm maturation . In the As shown in The protamination process involves the species-specific participation of two protamine families: protamine 1 (PRM1) and protamine 2 . The PRM1 is usually synthesized as a mature protein. PRM1 is composed of 50 amino acids with 3 main domains: a central arginine-rich domain; a high affinity for DNA, flanked by serine and cysteine residues domain; and the last containing threonine segments and several phosphorylation sites . Also, PProtamine substitution in mature bull spermatozoa would establish a strong and protected structure, believed to be a result of an evolutive mechanism due to high selection pressure. For this reason, it is important to measure protamination, as it would be a checkpoint to assure bull fertility. Immature spermatozoa inside the testis or in epididymal transit still show low protamine binding (CMA3 assay) which correlates with high rates of DNA susceptibility to fragmentation .Protamines have an important role in maintaining male fertility, as this hyper-compaction would preserve the integrity of sperm chromatin throughout the female tract until reaching the oocyte . At the Chromatin compaction is a late-spermatogenesis process with species-specific signaling, so the conformation and amount of each protamine in sperm chromatin will differ between species . Mammal In spermatozoa of some species like humans, the physiological replacement of protamines in spermatozoa is incomplete, generating a fraction of DNA (≅ 15%) still attached to nucleosomes . The mecSperm chromatin content is considered a unique mosaic for each species and even for breeds as the proportion of nucleoproteins varies among individuals, thanks to the presence or absence of regulatory transcription factors . These pin vitro reproduction techniques, such as intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF)  bound to DNA molecules when compared to humans, which have more DNA still attached to histones . Among ton (IVF) . Afterwaon (IVF) .Moreover, experiments using murine spermatozoa as a biological model also observed a complementary role of the binomial PRM1:PRM2 . These sIn bulls, chromatin compaction was attributed only to PRM1, while PRM2 was believed to be nonfunctional . RecentlTherefore, some studies mention the relationship of deleterious morphological defects with failures in protamination or higher rates of DNA fragmentation in bovine spermatozoa . Also, iSpermatozoa protamination in mammals occurs at the last epididymal passage and is part of the last structural modifications of the spermatozoa to acquire the hydrodynamic characteristics that allow it to fertilize the oocyte . HoweverThroughout spermatogenesis, canonical histones undergo post-transcriptional modifications (PTM) within the nuclear structure . These PProtamine genes could also be targeted by epigenetic changes, sometimes resulting in an inefficient translation. High variability in the 5' VLR (Variable Length Repeat) region of the human PRM2 gene could lead to dysregulated protamination . The preInside the testis parenchyma, there is an endocrine regulation of spermatogenesis that could reflect on sperm DNA compaction. The interaction between follicle-stimulating hormone (FSH) and testosterone plays a regulatory role in sperm chromatin, as FSH knock-out experiments reported changes in sperm chromatin and increased DNA fragmentation in murine spermatozoa . Also, FA controlled environment is important as some external conditions could reflect negatively on the spermatozoa. Exposure to noxious substances or stressful conditions like heat stress could increase sperm metabolism and consequently, the production of reactive oxygen species (ROS), as suggested in Nevertheless, heat stress can be present in different stages of spermatogenesis, being more deleterious in early spermatogenesis (spermatocyte and spermatid stages) irrupting transcription and transduction of various genes, yet not defined . PhysiolTherefore, some heat-injured spermatozoa continue along spermatogenesis with several alterations, resulting in a dysfunctional gamete with morphological alterations, mitochondrial misfunction, and a looser chromatin . Those aTo compensate for the excessive production of ROS, supplementation with vitamins (known for their antioxidant function) is used as a therapeutic alternative . MoreoveIn summary, in the sperm chromatin is happening some sequenced events: heat stress negatively modifies gene and protein expression which arises ROS production resulting in oxidative damage . NeverthProtamination status is not checked in routine semen evaluation, but experimental approaches consider direct or indirect evaluation. Direct evaluation has been validated already in humans and murine through absolute quantification of protamines by qPCR-RT, as the relative expression is not appropriate due to the reduced transcription activity in mature spermatozoa . It is amale factors, and the poor understanding of sperm trait interactions. Despite this, chromomycin A3 is described as a useful test to measure chromatin packaging and is correlated to bull fertility . Therefore, this review described the mechanisms that may impair protamination and consequently chromatin maturation. So far, it is acknowledged that external stimulus (ie: heat stress) and dysregulation of intrinsic factors may alter spermatogenesis, and gene expression and finally impair protamination. Also, the current methods (direct and indirect) of protamine evaluation are important but it is suggested that some functional studies are needed to achieve a better understanding of protamine dynamics in bulls.


Nicotiana tabacum) roots with glycine (Gly) amino acid, and glycilglycine (GlyGly) and glycilaspartic acid (GlyAsp) dipeptides (10−7 M concentration) significantly increased the development of root hairs. In the root, peptide accumulation was tissue-specific, with predominant localization to the root cap, meristem, elongation zone, and absorption zone. Peptides penetrated the epidermal and cortical cell and showed greater localization to the nucleus than to the cytoplasm. Compared with the control, tobacco plants grown in the presence of Gly, GlyGly, and GlyAsp exhibited the activation of WER, CPC, bHLH54, and bHLH66 genes and suppression of GTL1 and GL2 genes during root hair lengthening. Although Gly, GlyGly, and GlyAsp have a similar structure, the mechanism of regulation of root hair growth in each case were different, and these differences are most likely due to the fact that neutral Gly and GlyGly and negatively charged GlyAsp bind to different motives of functionally important proteins. Short peptides site-specifically interact with DNA, and histones. The molecular mechanisms underlying the effect of exogenous peptides on cellular processes remain unclear. Since these compounds acted at low concentrations, gene expression regulation by short peptides is most likely of epigenetic nature.Root hairs absorb soil nutrients and water, and anchor the plant in the soil. Treatment of tobacco ( Root hairs play diverse roles that are critical for plant growth and development: absorption of sufficient amounts of water and nutrients from the rhizosphere under drought and/or nutrient deficient conditions; interaction between the plant and soil microfauna; and anchoring the plant in the soil ,2,3. TheArabidopsis thaliana, root hair development is broadly divided into four stages: root hair cell specification, initiation, elongation (tip growth) and maturation [Plant cell differentiation is a complex process . Based oturation ,8. Depenturation ,10. In tturation ,12; the turation . The fatturation ,15. In sturation ,16.bHLH) transcription factor ROOT HAIR DEFECTIVE 6 (RHD6) and its close homolog RHD6-LIKE 1 (RSL1) [During root hair elongation, cells in root hair tips exhibit a unique growth pattern, which is regulated by the cytoskeleton and is accompanied by changes in the cell wall . Root ha1 (RSL1) ,23.bHLH superfamily consists of a large number of proteins found virtually in all organisms, including fungi, plants, and animals [bHLH transcription factors act as a link between the R2R3-MYB proteins, such as WEREWOLF  and GLABRA1 , and the WD-40 transcription factor TRANSPARENT TESTA GLABRA1 (TTG1) to form an activation complex [The  animals . The bHL complex ,26,27,28CPC, ETC, GL2, GL3/EGL3, TTG, and WER [CPC, GL2, and WER are preferentially expressed in N cells during seed germination, and regulate the expression of GL3/EGL2 in H cells [CPC, GL2, and WER expression. Interaction between these regulatory genes has been demonstrated previously [WER positively regulates CPC expression and GL2 appears to move into cells at position H and inhibit expression WER and CPC, which causes the cell to initiate hair formation.Previous studies have shown that the initiation of root hair differentiation is determined by interactions among six genes:  and WER . Among t H cells . Initiateviously . At posiGL2 gene is predominantly expressed in N cells, it is usually considered a negative regulator of root hair development, and its activation or inhibition determines the fate of root hair development. Therefore, GL2 is believed to represent an important genetic switch in determining the cell fate during the formation of root hairs [Since the ot hairs .DF1 inhibit the growth of hair roots in Arabidopsis. Both GTL1 and DF1 directly bind to the RSL4 gene promoter and regulate its expression, suppressing the growth of root hairs. Thus, this study reveals a basic regulatory module that precisely determines the degree of root hair growth through the orchestrated actions of opposing transcription factors [An important question is how plants determine the final size of growing cells. The RSL4 transcription factor promotes the growth of root hairs . Another factors .RSL4. In addition, environmental factors, such as nutrient availability, also affect root hair development. It has been suggested that hormones and environmental factors influence root hair initiation in a different manner than that occurring during normal development [Root hair growth is fine-tuned by various hormonal and environmental signals. Auxin is well known to enhance the growth of root hairs ,36. Exogelopment . TogetheEndogenous peptides, like phytohormones, play important roles in the regulation of numerous intercellular connections and physiological activities, and respond to various influences . Peptide−7 M in the medium significantly stimulate the growth and development of seedlings of Nicotiana tabacum. They stimulate the formation and growth of leaves and roots. Peptides modulate expression of genes of the KNOX and GRF families that are responsible for cell differentiation and encoding transcription factors [We have previously shown that dipeptides GlyGly and GlyAsp and the amino acid Gly at a concentration of 10 factors . The proNicotiana tabacum L.).The aim of this study was to investigate the effect of glycine (Gly) amino acid and GlyGly and Gly-aspartic acid (GlyAsp) dipeptides on the regulation of root hair growth in tobacco (−7 M) in the medium was presumably a result of their regulatory signaling role in the cell.The amino acid Gly and the dipeptides GlyGly and GlyAsp significantly stimulated the growth and development of tobacco seedlings. GlyAsp and GlyGly showed the most pronounced stimulating, which significantly accelerated the development of seedlings and the root system . The lenGly had a less pronounced growth stimulating effect the dipeptides .Compared to the control, Gly increased the total length of seedlings and the length of the main root increased by about 10–20% . HoweverThus, the stimulating effect of Gly and dipeptides was accompanied by the appearance of additional roots and greater seedling biomass compared with the control.The results revealed zoning of root staining . In the When treated with FITC-Gly, diffused staining was observed in the root cap and meristem and intense staining was observed in the elongation zone h, mainlyThus, FITC-labeled Gly and dipeptides stained the cell walls, cytoplasm, and nuclei in root cells, although the intensity of staining differed among cells. However, certain areas of the root showed no stained cells.The localization pattern of the peptides indicates that the FITC-conjugated peptides penetrate and accumulate in tobacco roots. However, the nature of the accumulation of these substances varies among the root tissues and cells. Thus, the appearance and accumulation of the studied peptides in the root is tissue-specific. This may be because cells in different root zones exhibit different competence levels for interaction, penetration and accumulation of peptides. Thus, the determination of the localization of peptides can be effectively used for the rapid assessment of the status of root cells under different growth conditions and under the influence of various factors.Since the elongation and root hair zones are mainly responsible for the absorption of nutrients, we investigated the intact parts of the tobacco root . The rooCPC, ETC, GL2, GL3/EGL3, TTG, and WER), three of which  are expressed in H cells, along with bHLH, which acts as a linker. The WER gene suppresses the expression of CPC and GL2 genes in H cells, thereby initiating the formation and growth of root hairs. The other complex includes WER, CPC, and GTL1 genes, where the latter inhibits the growth of root hairs.In plants, several systems of regulation levels (active complexes) presumably control root hair growth. In this study, we considered two active complexes. One of these complexes includes six genes  of tobacco roots was unaffected by Gly and GlyGly compared with the control. By contrast, treatment with GlyAsp increased the expression of bHLH66 by more than 2-fold in comparison with the control , then the expression level of GTL1 was 1.5–2.5-fold lower than the control. This was especially true for tobacco seedlings grown in the presence of Gly and GlyGly.Gly and both dipeptides inhibited the expression of ot hairs . HoweverWER, CPC, bHLH54, and bHLH66 genes and the inhibition of GTL1 and GL2 genes could probably explain the lengthening of the root hairs in tobacco seedlings grown in the presence of Gly, GlyGly, and GlyAsp compared with the control.The activation of Thus, despite the similar structure of peptides, their impact on root hair growth differed, most likely because the neutral Gly amino acid and GlyGly dipeptide and the negatively charged GlyAsp dipeptide bind to different motifs in functionally important proteins. Genes expression levels differed slightly between tobacco plants grown in the presence of GlyGly and Gly, most likely because of differences in the binding constants between them and protein motifs.Short peptides, such as GlyGly and GlyAsp, and the amino acid Gly, present in the soil as waste products of soil microorganisms, penetrate the plant roots. Experiments with FITC-labeled additives showed fluorescence in the root cap apex, root meristem and extension and absorption zones. FITC-labeled peptides and Gly penetrated into the plant cell and localized to different subcellular compartments, including the nucleus, cytoplasm and cell wall. Penetration of these compounds into the nucleus can be accompanied by a change in chromatin structure. Nutrients and water are transported through the root hairs and differentiating vessels, and are distributed throughout the plant. Plants absorb water from the soil by osmosis. Root hair cells are adapted to osmosis, as they have a large surface area and a large permanent vacuole.−7 M) is presumably due to the fact that they perform a regulatory signaling function in the cell like peptide hormones.The pronounced biological activity of these GlyGly and GlyAsp, and the amino acid Gly at such a low concentration in the medium  [The first plant peptide that was shown to have signaling activity was systemin, consisting of 18 amino acid residues, was isolated from tomato (ulentum) . It has ulentum) . The CLEulentum) .Another family of hormonal peptides that affect the development of the root system are peptides containing sulfated tyrosine . It is aThe secreted peptide RALF  with 5 kDa, has the ability to increase the pH of the medium in cultured cells . The RALPeptide hormones are the main regulators in plants, they participate in many aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. While the effect of exogenous peptides on animals has been studied for several years ,54, the −7 M) in this study, suggesting that the regulation of gene expression by short peptides is most likely of an epigenetic nature. A previous study showed that short exogenous peptides (GlyGly and GlyAsp) and Gly selectively alter the expression of genes encoding DNA methyltransferases and SNF2 proteins [DRM2 methyltransferase gene, which carries out de novo DNA methylation, by 2–4-fold.Although the molecular mechanisms underlying the effect of exogenous peptides on cellular processes remain poorly understood, one of the possible mechanisms is the regulation of gene transcription. GlyGly, GlyAsp, and Gly, for example, can bind to regulatory proteins to influence gene expression. These compounds act at low concentrations  cultivar Samsun were placed in flasks containing hormone-free Murashige–Skoog (MS) agar medium supplemented with or without 10−7 M Gly, GlyGly, and GlyAsp. Experiments were carried out in four replicates. After 28 days, the seedling fresh weight, seedling height, and root and seedling length were evaluated. The last parameter was measured under an Olympus BX51 microscope  furnished with the Cell program. The calculation of the main statistical parameters was carried out according to standard methods, and Statistica 6.0 and STATAN programs for statistical data processing were used.Seeds of tobacco (A solution of FITC (1 μg in 10 μL of 0.5 M sodium bicarbonate) was added to a solution of Gly, GlyGly, and GlyAsp in 0.01 M Tris-HCl buffer (pH 7.0) at a ratio of 1.2:1 . The rea−5 M; control FITC and FITC without peptides were used as controls [To determine the localization of peptides in root cells, the FITC-labeled dipeptides and Gly were used at a concentration of 10controls . The inccontrols .Roots stained with FITC-labeled peptides were fixed in 4% paraformaldehyde and embedded in LRW, as described previously . Then, sTotal RNA was isolated from tobacco roots, from which root hairs were carefully removed, using RNA-Extran RNA isolation reagent kit , according to the manufacturer’s instructions. The concentration of isolated RNA preparations was determined on NanoPhotometer IMPLEN.Then, cDNA was obtained using a set of reverse transcription reagents , according to the manufacturer’s instructions.Nicotiana tabacum genes was obtained from the National Center for Biotechnology Information (NCBI). Primers for these genes were designed using the NCBI Primer-BLAST web tool, and synthesized at Syntol. Real-time PCR was performed on CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using a set of RT-PCR reagents and SYBR Green . The relative expression level of genes was calculated using a calibration curve constructed with PCR products derived from the GAPDH gene. The efficiency of real-time PCR was calculated as E% = (10−1/s−1) − 100, where s is the slope of the dependence of the decimal logarithm of Ct values on the cDNA concentration. The efficiency of RT-PCR performed using primers corresponding to the genes of interest was 95–96%. The calculation of the main statistical parameters was carried out according to STATAN program on a significance level p < 0.05.Information on the primary structure of The equipment of the Center for Collective Use of the Federal State Budgetary Scientific Institution VNIISB RAS was used in this study.Nutrients and water are carried through the root hairs and root differentiating vessels and are distributed throughout the plant. The ability to regulate root hair growth can have a direct impact on sustainable organic farming through improved water and nutrient intake.Short peptides GlyGly and GlyAsp and Gly penetrate into the roots and their accumulation is tissue-specific, with predominant localization in the root cap, meristem, zones of extension and absorption. Peptides penetrate into the epidermal and cortical cells and show preferential localization in the nucleus than in the cytoplasm.Dipeptides and Gly have a marked effect on the development and elongation of tobacco root hairs. In the presence of peptides, the root hairs were longer, denser, and differed in the character of pubescence in comparison with the control. The mechanism of the action of dipeptides and Gly differed, depending on their structure and charge. Overall, the dipeptides GlyGly and GlyAsp and the amino acid Gly showed pronounced physiological activity, and can be considered as positive regulators of the formation of root hairs.It is suggested that GlyGly, GlyAsp, and Gly are a new class of plant growth and development regulators. The most studied mechanism of regulation of genome transcription is epigenetic regulation, which includes DNA methylation and modification of histone proteins.


Keratinocyte differentiation is an essential process for epidermal stratification and stratum corneum formation. Keratinocytes proliferate in the basal layer of the epidermis and start their differentiation by changing their functional or phenotypical type; this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes that play a pivotal role in epidermal development. Epidermal development and the keratinocyte differentiation program are orchestrated by several transcription factors, signaling pathways, and epigenetic regulators. The latter exhibits both activating and repressive effects on chromatin in keratinocytes via the ATP-dependent chromatin remodelers, histone demethylases, and genome organizers that promote terminal keratinocyte differentiation, and the DNA methyltransferases, histone deacetylases, and Polycomb components that stimulate proliferation of progenitor cells and inhibit premature activation of terminal differentiation-associated genes. In addition, microRNAs are involved in different processes between proliferation and differentiation during the program of epidermal development. Here, we bring together current knowledge of the mechanisms controlling gene expression during keratinocyte differentiation. An awareness of epigenetic mechanisms and their alterations in health and disease will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising target therapies. The epidermis constitutes the skin surface and is composed of specialized epithelial cells called keratinocytes, which originate from two pools of quiescent epidermal stem cells (qESC), one living in the basal layer of the interfollicular epidermis (IFE) and the other, bulge stem cells, located in the hair follicle of the sebaceous gland that gives rise to keratinocytes of the hair follicle (HF) lineage. From qESC, transient amplifying (TA) cells generate “mature” keratinocytes residing in the basal layer of the epidermis or in the hair bulge (region of the outer root sheath) . ProgeniIn the present review, we aim to summarize the available evidence on activating and repressive effects on chromatin that promote epidermal differentiation. In the first section, we review the normal functions of epigenetic mechanisms and their role in epidermal differentiation. The second section focuses on our current understanding of the different types of epigenetic changes in psoriasis and atopic dermatitis—as emblematic examples of inflammatory skin diseases—providing some specific examples of each.In summary, we provide an overview of the epigenetic mechanisms underlying epidermal differentiation in health and disease and we conclude with a discussion of the potential implications for clinical practice.To provide an overview of the current state of knowledge regarding the topic of epidermal differentiation, papers were selected from those included in the electronic databases PubMed/MEDLINE, Google Scholar, Scopus, and Web of Science over the past 20 years. The following search terms were used: keratinocytes, epigenetics, epigenetic regulators, methylation, histones, epidermal differentiation, epidermal differentiation complex, microRNAs, psoriasis, atopic dermatitis, inflammatory skin diseases, environmental factors. Each selected paper was analyzed, the data extracted, and presented in the main text to provide an overview of the main aspects of epigenetic regulation that influences the programming of epidermal keratinocytes.Epigenetics refers to changes in gene expression without alteration of the DNA sequence. These changes, which make up the so called “epigenome”, include modifications to DNA and the histone components of nucleosomes as well as expression of noncoding RNAs (ncRNAs). Since epigenetic signatures help determine if genes are turned on or off, it influences the production of encoded proteins, thus ensuring cell-type-specific expression . EpigeneHistone modification is another key epigenetic mechanism. Histone complexes are composed of two H2A-H2B dimers and a H3-H4 tetramer to form the nucleosome, and they may be chemically modified through the action of enzymes to regulate gene transcription. The most common modifications are the methylation of arginine or lysine residues, or the acetylation of lysine  Figure b. Methylation can modulate the interaction between transcription factors and other proteins with nucleosomes , while lMiRNAs are a group of short (~22 nucleotides), single stranded, non-coding RNAs that can regulate the expression of other protein-coding gene at the post-transcriptional level. MiRNAs function via base-pairing with complementary sequences within messenger RNA (mRNA); after this binding, mRNA molecules are silenced by one or more of the following processes: (i) mRNA cleavage, (ii) destabilization of the mRNA through de-adenylation of its poly(A) tail, and (iii) translational inactivation . In rareBelow, we review the mechanisms that control chromatin remodeling, with special emphasis on keratinocyte-specific gene expression programs in skin development , Mi-2/nucleosome remodeling and deacetylase (NuRD) complex, imitation switch (ISWI)/SNF2L, and INO80 . The SWIAdditionally, regulatory subunits of the SWI/SNF complex such as the actin-like 6A protein/Brahma-associated factor (ACTL6a/BAF53A), along with the catalytic subunits , are involved in suppressing differentiation and maintaining the progenitor state in epidermal cells . ATP-dependent chromatin remodelers of the CHD group are characterized by a chromodomain that specifically binds to methylated lysine residues; the ATPase subunits within this family include Chd1-9, but Chd3 and Chd4  have a prominent role in NuRD complex. The chromatin remodeler Mi-2β is crucial for the self-renewal of epidermal precursors during the earlier phases of embryogenesis, and its ablation leads to defective basal layer formation, whereas its loss during the later stages of embryogenesis leads to impaired induction and development of the hair follicles . Histone methylation is the modification of certain amino acids in a histone protein by the addition of one, two, or three methyl groups. The site-specific methylation and demethylation of histone residues are catalyzed by methyltransferases and demethylases, respectively. Histone methylation is generally associated with transcriptional repression; however, methylation of some lysine and arginine residues of histones results in transcriptional activation . TrimethIn contrast, the chromatin of an inactive gene is enriched in H3K9me2/3, H3K27me2/3 and H4K20me3 modifications . The JumHistone H4 mono-methylation at lysine 20 is regulated by Setd8 histone methyltransferase. Setd8 is a transcriptional target of c-Myc, a protein involved in cellular epidermal differentiation, and it is involved in many physiological processes, including cell cycle, chromatin condensation, apoptosis, tumorigenesis, and epithelial to mesenchymal transition . The losSuv39h1, a gene encoding a H3K9 histone methyltransferase, provoked a marked reduction in the level of tri-methylation at the ninth lysine residue of the histone H3 protein and promoted changes in the expression of several keratinocyte-specific genes [Suv39h1 knockout also led to induction of genes encoding differentiation markers such as keratin 10, desmoglein 1, S100A8, and late cornified envelope (LCE1) proteins, whereas expression of genes associated with undifferentiated keratinocytes, such as keratin 14 and S100A6, remained unaltered, or at most, slightly decreased [In HaCaT cells, spontaneously immortalized human keratinocytes, knockout of ic genes . Suv39h1ecreased . OverallOvol1 , which is regulated in turn by the PI3K-AKT signaling pathway [Ship1 activates the downstream PI3K-AKT pathway and enhanced Ovol1 expression in HaCaT cells, whereas overexpression of Ship1 led to decreased Ovol1 expression [Lysine demethylase Jarid1 family members  demethylase the fourth lysine residue of histone H3 and participates in multiple repressive transcriptional complexes, although the specific regulatory effects on chromatin remain undefined . The rol pathway . Jarid1bpression . Thus, Spression . Special AT-rich sequence-binding protein-1 (Satb1) is a global chromatin organizer and transcription factor and is critical for integrating higher-order chromatin architecture with gene regulation . Satb1 regulates gene expression by acting as a “docking site” for several chromatin remodelers and by recruiting corepressors or coactivators directly to promoter regions , thus esde novo DNA methylation enzyme that is enriched in undifferentiated epidermal progenitor cells, where it is required to retain proliferative potential and suppress differentiation; after the embryonic stages, it remains confined to the basal epidermal layer containing proliferating keratinocytes [DNMT1is both a maintaining and inocytes . Khavariinocytes . Epidermal depletion of DNMT1 coexists with overexpression of genes associated with cell cycle arrest such as Cdk inhibitors ; however, cyclin-dependent kinase 4 and 6 (Cdk4/6) partially rescues the effect of DNMT1 impairment on cell proliferation . These findings support the crucial role of DNMT1 in preserving epidermal progenitor cell identity and remodeling of this pattern during terminal differentiation . Some gene promoters remain active when methylated through the binding of CCAAT-enhancer-binding proteins (C/EBPα) that contribute to the opening of the chromatin structure with consequent gene activation, whereas 5-aza-cytidine promotes the inhibition of DNA methylation and prevents the expression of a subset of genes during calcium-induced differentiation in culture . 5-azacyFinally, 5-hydroxymethylcytosine (5hmC) is the first oxidative product in the active demethylation of 5-methylcytosine (5mC) a. Three Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone, resulting in a more closed chromatin structure and repression of gene expression . HDACs pFurther evidence for the role of HDACs was obtained after depletion of HDAC1/2 from the basal layer of mouse epidermis resulted in increased histone acetylation and enhanced keratinocyte proliferation, leading to hyperplasia in addition to apoptosis and hair follicle dystrophy . HDAC1/2 homozygous deletion caused hyperacetylation of c-Myc. Taken together, these findings indicate that HDAC1/2 play three distinct roles in adult epidermis: (i) deacetylation of p53 to promote progenitor cell proliferation, (ii) deacetylation of c-Myc to prevent premature differentiation, and (iii) restraint of p16 to repress senescence and allow long-term maintenance of progenitor cells [Zhu et al. demonstrated for adult mouse epidermis that homozygous epidermal HDAC1/2 codeletion—in association with a null p53 expression—partially restored epidermal proliferation, indicating that the effects of HDAC1/2 deletion on keratinocyte proliferation are mediated in part via increased p53 activity. Moreover, p16 deletion did not significantly rescue epidermal thickness, proliferation, and/or apoptosis of HDAC1/2 null epidermis, but its loss enabled survival of HDAC1/2-deficient keratinocytes by preventing their senescence. Finally, the same authors demonstrated that double or cells . Polycomb group (PcG) proteins are transcriptional repressors with a key role in stem cell identity and differentiation. PcG proteins are evolutionarily conserved and act within complexes, called Polycomb repressive complexes (PRCs).PRC1 mono-ubiquitinates histone H2A on lysine 119 (H2AK119Ub1) and PRC2 catalyzes trimethylation of lysine 27 on histone H3 (H3K27me2/3) . ChromobBmi-1 is another PRC1 component. It is expressed in the basal layers of human epidermis and is implicated in the control of cell survival by altering cell cycle regulatory protein expression, such as p16 and p19, and inhibiting apoptosis ,61. PRC2 is the most widely conserved PcG complex in multi-cellular eukaryotes and is composed by four core subunits with an intrinsic histone methyltransferase activity; the catalytic subunit, known as Ezh2 (enhancer of zeste homolog 2), contains the signature SET domain commonly found in lysine methyltransferases . It has MicroRNAs (miRNAs) are a group of short (~22 nucleotides), single stranded, non-coding RNAs, which can regulate the expression of other protein-coding gene at post-transcriptional level. In most cases, miRNAs interact with the 3′ UTR of target mRNAs to suppress expression , but theThere are several reports documenting the role of miRNAs in epidermal development and differentiation; for example, miR203, is induced in terminally differentiated keratinocytes, both in vitro and in vivo. Through the regulation of p63 expression, miR203 maintained the proliferative and differentiation potential of basal keratinocytes as well as precursor cells . BesidesA total of nine miRNAs—miR-203, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a—were found not only to be induced in terminally differentiated keratinocytes in vitro and in vivo but were also functionally involved in the differentiation process . Another miRNA, miR-214, when overexpressed, led to a reduction in epidermal thickness, lower keratinocyte proliferation rate, and hair follicle loss . These examples suggest that several miRNAs are involved in different processes between proliferation and differentiation in the program of epidermal development. In the field of dermatology, skin disorders notably affect skin integrity and impair epidermal differentiation. Epigenetic mechanisms have been extensively studied in cutaneous T-cell lymphoma (CTCL) , but in Psoriasis is a chronic, immune-mediated inflammatory skin disease with a multifactorial etiology involving accelerated proliferation and abnormal differentiation of keratinocytes . It is nEpigenetic changes, including DNA methylation, histone modification, and noncoding RNA regulation have been reported to be involved in the complex pathogenesis of psoriasis . Roberson et al. first identified global DNA methylation in psoriatic skin lesions when compared to controls, in which more than 1000 differentially methylated CpG islands (CGIs) were detected and, among them, twelve mapped to the epidermal differentiation complex or nearby genes upregulated in psoriasis. Moreover, the authors highlighted the reversible nature of DNA methylation, suggesting that this epigenetic mechanism is a dynamic actor in this disease . S100A9 , SELENBP1 (selenium binding protein 1), CARD14 (caspase recruitment domain family member 14), KAZN , and PTPN22 (protein tyrosine phosphatase non-receptor type 22), indicating the potential role of DNA methylation in regulating specific histopathological features commonly seen in psoriasis [Chandra et al., in an epigenome-wide DNA methylation study, found several differentially methylated loci, overlapped with different PSORS regions, involved in the regulation of pathogenetic gene expression, such as soriasis . SHP-1 (Src homology region 2 domain-containing phosphatase-1), a protein tyrosine phosphatase (PTPs) that regulates several cellular processes, including growth and cell differentiation [A candidate gene approach has been employed to define the promoter methylation profile of several psoriasis risk loci and, according to gene ontology, CpG hyper/hypomethylation coincided with genes involved in many processes impaired in the pathogenesis of psoriasis, such as cell cycle, apoptosis, immune system regulation, cell communication, and signal transduction . Loss ofntiation , and at ntiation , p15 andntiation . PDCD5 (programed cell death 5) and TIMP2  genes, which are involved in apoptosis and the maintenance of tissue homeostasis by suppressing the proliferation of quiescent tissues, respectively, thus regulating the proliferation of keratinocytes [SFRP4 (secreted frizzled related protein) gene, a negative regulator of the Wnt signaling pathway, has been found heavily hypermethylated in psoriasis skin lesions and its consequent downregulation contributes to epidermal hyperplasia [In addition, hypermethylation has been detected in the promoter sequences of inocytes . Moreoveerplasia .TLR-7 (Toll-like receptor 7) and IRAK1 (interleukin 1 receptor associated kinase 1) [Finally, genome-wide association studies have identified hundreds of hypermethylated genes in psoriasis, and among them are immune-associated genes, such as inase 1) .Rorc (RAR related orphan receptor C), which encodes the master Th17 transcription factor Rorγt and Th17 cytokine genes such as IL-17, IL-17f, and IL-22 [Evidence of unbalanced histone modification in psoriatic skin is also beginning to increase. H3K9 dimethylation is decreased in keratinocytes from psoriasis patients, correlating with IL-23 overexpression and supporting its relevance for this disease. Indeed, the actin polymerizing molecule N-WASP controls IL-23 expression in keratinocytes in response to TNF-α by regulating the degradation of the histone methyltransferases G9a and GLP and H3K9 dimethylation of the IL-23 promoter, leading to a chronic skin inflammation with an IL-23 inflammatory profile . Zhang end IL-22 .SOCS-3 , which is involved in inflammatory responses and keratinocyte functions [Changes in miRNA expression have also been found in psoriatic skin lesions and more than 250 miRNAs have been implicated in the pathogenesis of this skin disorder . MiR-203unctions . Subsequunctions . Moreoveunctions . MiR-146a is another microRNA that appears upregulated in psoriasis. It can promote TNF expression and is positively correlated with IL-17-driven inflammation in keratinocytes, revealing a key role with therapeutic potential in the pathogenesis of psoriasis ,93. In aSome miRNA clusters have also been also implicated in the pathogenesis of psoriasis; for example, cytokine-induced overexpression of the miR-17-92 cluster can promote keratinocyte proliferation, contributing to the development of psoriasis-like inflammation . Finally, serum levels of miR-33, miR-126, and miR-143 have been proposed as potential biomarkers for disease severity or the prognosis of psoriasis . Atopic dermatitis is one of the most common inflammatory skin diseases worldwide with a recurrent, chronic-relapsing clinical course characterized by an impairment of the skin barrier function supported by the (epi-)dermal immune system and the microbiome of the skin . Epigenetic modifications have also been recognized in this disease and are mainly mediated by DNA methylation and non-coding RNAs ,102 Fig. KRT6A [Regarding DNA methylation changes in atopic dermatitis, Rodriguez et al.  used an KRT6A . Both thKRT6A . A singlKRT6A , which bKRT6A . FurtherKRT6A  as well KRT6A ,108 thatKRT6A . Collectively, these findings have confirmed—in lesional skin of atopic dermatitis patients—an altered methylation pattern affecting key genes for keratinocyte differentiation, proliferation, and the immune response .TSLP  gene encodes a hemopoietic cytokine that plays a crucial role in promoting a T helper type 2 (TH2) cell response. It appears to be a central player in the development of asthma and atopic dermatitis and is being considered as a potential therapeutic target for the treatment of such diseases [TSLP methylation status, and the results showed—in lesional skin from patients with atopic dermatitis when compared to control—hypomethylation of the TSLP gene at the promoter level, confirming that DNA hypomethylation contributes to TSLP overexpression. The diseases . In a stdiseases , the autIn another study conducted by Olisova et al., DMSs were observed within genes involved in several atopic dermatitis-related processes, including immune response regulation, lymphocyte activation, cell proliferation, apoptosis, and epidermis differentiation, highlighting marked epigenetic involvement in the development of this disease . FLG) gene—essential for the regulation of epidermal homeostasis—in PBMCs of patients with atopic dermatitis, indicated that the association between loss-of-function mutations in the FLG gene and eczema was orchestrated by DNA methylation; in particular, at an 86% methylation level, filaggrin haploinsufficient subjects had about a 6-fold increased risk of eczema when compared to those with wild type FLG [Finally, a study investigating the relationship between genetic and epigenetic changes in the filaggrin , a damage-associated positive regulator of keratinocyte proliferation and migration that is the direct target of miR-10a-5p . Gu et aOther microRNAs that play a critical role in atopic dermatitis are the following: (i) miR-124 that regulates inflammatory responses in keratinocytes and chronic skin inflammation through the NF-κB pathway ; (ii) miInterestingly, miR-17-5p, miR-20a, miR-21, and miR-106b are upregulated in both atopic dermatitis and psoriasis skin lesions, whereas miR-122a, miR-133a-133b, miR-133b, miR-215, and miR-326 are downregulated in both diseases . The simThe central dogma of biology holds that cell information flows from DNA to RNA to proteins ; this coDNA methyltransferase DNMT1, histone deacetylases HDAC1/2, and Polycomb components  act as repressive chromatin regulators stimulating proliferation of progenitor cells, and some of these also inhibit premature activation of terminal differentiation-associated genes. In contrast, a group of chromatin remodelers such as histone demethylases, ATP-dependent chromatin remodeler Brg1, and genome organizer Satb1 promote terminal keratinocyte differentiation and also exhibit effects on cell proliferation. Finally, microRNAs are also involved in different processes between proliferation and differentiation in the program of epidermal development. As demonstrated by several studies concerning skin diseases, changes in the epigenetic signature in the epidermis can contribute to the pathogenesis of these diseases. More importantly, in contrast to genetic changes—which are difficult to reverse—epigenetic modifications are pharmaceutically reversible; thus, the emerging tools of epigenetics can be used as preventive, diagnostic, and therapeutic markers . In the past few decades, development of epigenetic drugs—so-called “epidrugs”—has achieved significant progress; thus, epigenetic therapy is a promising new therapeutical solution that targets the main causative epigenetic mechanisms involved in inflammatory/autoinflammatory diseases. Future efforts in this direction will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising tailored treatments.