Genomic imprinting, parent-of-origin-specific gene expression, is controlled by differential epigenetic status of the parental chromosomes. While DNA methylation and suppressive histone modifications established during gametogenesis suppress imprinted genes on the inactive allele, how and when the expressed allele gains its active status is not clear. In this study, we asked whether the active histone-3 lysine-4 trimethylation (H3K4me3) marks remain at paternally expressed genes (PEGs) in sperm and embryos before and after fertilization using published data. Here we show that mouse sperm had the active H3K4me3 at more than half of known PEGs, and these genes were present even after fertilization. Using reciprocal cross data, we identified 13 new transient PEGs during zygotic genome activation. Next, we confirmed that the 12 out of the 13 new transient PEGs were associated with the paternal H3K4me3 in sperm. Nine out of the 12 genes were associated with the paternal H3K4me3 in zygotes. Our results show that paternal H3K4me3 marks escape inactivation during the histone-to-protamine transition that occurs during sperm maturation and are present in embryos from early zygotic stages up to implantation. Genomic imprinting is regulated by differential epigenetic states between the parental alleles pretranscriptionally and controls development and maternal behavior in both eutherian and marsupial (therian) mammals . This prKCNQ1OT1, MEST, and PEG10 . The active histone-3 lysine-4 tri-methylation (H3K4me3) is found preferentially enriched on the promoter of the unmethylated allele at imprinted genes in mouse embryonic stem cells  and mousnd PEG10 . In mousansition . These eMethods section). Mouse round spermatids and sperm ChIP-seq data sets were analyzed to identify PEGs retaining the active H3K4me3 marks after the histone-to-protamine transition. Parental histone modifications were tracked up to the blastocyst stage to confirm retention of the paternal active status during embryogenesis. Combining ChIP-seq data with RNA-seq data from pre-implantation embryos derived from reciprocal crosses, we identified eight new transient PEGs associated with the H3K4me3 on paternal genome before and after fertilization. We suggest that H3K4me3 marks are retained by paternal histone transmission, correlating with the active status of PEGs after conception and up to implantation.Although paternally transmitted histone information is present at the subset of PEGs in sperm, the extent to which histone transmission may be associated with genomic imprinting after fertilization is still not defined. We hypothesized that paternally active chromatin with H3K4me3 remains in active sperm throughout the histone-to-protamine transition and even after fertilization provides active epigenetic status of PEGs during paternal genome activation in mouse preimplantation embryos. To address this hypothesis, publicly available mouse sequencing datasets were analyzed (see https://www.ncbi.nlm.nih.gov/sra) or DDBJ SRA . All ChIP-seq, RNA-seq, and WGBS data used in this study is summarized in Publicly available raw sequencing datasets were downloaded from either NCBI SRA  with default settings to remove adaptor sequences, low-quality reads, and less than 20-bp reads. The trimmed reads were aligned to the mouse reference genome (mm10) using Bowtie2  . Peaks of H3K4me3 and H3K27me3 at the known PEGs were visualized with MACS2 output using Spark (v2.6.2) (https://genome.ucsc.edu).To confirm active histone retention at the known PEGs throughout the histone-to-protamine transition in mice, ChIP-seq data using mouse sperm and round spermatids were analyzed . All ChI_016368) . PCR dup_016368)  with the_016368) . After c_016368) . The cov(v2.6.2) . Informahttps://github.com/FelixKrueger/TrimGalore) with default settings. The trimmed reads of MII oocytes, maternal pronucleus, and paternal pronucleus of a pronuclear stage 3 (PN3) zygote were aligned to the mouse reference genome (mm10) using Bowtie2 (v2.3.4.3) (http://asia.ensembl.org/index.html). As for early embryos from PN5-stage zygotes to inner cell mass (ICM), the trimmed reads were aligned to mm10 with Bowtie2 followed by assigning reads to each parental allele by SnpSplit (v0.3.4) (http://www.sanger.ac.uk/science/data/mouse-genomes-project). Thereafter, the trimmed reads of early embryos were aligned to the N-masked mouse reference genome using Bowtie2 with a parameter --end-to-end to avoid soft clipping. The mapped reads were then split into either the PWK/PhJ allele or the C57BL/6N allele by SnpSplit (v0.3.4). As the two replicates were highly correlated to each other (Pearson’s correlation >0.96), the two replicates were then pooled together for each stage for the downstream analysis. Bedgraph coverage tracks were generated from the allele-splitted reads using Deeptools (v3.3.1) (https://igv.org/app/) were combined to define regions including promoter CpG islands for counting reads by FeatureCount (v2.0.1)   with def2.3.4.3)  with the(v0.3.4) . Before (v3.3.1) . The all(v2.0.1) . Thereafhttps://github.com/FelixKrueger/TrimGalore) with default settings. Before mapping reads to the mouse genome, all SNP sites were N-masked with SnpSplit (v0.3.4) (http://www.sanger.ac.uk/science/data/mouse-genomes-project). Thereafter, the trimmed reads were aligned to the N-masked mouse reference genome using HISAT2   x C57BL/6N (Mother), to confirm its paternal expression. If the PEG candidates also had log2FC >3 and FDR <0.001 in the data from the second study, the genes were considered as PEGs. The PEGs were further examined using RNA-seq data of two-cell embryos derived from androgenotes, parthenogenotes, and amanitin-treated embryos  with default settings. The trimmed reads were mapped against the mouse reference genome (mm10) with default settings. The mapped reads of each biological replicate of each group  were then passed to HTSeq  .To identify H3K4me3 transmission associated-paternally expressed genes in mouse early embryos, RNA-seq data using mouse embryos were analyzed  and comb(v0.3.4)  based on_015530)  with par_015530) , RNA-seq_015530)  with par_015530)  based on_015530) . The ass embryos . All RNA_005514)  to estim_005514)  based onhttps://github.com/FelixKrueger/TrimGalore) with parameters --clip_r1 9 --clip_r2 9 --paired. The trimmed paired-end reads were separately aligned to the mouse genome as single-end reads with Bismark  (http://www.nodai-genome.org/mouse.html?lang=en) (https://genome.ucsc.edu).To characterize the mouse methylome before and after fertilization, whole-genome bisulfite sequencing (WGBS) data using mouse oocytes and embryos were analyzed . Raw WGB_005604)  with thelang=en) . CpG methttp://www.geneimprint.com/site/genes-by-species.Mus+musculus and https://www.mousebook.org/imprinting-gene-list)] were confirmed to have not only the active H3K4me3 but also the suppressive H3K27me3 marks .We asked if changes in histone information at PEGs occurred during the histone-to-protamine transition. We examined the active (H3K4me3) and inactive (H3K27me3) chromatin status using publicly available ChIP-seq datasets of mouse round spermatids  in the paternal pronucleus  had a traceable parental SNP information around the histone-retained regions in sperm. Promoters of 16 out of the 18 genes showed paternal enrichment of the active H3K4me3 modification at all stages up to the blastocyst stage   except at the early two-cell stage . At least 16 known PEGs including the two DNA methylation-independent imprinted genes, Jade1 and Sfmbt2, retained the paternally skewed H3K4me3 status throughout the global wave of epigenetic reprogramming during early embryogenesis.Although histone information is dynamically reprogrammed due to the global wave of epigenetic reprogramming during early embryogenesis , the patd Sfmbtl . Using md Sfmbtl , showingonucleus . We alsore test) . PEGs, PNnat, Kcnq1ot1, Zrsr1, Peg13, and Jade1 showed paternal expression . The other two genes showed biallelic expression (Log2FC <1). One of the two genes, Gpd2, was present before zygotic genome activation based on the amanitin-treated two-cell embryos. It was biallelically expressed (FDR ∼0.1) due to the presence of transcript before zygotic genome activation. Although there was almost no expression in the amanitin-treated two-cell embryos, the other gene, Prkcz, also had biallelic expression (FDR ∼0.5). As the high FDR and this gene showed paternal expression after the late two-cell stage in our analysis, we could not conclude its paternal expression based on the differential expression analysis between androgenotes and parthenogenotes.Eight out of the 16 known PEGs showed paternal expression during the major zygotic genome activation (from the early two- to four-cell stage). It is, therefore, possible that there are many more imprinted genes that also have similar paternal expression during paternal genome activation. To identify PEG candidates during zygotic genome activation, RNA-seq data  derived from a reciprocal cross, PWK/PhJ (Father) x C57BL/6N (Mother) and C57BL/6N (Father) x PWK/PhJ (Mother), were analyzed . After cKcnq1ot1, Impact, Gab1, Jade1, Etv6, and Tle3, were included in the 18 genes. So, the remaining 12 genes out of the 18 genes were newly described PEGs associated with the sperm H3K4me3  were confirmed to have retained H3K4me3 in sperm . The six H3K4me3 .cis-elements. Using UCSC genome browser, we asked whether common repetitive elements are present at promoters of the 13 newly described PEGs. As a result, seven out of the 13 new PEGs had ERV motifs within 2.5 kb from their transcription start sites. Atxn10, Cry2, and Klf3 had ERVL-MaLR family motifs, Prkcz, Sez6l, and Hk1 had ERVK family motifs, and 1700013H16Rik had ERV1 motif.Since early mammalian zygotic transcription includes repetitive elements including endogenous retroviruses (ERVs) , it is pJade1, Gab1, Etv6, and Tle3 are known to be associated with the maternal suppressive histone modification, H3K27me3   .Impact and Kcnq1ot1 had paternal expression up to the blastocyst stage. However, all of the seven newly identified PEGs lost their imprinted gene expression as embryogenesis proceeded. All of the genes also showed biallelic expression in placental tissues derived from a reciprocal cross. Based on their transient allelic expression, we categorized them as transient PEGs. A newly identified transient PEG, Prkcz, lost differential H3K4me3 status at the eight-cell stage as the allelic ratio became about 49%. From the eight-cell stage, Prkcz showed biallelic expression. as Also, Prkcz, four newly identified transient PEGs, Gpd2, Klf3, Sigirr, and Cry2, began to lose paternally skewed expression when the paternal allelic ratio of H3K4me3 became about 60%. All of the genes lost imprinted gene expression coinciding with the developmental stages when the gene loci reprogrammed their differential H3K4me3 status between the parental alleles  x C57BL/6N (Mother), were analyzed. Known PEGs such as  alleles .Before the histone-to-protamine transition during sperm maturation, promoters of the majority of known PEGs had bivalent chromatin status marked by both H3K4me3 and H3K27me3. Even after the histone eviction, the active H3K4me3 and the inactive H3K27me3 were present at more than half of the known PEGs in the sperm. Since some of the PEGs only retained the active H3K4me3, retaining this modification in the sperm is a common feature of these PEGs, regardless of their known maternal imprinting status (either H3K27me3 or gDMR) in mice. Tracking the paternal histone information confirmed that at least 16 known PEGs with informative SNPs had paternal H3K4me3 enrichment during embryogenesis. By analyzing reciprocal crosses, the 13 new PEGs during zygotic genome activation were identified in this study. Twelve out of the 13 genes were associated with the paternal H3K4me3 in sperm, and 9 out of the 12 genes were associated with the paternal H3K4me3 in the PN3 zygote. By combining RNA-seq and ChIP-seq using parental SNP information, we confirmed that seven out of the nine genes lost their imprinted expression at different stages of early embryogenesis when differential H3K4me3 status was reprogrammed. Analyses of suppressive modifications including DNA methylation suggested that the majority of the transient PEGs are likely to be controlled by differential histone modification-based imprinting. By correlating the paternal H3K4me3 retention and the imprinting status of PEGs, our results suggest that histone retention with the active H3K4me3 in sperm may occur during the early stages of mouse genomic imprinting.Xist, which acts as a cis-acting factor to inactivate the paternally inherited X chromosome  in the paternal pronucleus. The secondary DMRs are acquired during postimplantation development  and after fertilization. Using parental SNP information, we confirmed that seven out of eight new transient PEGs developed biallelic expression as embryogenesis proceeded, suggesting that if they have a function, it can only be in the early stages of preimplantation development. For example, one of the new transient PEGs identified in this study, utophagy . Autophautophagy . Paternang cells , and Prkng cells . Prkcz mamically . It is pLa cells  and Atxnn stages . Normal n stages ; a phenon stages ]. Furthe


Arc/Arg3.1, were different. Here, we review the roles of MKL/MRTF in the nervous system, with a special focus on the MKL/MRTF-mediated fine-tuning of neuronal morphology and gene transcription. In the concluding remarks, we briefly discuss the future perspectives and the possible involvement of MKL/MRTF in neurological disorders such as schizophrenia and autism spectrum disorder.Accumulating evidence suggests that the serum response factor (SRF) cofactor megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF) has critical roles in many physiological and pathological processes in various cell types. MKL/MRTF molecules comprise MKL1/MRTFA and MKL2/MRTFB, which possess actin-binding motifs at the N-terminus, and SRF-binding domains and a transcriptional activation domain (TAD) at the C-terminus. Several studies have reported that, in association with actin rearrangement, MKL/MRTF translocates from the cytoplasm to the nucleus, where it regulates SRF-mediated gene expression and controls cell motility. Therefore, it is important to elucidate the roles of MKL/MRTF in the nervous system with regard to its structural and functional regulation by extracellular stimuli. We demonstrated that MKL/MRTF is highly expressed in the brain, especially the synapses, and is involved in dendritic complexity and dendritic spine maturation. In addition to the positive regulation of dendritic complexity, we identified several MKL/MRTF isoforms that negatively regulate dendritic complexity in cortical neurons. We found that the MKL/MRTF isoforms were expressed differentially during brain development and the impacts of these isoforms on the immediate early genes including  Of note, the authors performed microarray analysis using MRTF brain-specific double KO mice and found MRTF might be involved in Arc/Arg3.1 gene induction in vivo  analyses demonstrated the involvement of MKL2/MRTFB in the BDNF-induced activation of SARE in the Arc/Arg3.1 gene in cortical neurons although the involvement of MKL1/MRTFA might be very small, if any  (Holt et al., de novo mutation of the MKL2/MRTFB gene, which caused an amino acid substitution in a patient with ASD (Neale et al., A series of brain-specific SRF KO mice revealed that SRF dysfunction might be involved in neuropathology. Interestingly, neuropathological phenotypes were observed in adult SRF deletion: hyperactivity syndrome in the dopamine system (Parkitna et al., AT wrote the manuscript. DI assisted in making the figures. All authors edited and revised the manuscript and approved the final version of the manuscript for publication. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N‐cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E‐box element within N‐cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist‐related tumour metastasis.Ajuba is a multiple LIM domain‐containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene  During the EMT, Twist induces the transcription of genes encoding mesenchymal markers, such as fibronectin and N‐cadherin.22.1Cat#11005500T8) supplemented with 10% fetal bovine serum  and incubated at 37°C with 5% CO2.HEK 293T, SW1116 and SW480 cells were obtained from ATCC and authenticated by DNA typing, and no signs of mycoplasma contamination were found. The cells were seeded in Dulbecco's modified Eagle's medium  were used for the point mutations of the Twist box of Twist and E‐box of N‐cadherin‐Luc reporter. For shRNA in SW1116 and SW480 cells, the following oligonucleotides were inserted into pLKO.1‐puro vector: 5'‐GCTCCTTATCTGTCTGA ‐GAAT‐3' for human Ajuba and 5'‐GCATTCTGATAGAAGTCTGAA‐3' for human Twist. All inserted fragments and cDNA of truncations and mutants were verified by DNA sequencing.Plasmids of pMEX‐Myc‐Ajuba and their truncations have been described previously.2.3Cat# 06365787001). Viral supernatant was collected at 24 ~ 48 hours post‐transfection, passed through a 0.45 µm filter. When growing to 40 ~ 60% confluence, SW1116 and SW480 cells were infected with viral supernatants. Forty‐eight hours later, 2 μg/ml puromycin was added to the medium and the stable cells were selected.Lentiviral supernatants for shRNA were produced by cotransfecting 293T cells with the construct pLKO.1‐shAjuba or pLKO.1‐shTwist and packing plasmids pMD2.G and psPAX2 by using X‐tremeGENE 9 DNA Transfection Reagent  as described.Cat#A0919) and Co‐IP assays were performed with either α‐Myc or α‐Flag antibodies. In all Co‐IP experiments, 3% inputs were loaded as controls for Western blot. Western blot assay was performed according to the previous description.Cat#MA1‐980, 1:2000), anti‐Flag , anti‐HA , anti‐CBP , anti‐acetyl‐H3, anti‐Ajuba  and anti‐Twist .The transient transfection was performed using Lipofectamine 2000 . qRT‐PCR assays were carried out using the SYBR Green PCR Master Mix . Semi‐quantitative PCR assays were used to detect the level of N‐cadherin and β‐actin. The level of β‐actin was used as the endogenous control. The specific primers used were as follows: 5'‐TACCAGGACGAGCTAACAGC‐3' (forward) and 5'‐GCACTTGATA‐CAGGTGCCGAA‐3' (reverse) for Ajuba; 5'‐GCCGGAGACCTAGATGT‐CATT −3' (forward) and 5'‐TTTTAAAAGTGCGCCCCACG‐3' (reverse) for Twist; 5'‐ACAGTGGCCACCTACAAAGG‐3' (forward) and 5'‐CCGAG‐ATGGGGTTGAT‐AATG‐3' (reverse) for N‐cadherin; and 5'‐TCACCAACTG‐GGACGACAT‐3' (forward) and 5'‐CACAGCCTGGATAGCAACG‐3' (reverse) for ꞵ‐actin. The fold changes were shown as means ± SD in three independent experiments.Total RNA from cells was prepared using TRIzol reagent (Life Sciences) following the manufacturer's protocol. Complementary DNA was synthesized with 2 µg of total RNA using SuperScript Ⅳ RT Reagent Kit  was used as a negative control. The precipitated DNA fragments were detected by qRT‐PCR with primer set 1: 5'‐GGAAGCAGAGCAGTTTACGC‐3' and 5'‐ATCGGCTGCTTAGTCTGG‐AA‐3'. The 220‐bp amplicon flanks the E‐box located in intron 1 of N‐cadherin gene. Primer set 2: 5'‐TGCGTCCTTAGTTTGCTGTG‐3' and 5'‐GGGCAGGAACTTGATTGGTA‐3', was used to amplify a 187‐bp fragment flanking 5000‐bp region upstream of the transcription start site.SW480 cells were stably transfected with the indicated plasmids and subjected to ChIP assays. The method has been described previously.2.7Cat#11668019). Twenty‐four hours post‐transfection, cells were harvested and the luciferase activity assays were carried out following the kit's instructions . Three independent experiments were carried out in triplicate.293T cells were seeded with a 12‐well plate. The pRL‐SV40 vector (20 ng) and pGL3‐N‐cadherin‐Luc reporter (200 ng), along with Ajuba‐ and/or Twist‐ and CBP‐encoding plasmids, were transiently cotransfected into 293T cells with Lipofectamine 2000 reagent  a highly conservative Twist domain; (2) a glycine‐abundant domain; (3) a basic helix‐loop‐helix motif; and (4) the Twist box region (or WR region).3.3N‐cadherin.N‐cadherin was selected to assess the transcription activity of Twist. In the absence of Ajuba, the expression of N‐cadherin in SW1116 and SW480 was decreased in different degrees  was linked to pGL3‐basic luciferase plasmid to produce an N‐cadherin‐Luc reporterNext, to verify whether Twist and Ajuba directly bind the endogenous E‐box region of N‐cadherin, the ChIP assay was performed with Twist and Ajuba antibodies in SW480 cells. The precipitated DNA was amplified by qRT‐PCR using primers flanking the E‐box region of N‐cadherin, and the region ~5000bp upstream of N‐cadherin transcription start site was used as a negative control. Our results showed that both Ajuba and Twist bound to the E‐box region of N‐cadherin, suggesting that Ajuba formed a complex with Twist to bind to the E‐box region of N‐cadherin. In addition, knockdown of Ajuba greatly reduced the enrichment of N‐cadherin promoter DNA by Twist antibody Figure . Collect3.4It was reported that Ajuba can enhance the interaction between p300/CBP and PPARγ to activate the latter target gene transcription.To further verify the assembling of Ajuba, Twist and CBP ternary complex at endogenous chromatins, the ChIP assay was carried out in SW480‐shNC and SW480‐shAjuba cells using Twist, Ajuba, CBP and H3‐Ac antibodies respectively. We used the primer sets flanking the E‐box site to measure precipitated DNA fragments by qPCR. In SW480‐shNC cells, Twist, Ajuba and CBP were identified to bind to Twist target promoter in different degrees, and acetylated histone maintained at the middle level represented by H3‐Ac antibody Figure . Surpris4Herein, we found that Ajuba can interact with the Twist box of Twist via its LIM domain. Furthermore, we demonstrated that Ajuba recruits CBP/p300 and Twist to the target chromatins to augment histone acetylation to induce the transcription of N‐cadherin. Together, our results showed that Ajuba is a key coactivator for Twist.Structurally, Ajuba contains the N‐terminal preLIM domain and the C‐terminal three tandem LIM domains. The LIM domains supply a scaffold for various protein‐protein interplays.Twist, a major marker of EMT, can facilitate cell motility and invasive activity, and enhances some features of cancer cells via regulating a series of target gene expression.Mechanistically, the previous investigation showed that Ajuba can function as a coactivator for PPARγ and ERα by recruiting CBP/P300.In summary, our results not only enriched the research on the tumour metastasis mechanism of Twist and Ajuba, but suggested that Ajuba/Twist/CBP/p300 pathway may be a new target for therapeutics to cancer metastasis.The authors declare no conflict of interest.Zhaoxia Wu : Conceptualization ; Methodology . Xiuqun Zou: Methodology ; Supervision . Ying Xu: Resources ; Validation . Fengli Zhou: Investigation . Rong Kuai: Investigation . Ji Li: Software . Daming Yang: Data curation ; Formal analysis . Yimin Chu: Supervision ; Writing‐original draft . Haixia Peng: Data curation ; Funding acquisition ; Writing‐review & editing .


BUR6, MED6, SPT10, SUA7, are listed for SAF1 in the genome database of Saccharomyces cerevisiae. Here in this study, we carried out an in-silico analysis of gene expression and transcription factor databases to understand the regulation of SAF1 expression during stress for hypothesis and experimental analysis.Stress response is mediated by the transcription of stress-responsive genes. The F-box motif protein Saf1p is involved in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1p upon nutrient stress. The four transcription regulators, SAF1 expression when cells were treated with stress agents such as Clioquinol, Pterostilbene, Gentamicin, Hypoxia, Genotoxic, desiccation, and heat. The increase in expression of SAF1 during stress conditions correlated positively with the expression of RLM1, encoding the Rlm1p transcription factor. The expression of AAH1 encoding Aah1p, a Saf1p substrate for ubiquitination, appeared to be negatively correlated with the expression of RLM1 as revealed by an analysis of the Yeastract expression database. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes together may contribute to stress tolerance. The experimental analysis of cellular growth response of cells lacking both SAF1 and RLM1 to selected stress agents such as cell wall and osmo-stressors, by spot assay indicated stress tolerance phenotype similar to parental strain however sensitivity to genotoxic and microtubule depolymerizing stress agents.An analysis of the GEO profile database indicated an increase in silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.Based on in-The online version contains supplementary material available at 10.1186/s41021-021-00218-x. Saccharomyces cerevisiae cells from in and out of the quiescence phase due to nutrients availability or stress condition remains an active research area for both basic research and biotechnological purpose. Upon nutrient deprivation, yeast cells stop dividing and enter into a stationary phase. This transition leads to glycogen accumulation and reduced cell wall porosity which makes cells resist stressors which increases survival probability. The mutants with cell cycle transition defects  that are not able to enter into the stationary phase showed sensitivity to stress condition and may die due to starvation [Saf1-E3 ligase in S.cerevisiae recruits Aah1p (adenine deaminase) for ubiquitin-mediated degradation upon nutrient deprivation condition [AAH1 expression is down regulated during the transition from proliferative state to quiescence state by Srb10p and Srb11p however regulation at the post-transcriptional level of Aah1p requires SCFSaf1-E3 ligase activity. The SCFSaf1-E3 ligase also requires for degradation of the unprocessed vacuole and lysosomal proteins [PRB1), protease C (PRC1), and Ybr139w reported to be targets of Saf1p for proteasomal degradation [SAF1 is regulated by regulatory transcription factors  reported in a study on chromatin immunoprecipitation-chip analysis upon heat treatment [SPT10 which encodes, a histone H3 acetylase, reported in a microarray RNA expression-based study [SAF1 exhibits decreased death rate at elevated temperature [The natural population of eukaryotic cells, mostly microorganisms remains non-dividing state and proliferate upon nutrients availability . The proarvation . The stuarvation  listed tarvation , 5 implyarvation  and the ondition . The adeproteins  and threradation . The expperature .Saccharomyces cerevisiae RLM1/YPL089C  is a MADS-box  family transcription factor which is phosphorylated and activated by MAP-kinase Slt2p [S.cerevisiae. Deletion of RLM1 in S.cerevisiae results in resistance to cell wall disrupting agents [S.cerevisiae RLM1 homologs have been reported from other filamentous fungi such as Aspergillus niger [Aspergillus fumigatus [and Candida albicans [The se Slt2p . The Rlmse Slt2p  indicatig agents . S.cerevus niger , Aspergiumigatus , and Canalbicans .http://www.yeastract.com). It is a  repository of curated associations between transcription factors and genes in Saccharomyces cerevisiae [http://www.ystrexdb.com/) allows analysis of gene-specific expression patterns during stress. The database has a collection of genome-wide expression data on stress response in S.cerevisiae [The gene expression omnibus (GEO) profile database at the National Centre for Biotechnology Information (NCBI) is a repository of genome-wide gene expression of different model organisms and can be mined for gene expression status using both experiment-centric and gene-centric approach , 20. Therevisiae . The Yearevisiae .SAF1 during stress conditions, and yeast transcription databases were investigated for  the association of transcription factors with the SAF1 expression during stress. The regulatory association analysis was carried out to understand, transcriptional regulation of target genes by transcription factors during stress for hypothesis followed by experimental analysis. We report RLM1 as a transcription factor regulating the expression of SAF1 during stress through in-silico analysis. Further, we hypothesized that ablation of both genes may contribute to stress tolerance. We report that the loss of SAF1 and RLM1 together leads to stress resistance in S. cerevisiae to selected agents.In the present study, we studied the Gene Expression Omnibus profile database (GEO) for the expression status of Saccharomyces cerevisiae strain, BY4741 (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and JC2326 -146) and their deletion derivatives used in the study are mentioned in the (Table https://www.ncbi.nlm.nih.gov/geoprofiles/) was searched gene-specific keyword (SAF1) to study the expression profiles during stress in S. cerevisiae.The GEO profile database at  was searched for global transcription factors during normal and stress conditions for SAF1 and AAH1, further analysis for regulatory association of SAF1 and AAH1 with common transcription factors (TF) was carried out during stress condition.For the search of transcriptional regulators of a gene in http://www.ystrexdb.com/) was analyzed for the expression status of SAF1, AAH1, and RLM1 by using an advanced search tool at default cut-off (log2 Fold Change > 1.5) and  under all experimental classes. The results on the expression status of genes included the statistical values and biological features for specific conditions.The Yeast stress expression database (To compare the growth rate between WT (BY4741) and its deletion derivatives in YPD broth and solid media standard protocols were  adopted. For growth kinetics, a single colony of each strain was inoculated in 25 ml YPD broth and grown overnight at 30 °C. The next day each culture was  diluted to 0.05 optical density (OD) at 600 nm and allowed to grow up to 14 h. The OD after every 2 h for each culture was measured using TOSHVIN UV-800 SHIMADZU spectrophotometer. The experiment was carried out in triplicates and  the average OD was plotted against each time point to compare the growth rate. To compare the growth on solid media each strain was streaked on YPD + agar plates followed by incubation at 30 °C for 2–3 days.To compare the morphological features between WT and deletion derivative yeast strains, phase contrast microscopy was carried out on log-phase grown cultures in YPD medium at 30 °C using Leica DM3000 microscope at × 100 magnification. To study the chitin distribution in the WT and mutant strains Calcofluor white staining method was adopted. Briefly, WT and mutant strain were grown overnight at 30 °C and the next day a small aliquot was inoculated in fresh YPD medium in 1:10 ratio for growth till log phase. The cells were suspended in 100 μl of the solution containing Calcofluor white (50 μg/ml solution) fluorescent dye, after staining cells were observed under × 100 magnification of fluorescence microscope using ultraviolet filters as per instructions mentioned in the manual of Leica DM3000 fluorescence microscope.600 ~ 0.8) at 30 °C. Yeast cells were washed with distilled water and suspended in 1X PBS . Further, fixation was done by the  addition of 70% ethanol before DAPI staining. Cells were washed with 1X PBS buffer then again centrifuged for 1 min at 2500 rpm. DAPI stain (1 mg/ml stock) to  a final concentration of 2.5 μg/ml was added and incubated for 5 min at room temperature and visualized under a fluorescent microscope with 100 X magnifications. A total of 200 cells were counted and grouped according to  0, 1, 2, and multi nuclei per cell, more than two nuclei per cell indicated the nuclear migration defect.To study the nuclear migration defects in WT and its deletion derivatives, assay described , 25 was 2O2 as each separate agents. The plates were incubated at 30 °C for 2–3 days and cellular growth of the WT and mutants were recorded. The plates with spot assays were analyzed as per the method mentioned [− 1, 10− 2 and 10− 3 dilutions were analyzed for quantification.For comparative assessment of WT and its deletion derivatives for cellular growth response to stress agents, standard semi-quantitative spot assays were performed. Briefly, wild type (BY4741) and its deletion derivatives strains were grown in the 25 ml YPD  medium overnight at 30 °C. The next day the cultures were diluted and grown in fresh YPD medium for 3–4 h to reach the  log phase (OD600 0.8–1.0). The cultures concentration was adjusted by dilution  serially diluted, from each dilution, an aliquot of 3 μl volume was spotted onto YPD agar plates, without stress agents and plated containing Hydroxyurea (HU), Methyl methanesulfonate (MMS), Nocodazole, Benomyl, Calcofluor White, SDS, NaCl, Glycerol, DMSO, Hentioned  for quanJC2326 was used for the construction of deletion derivatives. The reporter and its deletion derivatives contain a single Ty1 element marked with indicator gene HIS3AI. The indicator gene HIS3 is interrupted by  an artificial intron (AI) in an orientation opposite to the HIS3 gene transcription. During transcription of marked Ty1 with this arrangement followed by splicing and reverse transcription, generates cDNA with the functional HIS3 ORF which upon integration into the genome, generates HIS3 positive phototrophs. The quantitative measurement of the HIS3 positive colonies was considered as  the frequency of retromobility. To measure the Ty1 retrotransposition frequency in the WT  and the deletion derivatives, a single colony of each strains was inoculated into 10 ml YPD broth and grown overnight at 30 °C. The overnight grown cultures were again inoculated in 5 ml YPD at 1:1000 dilutions. The cultures were grown for 144 h at 20 °C. The culture was serially diluted and plated on minimal media (SD/His− plates) followed by incubation at 30 °C for 3–7 days. The three independent replicates of each strain were  used for serial dilution and plated on SD/His− plates. The numbers of HIS+ colonies were counted from each plate and plotted for comparative assessment of Ty1 retrotransposition.To assess the Ty1 retro-mobility in WT and its deletion derivatives assay was adopted mentioned in , 28. TheP-value is less than 0.05 indicated significance. To compare the statistical significance between groups and within groups one-way ANOVA and posthoc Bonferroni analysis was performed.Statistical significance of observations was determined using the paired student t-test. SAF1 when cells of S.cerevisiae were treated with Clioquinol, Pterostilbene, Gentamicin, hypoxia, genotoxic, desiccation, and heat stress agents. The summary of the results is  mentioned and Table The analysis of the GEO profile database showed elevated expression of in-silico analysis using the YEASTRACT database (http://www.yeastract.com) for the association of global transcription factors with SAF1 during normal and stress conditions revealed a total of three transcription factors i.e. Rlm1p, Spt23p, and Cin5p  showed a 2-fold elevated expression of SAF1 and RLM1 upon stress condition  is a fluorochrome stain, which binds to chitin, and perturbs the cell wall whereas SDS is a membrane disrupting agent. The cells lacking both f1∆ Fig. A when stLM1 Fig. B. The spm1∆ Fig. C, D. Fur2O2) and Dimethyl sulfoxide (DMSO) both are oxidative stress agents [SAF1 and RLM1 showed tolerance to 2 mM H2O2, 8% DMSO  and Nocodazole (50 μg/ml) in the spot assay  elicits DNA damage in cells, which results in activation of DNA damage repair pathways . The repSAF1 and RLM1 prompted us to study the nuclear status in the cells using DAPI staining followed by fluorescence microscopy The DAPI stained cells showed compact nuclei as single dot and multi-nuclei feature , oxidative stressors (DMSO and hydrogen peroxide), osmo-stressors (Glycerol and NaCl) compared to parental strain however, cells showed sensitivity to genotoxic stressors (HU) and microtubule inhibitors (Nocodazole and Benomyl) suggesting, the role of other alternate pathways constituted SAF1 and RLM1 as part in response to genotoxic stress. This is supported by the fact that cells lacking both SAF1 and RLM1 together exhibit an elevated level of Ty1 retrotransposition and multi-nuclei phenotype. It must be noted that the lack of RLM1 and SAF1 may contribute to the chronic presence of adenine deaminase, Aah1p encoded by AAH1.The adenine deaminase enzyme (Aah1p) is crucial for purine metabolism and is involved in the conversion of adenine to hypoxanthine [Further, based on xanthine  and imbaxanthine .AAH1 during heat stress when the expression of RLM1 and SAF1 was minimal [SAF1 and RLM1 could be due to sustained expression of AAH1. Understand the mechanism of general stress tolerance by cells lacking SAF1 and RLM1 requires future studies, however, the  present study suggests a model  database  showed t minimal  Fig. 8)AAH1 duridel Fig.  where thsilico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.Based on in-Additional file 1. Supplementary Figures from S1 to S7.


Tools for sequence-specific DNA binding have opened the door to new approaches in investigating fundamental questions in biology and crop development. While there are several platforms to choose from, many of the recent advances in sequence-specific targeting tools are focused on developing Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated (CRISPR-Cas)-based systems. Using a catalytically inactive Cas protein (dCas), this system can act as a vector for different modular catalytic domains (effector domains) to control a gene's expression or alter epigenetic marks such as DNA methylation. Recent trends in developing CRISPR-dCas systems include creating versions that can target multiple copies of effector domains to a single site, targeting epigenetic changes that, in some cases, can be inherited to the next generation in the absence of the targeting construct, and combining effector domains and targeting strategies to create synergies that increase the functionality or efficiency of the system. This review summarizes and compares DNA targeting technologies, the effector domains used to target transcriptional control and epi-mutagenesis, and the different CRISPR-dCas systems used in plants. Over the past three decades, the development of tools that can bind to DNA in a sequence-specific manner has led to technologies that can specifically target and regulate gene transcription and epi-mutagenesis. Due to its ease of use, in the most recent wave of developments, tools based on the Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR–Cas) using a catalytically inactive Cas protein have come to the forefront with an array of attachments allowing for the targeted transcriptional control or epi-mutagenesis of a specific locus; pushing the functionality of CRISPR–Cas beyond gene editing.These constructs rely on either the direct recruitment of basal transcription machinery or the targeting of epigenetic factors to manipulate transcription of nearby genes. While the recruitment of basal transcription machinery requires the presence of the targeting construct, changes in DNA methylation can in some cases be mitotically and meiotically inherited allowing for this targeted epi-mutagenesis to be maintained in the following generations in the absence of the targeting construct . The herIn plants, targeted manipulation of transcriptional control and epimutagenesis have already been used to increase resistance to drought, manipulate plant developmental phenotypes, increase our understanding of interactions between essential proteins, and further our understanding on how plants add, maintain and use DNA methylation . As we cIn this review, we will cover the recent discoveries and advancements in targeted transcriptional control and epi-mutagenesis using CRISPR–dCas-based targeting technologies in plants.Eukaryotic DNA is packaged into ∼146 base pair (bp) DNA segments that wrap around a histone octamer known as a nucleosome . NucleosArabidopsis thaliana), CRCs, such as the SWITCHING DEFECTIVE 2/SUCROSE NON-FERMENTING 2 (SWI2/SNF2) proteins use ATP hydrolysis to alter the structure or positioning of nucleosomes, which in turn mediate the accessibility of the chromatin to transcription factors and other regulatory proteins , has also been associated with gene regulation. Often, the presence or absence of DNA methylation is associated with repression and activation, respectively, of nearby genes or transposable elements. In plants, DNA methylation is maintained by four different DNA methylation maintenance pathways and can be established de novo by the RNA-directed DNA methylation (RdDM) pathway . The canDNA methylation is a mitotically and meiotically heritable mark . MaintenTargeted manipulation of epigenetic marks or gene expression requires a way to specifically and ectopically recruit molecular components capable of transcriptional control or epi-mutagenesis to the site of interest on the genome. Several targeting technologies are reviewed in the following section.Both siRNAs and miRNAs have been broadly applied for transcriptional and post-transcriptional gene silencing, not only because small RNA-mediated gene silencing is sequence specific and efficient, but also because they can cause partial loss-of-function alleles that can overcome the lethality of certain null mutants . EndogenWhile synthetic siRNAs and miRNAs can target gene silencing, they cannot recruit other factors or be used for targeted activation, and are prone to off-target effects  Table 1Table 1. Zinc Fingers (ZFs) are one of the earliest and best characterized tools for targeting effector domains to specific regions of a genome. These proteins typically contain a classical C2H2 ZF structure with two β-sheets and one α-helix maintained by hydrophobic interactions and a zinc ion . Each fiLike ZFs, TRANSCRIPTION ACTIVATOR-LIKE EFFECTORS (TALEs) contain specific amino acid sequences that allow for the programmable recognition of specific DNA sequences . The TAL–Cas systems can be used to target specific regions of a genome. These systems are naturally occurring in bacteria and archaea, and evolved as a type of adaptive immune system  gene, this was not observed when the DNMT3A-SunTag system targeted the Krüppel-like factor 4 gene, indicating a locus-specific effect  to a single locus allowing for the visualization of the target . This wac effect . The infc effect . For exaLike the SunTag system, the MS2 system is a CRISPR–dCas9-based system that gives the user the ability to target multiple effector proteins to a specific locus . HoweverThere are an array of different effector domains that can be used to activate transcription in targeted systems. This can be achieved directly by attracting basal transcription factors, or indirectly by adding active histone marks or removing repressive marks. Targeting of the modular activating domain of the herpes simplex virus VP16 to a specific locus has been extensively described as an efficient way to activate transcription .The VP16Directly targeting transcriptional activation can also be achieved through targeting the highly conserved plant-specific acidic-type activator domains found in the APETALA2 family of proteins, known as the EDLL domains. The EDLL domain of AtEFR98 is frequently used as a modular component to activate gene expression . This EDIn addition to the direct activation of transcription by recruiting activator domains, another option is recruiting domains capable of adding active or removing repressive epigenetic marks, thereby activating gene expression indirectly. In plants, targeting transcriptional activation through H3K27 acetylation using the p300 domain from humans or the catalytic domain of the plant-specific ARABIDOPSIS HISTONE ACETYLTRANSFERASE OF THE CBP FAMILY 1 (HAC1) can activate transcription of targeted genes; however, at least in the case of p300, a higher level of activation is achieved when VP64 is used . RemovinUnlike recruiting the basal transcriptional machinery, which can cause unwanted overexpression, using epigenetic marks to control transcription only facilitates the accessibility of the target promoter to the transcriptional machinery. This highlights an advantage to the manipulation of epigenetic marks over the targeting of an activator domain. In addition to this, altering epigenetic marks can also facilitate the accessibility of targeted activator domains and thus, achieve a synergistic effect when they are combined .While many studies have attempted to compare single activator domains to determine the best one to use for targeted activation, such studies will be heavily influenced by a number of unrelated factors, including the choice of targeting system and target site. While an activator domain might appear superior in a specific system or at a specific target site, this cannot be extrapolated to every possible scenario that exists in every plant genome, and thus a variety of tools are needed. However, a consistent trend seen in the development of these tools is that the ones that are capable of targeting multiple activator domains to a single locus have consistently been shown to perform better than those targeting a single domain . While tFWA promoter using the SunTag targeting system also resulted in the loss of DNA methylation, suggesting that simultaneously targeting mechanisms that remove DNA methylation and directly activate of gene expression could work synergistically in plants as it does in animals  domains, such as those found in SUPERMAN or BODENLOS. Outside of the targeting systems discussed here, these domains have been used extensively as a way to study highly redundant genes, as these repressor domains are dominant over activator domains, including VP16, and can be used to turn a constitutive activator into a constitutive repressor , DOMAINS REARRANGED METHYLTRANSFERASE (DRM) can add DNA methylation to a target promoter, resulting in transcriptional repression  . This is achieved by targeting CRISPR–dCas-based systems to the TSS region or downstream of the transcriptional start site of a gene and has been well demonstrated in mammalian cells . Howeverea mays) . In planea mays) .While a number of platforms have been developed for targeted transcriptional control and epi-mutagenesis in plants, including small RNAs, ZFs, and TALEs, development of CRISPR-based systems have recently become the dominant focus due to their ease of use and flexibility. Recent developments in CRISPR–dCas systems have focused on increasing efficiency and functionality, including creating systems capable of recruiting multiple copies and multiple types of effectors to a target site. In addition to this, progress has also been made in the development of additional and optimized delivery systems for these tools (Box 1). The development of CRISPR–dCas systems for targeted transcriptional control and epi-mutagenesis is still in its infancy and there are many ways in which these tools can be improved (see Outstanding Questions). Their recent development means that these tools have only been utilized in a few studies outside of the ones creating or optimizing them; however, examples are available and demonstrate the usefulness of these systems in answering basic questions and bioengineering . The furADVANCESRecent advancements in DNA targeting systems have largely focused on the development of CRISPR–dCas systems to include peptide tails and RNA binding proteins, allowing these systems to recruit multiple copies of any given effector protein.Effector protein domains capable of manipulating DNA methylation have been successfully deployed and, in some cases, changes have been shown to be heritable to the next generation in the absence of the targeting construct.Fusion and co-targeting strategies using common modular activator domains can synergistically activate transcription to levels higher than targeting with a single type of activator domain.CRISPR-dCas systems can be used to repress genes by targeting of modular repression domains, DNA methylation, or CRISPRi.OUTSTANDING QUESTIONSWhat effector domains can be used synergistically through direct fusion or co-targeting to create artificial protein complexes to regulate the chromatin environment through histone marks, DNA methylation, and basal transcription factors to ensure heritability of the transcriptional modification, allowing the targeting construct to be segregated out in later generations without reversion?Can tissue-specific expression of advanced CRISPR–dCas systems improve targeting efficiency, as has been the case with ZF and CRISPR–Cas9 constructs?How can we create tools which allow for the targeting of different effectors to separate sites in the genome to provide the ability to adjust entire transcriptional networks by activating and repressing specific components simultaneously?Can these tools be delivered to plants using a non-transgenic approach?BOX 1 Advances in the delivery of targeting systemsAgrobacterium tumefaciens-mediated gene transfer or particle bombardment (Generally, the preferred mode of delivery for CRISPR–Cas systems in plants has been through generating stable transgenic plants by bardment . Howeverbardment . Transiebardment . So far,bardment . Deliverbardment . Viral dbardment . Recent bardment . The secbardment . However


Trypanosoma brucei, genes are assembled in polycistronic transcription units (PTUs). Boundaries of PTUs are designated transcription start sites and transcription termination sites (TTSs). Messenger RNAs are generated by trans-splicing and polyadenylation of precursor RNAs, and regulatory information in the 3′ un-translated region (UTR), rather than promoter activity/sequence-specific transcription factors, controls mRNA levels. Given this peculiar genome structure, special strategies must be utilized to control transcription in T. brucei. TTSs are deposition sites for three non-essential chromatin factors—two of non-canonical histone variants (H3v and H4v) and a DNA modification . This association generated the hypothesis that these three chromatin marks define a transcription termination site in T. brucei. Using a panel of null mutants lacking H3v, H4v, and base J, here I show that H4v is a major sign for transcription termination at TTSs. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J absence, accompanied by accumulation of sub-G1 population. Thus, I propose that the coordinated actions of H3v, H4v, and J provide compensatory mechanisms for each other in chromatin organization, transcription, replication, and cell-cycle progression.In  Trypanosoma brucei is a parasitic protist that causes African sleeping sickness in humans and related diseases in animals, predominantly in sub-Saharan Africa. Disease transmission to mammals occurs by infected tsetse fly bites. To adapt in two different hosts, T. brucei undergoes stages of life cycle-specific differentiation. In the insect vector, trypanosomes proliferate as a procyclic form (PF) in the midgut and migrate to the fly’s salivary gland and differentiate into a non-proliferative metacyclic form. The infected fly injects the metacyclic form T. brucei into the mammalian host’s bloodstream. After differentiating into a bloodstream form (BF), trypanosomes proliferate in the host’s bloodstream and extracellular spaces. BF trypanosomes are transferred during a blood meal and differentiate into a PF and repeat the life cycle  regulation of transcription initiation and termination at boundaries of PTUs, (2) processing of precursor RNAs (trans-splicing and polyadenylation) that generates mature mRNAs, and (3) regulating individual mRNA using regulatory information present in the 5′ and 3′ un-translated regions (UTRs) and trans-acting elements like RNA-binding proteins (T. brucei. Transcription start sites (TSSs) feature acetylated H4 (H4K10ac), methylated H3 (H3K4me3), two essential histone variants (H2Az and H2Bv), and a kinetoplastid-specific DNA modification, base J  feature three non-essential factors, two histone variants (H3v and H4v) and base J , several of expression site-associated genes, 70-bp repeats, and a VSG gene located immediately upstream of the telomere repeat. There are six MESs, each containing a VSG immediately upstream of the telomere repeat. MES VSGs are expressed specifically in the metacyclic stage by RNA pol I. T. brucei has about 60 small linear minichromosomes (about 30–150 kb in size). They are organized with an inverted 177-bp repeat in the middle and telomeres at the ends. Some minichromosomes have a VSG located immediately upstream of the telomere repeat, but no promoter is present  blo present . The rem present . Trypano. brucei , but onl. brucei . The lev. brucei , indicatIn contrast to TSS chromatin marks, TTS marks are non-essential . I hypotT. brucei mutants that were deleted for one or two genes encoding TTS mark proteins. T. brucei may require three non-essential chromatin marks  that coincide at TTSs, which may functionally “signal” RNA polymerase II to stop. Thus, the simultaneous absence of all three marks would cause severe transcription termination defects, which lead to cell growth defects. To examine whether these TTS marks have a synthetic lethal genetic interaction, I generated a full set of TTS mark knockout (KO) mutants. All experiments were performed in BF trypanosomes, as J modification is detected only in BF stage. I had previously generated H3vΔ, JΔ, and H3vΔ JΔ strains . Reads per million mapped reads (RPM) values were generated separately from forward and reverse reads, and fold changes compared to WT were plotted. Chromosome 10  is shownNo further increase in antisense transcription was found in H4vΔ JΔ compared to H4vΔ strain . The samP values were generated from Kruskal–Wallis one-way analysis of variance test across all genotypes and from Mann–Whitney U test comparing mutants to WT for CDS levels. Location of CDSs with up-regulated antisense transcription confirmed that the increases in antisense transcripts are global and evenly distributed throughout all PTUs (Reads per kilobase per million mapped reads (RPKM) values of all, sense, or antisense reads mapping to 8,428 coding sequences  are presented in all PTUs . It is pT. brucei, we do not know whether antisense transcripts detected from this seq method are products of polyadenylation and trans-splicing as the sense transcripts.We demonstrated that antisense transcription was significantly increased at and near TTSs in J null cells using polyA-selected stranded RNA-seq experiments . In the Since JΔ increased antisense transcripts near termination sites in the polyA selection method, while JΔ did not exhibit significant changes in antisense transcript levels in rRNA-depletion method , it is pP values were generated from Kruskal–Wallis one-way analysis of variance test across all genotypes and from Mann–Whitney U test comparing mutants to WT for CDS levels.A transcriptome analysis of 8,428 CDSs showed that antisense transcription was 1.69-fold higher in the triple-KO mutant compared to WT . Statistvia RNAi interference by formation of dsRNA. However, levels of sense transcripts were not significantly changed in the Tet-treated TKO in both RNA-seq experiments, suggesting that increased antisense transcription does not substantially interfere with sense transcript stability.Levels of antisense transcripts increased in the Tet-treated TKO cells in both RNA-seq experiments. One would expect that increased levels of antisense RNA could disrupt the transcriptome T. brucei as in other eukaryotes  values of VSG CDSs were compared between WT and KO mutants for BES and MES VSGs  and a few of TTSs. About 40 (∼25%) of ORC1-binding sites are early replicating origins , RPA1, forms nuclear foci during replication stress and DNA damage in eukaryotes, including . brucei . Tet-trePA1 foci . CollectH3vΔ H4vΔ cells generated by conditional KO showed defects in growth, cell cycle, and DNA replication within 2 days . Growth Because H4v is the main factor for transcription termination control, I did not suspect major changes in transcription profiles, as H4v was already deleted in these DKO and TKO strains. Interestingly, transcription termination defects were partially alleviated in the DS5 clone, while no major change was observed in DS2 . The levTo ensure that the phenotypes observed in the Tet-induced TKO strain were due to the H3v removal and not some artifacts, I conducted complementation experiments. I re-introduced an “un-floxed” WT-H3v allele in the TKO strain  is enriched at TTSs and interacts with PAF1 complex in . brucei . PAF1 co. brucei . Interesrentolae , and JBP at TTSs . In plan at TTSs . FurtherH3v and H4v deletion led to problems in DNA synthesis and growth in the WT and J null background. However, about 5.6 and 3.1% of Tet-treated DKO and TKO cells produced viable clones, respectively. Since the high rate suggested another mechanism than genetic suppression, I hypothesized that epigenetic adaptation may underlie this effect. The initial outcome of H3v removal in the DKO and TKO strains could leave TTS regions unprotected by nucleosomes, which can be detrimental to the cells. In mice, cells with less nucleosomes accumulated more DNA damage, and nucleosome-occupied regions had more protection from irradiation-induced DNA damage . TherefoT. brucei mutant is an unusual phenotype for a checkpoint-defective mutant. Sub-G1 cells could arise through an unequal segregation of chromosome and/or uncoupling of mitosis/cytokinesis and replication completion. Alternatively, the sub-G1 cells could arise as a secondary effect of cell sickness and/or death. Whether JBP proteins have direct roles in cell-cycle control in the absence of H3v and H4v remains to be elucidated.Sub-G1 population increase was more pronounced in Tet-treated TKO, than DKO, indicating that J may have roles in cell-cycle control. In other eukaryotes, activation of cell-cycle checkpoint upon DNA damage or replication stress leads to cell-cycle arrest at G1 or G2/M, or delays in S-phase progression . Thus, tTrypanosoma brucei JBP proteins prompted the discovery of mammalian ten eleven translocation (TET) enzymes that convert 5-methylcytosine (5mC) to hydroxy-5mC (5hmC) [T. brucei JBP proteins converting methyl-U (T) to hmU]. TET enzymes are important factors for reprogramming during development and differentiation  is required for cell-cycle control in the H3vΔ H4vΔ condition, its cellular and molecular functions are still unclear. In addition to this discrepancy between BF and PF trypanosomes, the base J’s role is more important in transcription termination in Leishmania in which JBP1 is essential. This could be due to that Leishmania does not have an obvious H4v homolog. The cellular function of site-specific base J in T. brucei remains unclear, and further studies are needed to understand its roles, perhaps using non-targeted screening methods, e.g., mass spectrometry, to identify JBP1 or JBP2 interacting proteins or synthetic lethality screening in JΔ trypanosome cells.ntiation . TET1/2-ntiation . Regionsntiation . DiffereH3v, but not H4v, binds telomere repeats . ConsistT. brucei (the Lister 427 antigenic type MITat1.2 clone 221a) was cultured in HMI-9 at 37°C  conjugated with herpes simplex virus thymidine kinase gene (HSVTK or TK) was used. After deleting both alleles of a gene, tetracycline (Sigma-Aldrich) can be added to express Cre to remove the floxed HYG-TK and PUR-TK markers, so these selection markers can be reused to delete the next gene. To generate a conditional null mutant, a “conditional KO (cKO or floxed)” allele was introduced using a cKO vector, which is the same as the KO vector except that H3v-Ty1, H3v-HA, or Flag-H4v gene is inserted in between of the 5′ loxP and a selection marker gene. Cre can remove a region between two loxP sites , thereby generating cells that no longer express H3v or H4v gene upon tetracycline addition. WT (HSTB-904), JΔ (HSTB-778), H3vΔ (HSTB-881), and H3vΔ JΔ (HSTB-868) were generated previously . In thisH4vΔ strain: One H4v allele in the wild-type strain (HSTB-904) was deleted with pKP26 vector containing an H4v-KO-HYG-TK cassette (“H4v-KO” indicates that the cassette contains homology arms for H4v deletion), creating an H4vΔ/+ heterozygote (HSTB-1039). The second H4v allele in HSTB-1039 was deleted with pKP27 containing an H4v-KO-PUR-TK, creating an H4vΔ/Δ homozygote (HSTB-1051). Selection markers in HSTB-1051 were removed by Cre expression (HSTB-1067).H4vΔ JΔ strain: One H4v allele in the JΔ strain (HSTB-778) was deleted with pKP26 (H4v-KO-HYG-TK), creating an H4vΔ/+ JΔ (HSTB-1041). The second H4v allele in HSTB-1041 was deleted with pKP27 (H4v-KO-PUR-TK), creating an H4vΔ/Δ JΔ (HSTB-1055). Selection markers in HSTB-1055 were removed by Cre expression (HSTB-1069).H4vΔ H3vΔ strain: One H4v allele in the H3vΔ strain (HSTB-881) was deleted with pKP26 (H4v-KO-HYG-TK), creating an H4vΔ/+ H3vΔ (HSTB-1043). The second H4v allele in HSTB-1043 was deleted with pKP27 (H4v-KO-PUR-TK), creating H4vΔ/Δ H3vΔ strains .An attempt to generate H4vΔ H3vΔ JΔ strain: One H4v allele in the H3vΔ JΔ strain (HSTB-868) was deleted with pKP26 (H4v-KO-HYG-TK), creating H4vΔ/+ H3vΔ JΔ (HSTB-1045). pKP27 (H4v-KO-PUR-TK) was transfected in HSTB-1045. Clones were obtained initially but did not expand further.Floxed H3v-Ty1 in H4vΔ strain (DKO with a floxed H3v-Ty1): One H3v allele was deleted in the H4vΔ strain (HSTB-1067) with pDS88 (H3v-KO-HYG-TK), creating an H3vΔ/+ H4vΔ strain (HSTB-1074). Cre was induced to remove the HYG-TK selection marker (HSTB-1078). The remaining H3v allele in the H3vΔ/+ H4vΔ strain (HSTB-1078) was replaced with a floxed H3v-Ty1-PUR (pKP33 vector), creating a floxed H3v-Ty1/Δ H4vΔ strain . The H3v-Ty1 allele can be removed by tetracycline addition, generating H3vΔ H4vΔ double-KO cells (Tet-treated DKO).Floxed H3v-Ty1 in H4vΔ JΔ strain (TKO with a floxed H3v-Ty1): One H3v allele was deleted in the H4vΔ JΔ strain (HSTB-1069) with pDS88 (H3v-KO-HYG-TK), creating an H3vΔ/+ H4vΔ JΔ strain (HSTB-1076). Cre was induced to remove a HYG-TK selection marker (HSTB-1080). The remaining H3v allele in the H3vΔ/+ H4vΔ JΔ strain (HSTB-1080) was replaced with a floxed H3v-Ty1-PUR (pKP33 vector), creating a floxed H3v-Ty1/Δ H4vΔ JΔ strain . The H3v-Ty1 allele can be removed by tetracycline addition, generating H3vΔ H4vΔ JΔ triple-KO cells (Tet-treated TKO).Floxed H3v-HA in H4vΔ JΔ strain (TKO with a floxed H3v-HA): The remaining H3v allele in the H3vΔ/+ H4vΔ JΔ strain (HSTB-1080) was replaced with a floxed H3v-HA-PUR-TK (pDS84 vector), creating a floxed H3v-HA/Δ H4vΔ JΔ strain (HSTB-1142). The H3v-HA allele can be removed by tetracycline addition, generating H3vΔ H4vΔ JΔ triple-KO cells.Floxed Flag-H4v in H3vΔ JΔ strain (TKO with a floxed Flag-H4v): The remaining wild-type H4v allele in the H4vΔ/+ H3vΔ JΔ strain (HSTB-1045) was replaced with a floxed Flag-H4v-PUR-TK (pKP49), creating a floxed Flag-H4v/Δ H3vΔ JΔ strain (HSTB-1098). The Flag-H4v allele can be removed by tetracycline addition, generating H4vΔ H3vΔ JΔ triple-KO cells.TbRPA1 protein: One allele of RPA1 was epitope tagged with 3xHA by one-step PCR integration using a pMOTag-3H vector , 5 μg/ml blasticidin (InvivoGen), 1 μg/ml phleomycin (InvivoGen), 5 μg/ml hygromycin (InvivoGen), 0.1 μg/ml puromycin (InvivoGen), 1 μg/ml tetracycline (Sigma-Aldrich), and 35 μg/ml ganciclovir (Sigma-Aldrich). T. brucei strains, plasmids, and oligonucleotides used in this study are listed in Bloodstream-form Five to 10 million cells were collected and suspended in the Laemmli buffer and separated on an SDS-PAGE gel (Bio-Rad). Following transfer to nitrocellulose membrane , the proteins were analyzed using mouse anti-Ty1, mouse anti-HA (Sigma-Aldrich), mouse-anti-Flag (Sigma-Aldrich), mouse anti-VSG3 , rabbit anti-VSG3, rabbit H3 (Abcam), or mouse anti-tubulin  antibodiAbout 10 million cells of WT, DKO, and TKO strains treated or untreated with tetracycline were collected and fixed with ice-cold 70% ethanol. To stain DNA, fixed cells were incubated with 50 μg/ml PI (Sigma-Aldrich) and 200 μg/ml RNase A (Sigma-Aldrich) in phosphate-buffered saline  at 37°C for 30 min. Cell-cycle progression was analyzed using LSR II or Via (BD Biosciences), FACSdiva software (BD Biosciences), and FlowJo software package (FlowJo).Bromo-26deoxyuridine pulse experiments were performed as described previously , with soVariant Surface Glycoprotein switching from VSG2 to VSG3 was examined by staining cells with antibodies conjugated with different fluorophores. VSG2-expressing cells containing 0, 0.01, 0.1, 1, or 10% of VSG3-expressing cells and Tet-untreated or treated WT, DKO, and TKO cells were prepared. Six million live cells were stained with mouse anti-VSG2 antibody  conjugated with Dylight 488 (Abcam) and mouse anti-VSG3 antibody  conjugated with Dylight 650 (Abcam). One million cells were analyzed with Via (BD Biosciences).WT, KO mutants, Tet-untreated DKO and TKO, and Tet-treated (for 2 days) DKO and TKO cells in triplicate, three DS clones, and three TS clones were prepared. About 50 million cells were collected, and total RNA was extracted using the RNA STAT-60 (Tel-Test) according to the manufacturer’s protocol, quantified on a NanoDrop2000c, and further cleaned using RNeasy Kit (Qiagen). rRNA was removed using the Ribo-Zero kit (Illumina), and stranded RNA-seq libraries were prepared using random hexamer and NEB Directional RNA Library Prep Kit (NEB) and sequenced on the NovaSeq 6000 PE150.1 , aligned with Bowtie 2  as described previously . RPM val4 . RPKM va4  were disWT, JΔ, H4vΔ, H4vΔ JΔ, and Tet-treated or untreated TKO strains were grown in triplicate. About 50 million cells were collected, and total RNA was extracted using the RNA STAT-60 (Tel-Test) according to the manufacturer’s protocol, quantified on a NanoDrop2000c, and further cleaned using RNeasy kit (Qiagen). RNA-seq libraries were prepared from 500 ng of RNA samples using an oligo dT-based method with 15 cycles of PCR amplification with the Illumina TruSeq mRNA stranded kit (Illumina) and sequenced on the Illumina HiSeq 2000 v4 (50-bp single-end read). Reads were analyzed for transcription profile  and gene6/ml density were fixed with ice-cold 70% ethanol and DNA stained with 50 μg/ml PI in PBS. Cells in G1, early S, late S, and G2 phases were FACS-sorted (BD Aria3: BD Biosciences), and genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen). About 10 ng of genomic DNA was fragmented using the NEBNext dsDNA Fragmentase (NEB), and sequencing libraries were generated using the NEBNext Ultra DNA Library Prep Kit (NEB) and NEB Multiplex Oligos (NEB) for Illumina according to the manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 2000 sequencer (50-bp single-end read). Read quality was analyzed as above, quality checked using FastQC, trimmed using Trim galore, and aligned with Bowtie 2 to the Lister 427 genome. About 3–15 million reads were analyzed using the SeqMonk algorithm. To examine replication initiation profiles, chromosomes were binned at 10-kb resolution with 2.5-kb step and RPM values were generated with SeqMonk  was treated with tetracycline for 0, 1, and 2 days. Cells were fixed with 0.5% paraformaldehyde  for 10 min, permeabilized with 0.2% NP-40 (Sigma-Aldrich) in PBS, and incubated with mouse anti-HA antibody and then with the secondary antibody conjugated with Donkey-anti-mouse Alexa 567 (Invitrogen Molecular Probes). DNA was stained with 0.5 mg/ml DAPI (Sigma-Aldrich). Images were captured using a Zeiss Axioplan 2 fluorescence microscope and edited with Adobe Photoshop.Floxed H3v-Ty1 TKO strain expressing https://www.ncbi.nlm.nih.gov/sra .The datasets generated in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in H-SK designed the study, performed the experiments, analyzed data, wrote and revised the manuscript, and submitted the manuscript.The author declares 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.