Candida albicans to gain further insight into the interplay of chromatin accessibility and gene expression mounted during fungal adaptation to oxidative stress. The ATAC-seq workflow not only facilitates the robust detection of genomic regions with accessible chromatin but also allows for the precise modeling of nucleosome positions in C. albicans. Importantly, the data reveal genes with altered chromatin accessibility in upstream regulatory regions, which correlate with transcriptional regulation during oxidative stress. Interestingly, many genes show increased chromatin accessibility without change in gene expression upon stress exposure. Such chromatin signatures could predict yet unknown regulatory factors under highly dynamic transcriptional control. Additionally, de novo motif analysis in genomic regions with increased chromatin accessibility upon H2O2 treatment shows significant enrichment for Cap1 binding sites, a major factor of oxidative stress responses in C. albicans. Taken together, the ATAC-seq workflow enables the identification of chromatin signatures and highlights the dynamics of regulatory mechanisms mediating environmental adaptation of C. albicans.Human fungal pathogens often encounter fungicidal stress upon host invasion, but they can swiftly adapt by transcriptional reprogramming that enables pathogen survival. Fungal immune evasion is tightly connected to chromatin regulation. Hence, fungal chromatin modifiers pose alternative treatment options to combat fungal infections. Here, we present an assay for transposase-accessible chromatin using sequencing (ATAC-seq) protocol adapted for the opportunistic pathogen  Candida albicans is an extraordinary example of how a pathogen can occupy multiple host niches to persist and survive. C. albicans lives as a harmless commensal in the majority of humans, colonizing mucosal surfaces and epithelial barriers, especially the intestinal tract. However, severe immunodeficiency or a dysregulation of the host microbiota turns C. albicans into an invasive pathogen that can infect virtually any tissue or organ in the human body [C. albicans chromatin-modifying factors play important roles in fungal virulence. For instance, the functions of the histone acetyl transferases (HATs) Gcn5, Hat1, and Rtt109 are crucial for morphogenesis and virulence [SET3 abrogates fungal virulence [Candida spp. such as C. glabrata or C. auris [Human fungal pathogens respond to host immune defense through numerous mechanisms, including chromatin-mediated adaptive gene expression ,2,3. Forman body ,11. In tman body ,13,14. Girulence ,17,18,19irulence . Hence, C. auris ,23,24,25Several methods including chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digestion of chromatin coupled with next-generation sequencing  or DNase-seq have been employed to analyze chromatin accessibility and nucleosome positioning to reveal regulatory mechanisms ,28,29,30C. albicans. We developed a modified protocol and bioinformatics workflow that enables the sensitive detection of changes in the global chromatin landscapes in response to environmental stress. We have chosen oxidative stress as environmental cue, because it triggers genome-wide changes in gene expression [C. albicans challenged with hydrogen peroxide (H2O2) and genome-wide binding data of the oxidative stress transcriptional regulator Cap1 are available [C. albicans. With this approach, we demonstrate that H2O2-treatment of fungal cells increases chromatin accessibility in upstream regions of genes associated with the oxidative response, and we show that those genes tend to be transcriptionally upregulated. Moreover, signatures of accessible chromatin regions enable the prediction of putative novel regulators of stress signaling that are not yet linked to transcriptional control. In addition, genomic regions with an elevated ATAC-seq signal are enriched in binding sites for the key regulator Cap1, demonstrating the potential for de novo motif discovery of regulatory factors. In summary, we show the versatility of ATAC-seq chromatin profiling in C. albicans, especially when combined with complementary next-generation sequencing data such as RNA-seq. This approach uncovers dynamic and complex regulatory mechanisms during environmental adaptation of pathogens.Here, we aimed to adapt the original ATAC-seq protocol  for the pression ,16 and bpression ,44. Morevailable , which wC. albicans mating type-like loci (MTL) a/α clinical isolate SC5314 [The e SC5314  was usedC. albicans was first grown overnight to an optical density at 600 nm (OD600) of approximately 1 in YPD at 30 °C and was then treated with 1.6 mM H2O2  for 15 min while shaking at 30 °C. As control, cells without H2O2 treatment were cultured in parallel. Cells were harvested and directly subjected to spheroplasting and tagmentation (see below).To induce oxidative stress, C. albicans grown in YPD +/− H2O2 was based on a previously published protocol used for Saccharomyces cerevisiae [7C. albicans cells were washed 1× in sorbitol buffer . After cell harvesting, the pellet was then resuspended in 330 µL sorbitol buffer and 192 µL of this suspension was mixed with 8 µL of 50 mg/mL 100T Zymolase  and incubated for 5 min at 30 °C. Spheroplasting efficiency was monitored by diluting a 10 µL aliquot of the spheroplasting reaction into 1 mL distilled water and OD600 measurement. After 5 min of spheroplasting, the OD600 dropped by >90% with respect to the initial OD600. To sustain oxidative stress for H2O2-treated cells, 1.6 mM H2O2 was added to the spheroplasting reaction. Hence, the total time of H2O2 exposure was 20 min. Spheroplasts were then harvested for 5 min at 2000× g at 4 °C and washed 1× in ice-cold sorbitol buffer without DTT. The pellet was resuspended in 500 µL ice-cold sorbitol buffer without DTT and spheroplasts were counted on a CASY® cell counter. Five million spheroplasts were transferred into a tube on ice, harvested for 2 min at 2000× g at 4 °C and the resulting cell pellet was used for subsequent tagmentation.Nuclei preparation for tagmentation from revisiae . BrieflyC. albicans as described earlier [To control for the sequence bias of the Tn5 transposase (TDE1) , gDNA wa earlier  with sli earlier  using a 2O  and incubated at 37 °C for 30 min. The tagmentation reaction was immediately purified using a Qiagen MiniElute PCR purification kit and elution was done in 12 µL elution buffer provided by the kit.Five million fungal spheroplasts or 0.5 ng naked gDNA were resuspended in the tagmentation reaction mix , 0.50 µL 50× protease inhibitor cocktail , 0.25 µL 1% Digitonin  and 10.25 µL nuclease-free distilled H2O (ThermoFisher Scientific). The qPCR was performed in a Realplex Mastercycler  with the following cycling conditions: 72 °C 5 min, 98 °C 30 s, 25 cycles of 98 °C 10 s, 63 °C 30 s, 72 °C 1 min and a final hold step at 10 °C. The cycle number for optimal ATAC-seq library amplification was determined as in [2O (ThermoFisher Scientific). The PCR reaction was then incubated in a PCR thermocycler using the following conditions: 72 °C 5 min, 98 °C 30 s, x cycles (depending on the test qPCR results) of 98 °C 10 s, 63 °C 30 s, 72 °C 1 min, and a final hold step at 10 °C. All ATAC-seq libraries from this study were PCR amplified using 11 cycles. For multiplexing, a different Nextera Index primer 2 barcode was used for each sample. See ATAC-seq library preparation was based on the protocol published by the Greenleaf Lab  with mined as in . The enrThe amplified ATAC-seq libraries were immediately purified and size selected with a double-sided solid-phase reversible immobilization (SPRI) approach (0.5×/1.4×) using AMPure XP beads  according to the manufacturer’s instructions. Final DNA elution was done with elution buffer provided in the Qiagen MiniElute PCR purification kit. The final ATAC-seq libraries were quantified using the fluorescent dye QuantiFluor (Promega) according to the manufacturer’s manual. The yield for each ATAC-seq library sample was between 60 and 90 nM for replicate 1, replicate 2 and around 13 nM for replicate 3.The quality of purified libraries was analyzed on a Bioanalyzer High Sensitivity DNA chip  and pooled in equimolar ratios for sequencing. Sequencing was done in 75 bp paired-end read mode on a HiSeq 3000/4000 system at the Biomedical Sequencing Facility  at the Center of Molecular Medicine (CeMM) in Vienna, Austria.ATAC-seq libraries were prepared from three biological replicates for each condition . Trimmed reads were then aligned to the haploid C. albicans SC5314 genome  using NextGenMap v0.5.5 [https://broadinstitute.github.io/picard/, settings: MarkDuplicates REMOVE_DUPLICATES=true VALIDATION_STRINGENCY=LENIENT; Broad Institute, https://broadinstitute.github.io/picard/) and mitochondrial reads were removed using the “intersect” tool from BEDTools with –v settings (https://github.com/arq5x/bedtools2). The average number of mapped reads per conditions for each chromosome and the size of each chromosome in comparison are shown in C. albicans were retrieved from the National Center for Biotechnology Information (NCBI). The ATAC-seq fragment length distribution from properly paired reads is represented in Quality control of raw sequencing read files (.bam) was done using fastQC v0.11.8 . Illuminp v0.5.5  only keeAligned BAM files were split according to the fragment lengths of the sequencing read pairs as done previously . Read frC. albicans promoter regions (transcription start site (TSS) −/+ 1000 bp). Promoter regions were extracted using the “promoters” function from the Bioconductor GenomicRanges package [https://github.com/ENCODE-DCC/kentUtils). The NucleoATAC result was further compared with the nucleosome-free ATAC-seq reads (see above) and published MNase-seq data for C. albicans grown in YPD [C. albicans transcripts, a coverage matrix was computed using the deepTools2 “computeMatrix” function . MNase-seq reads were extended to 100 bp prior to coverage plotting as described in the original publication [Nucleosomal occupancies were predicted using NucleoATAC  with def package . The Nucn in YPD . MNase-sn in YPD , settingn in YPD  over allunction . The full analysis data and the RNA-seq regulation of clustered genes are provided in Differential read coverage (log2-ratio) in nucleosome-free ATAC-seq peaks between Hunction . For the ATAC-seq peak annotation, the genomic annotation from C. albicans assembly 22 (version A22-s07-m01-r88) was downloaded from the Candia Genome Database  and was merged with the 5′UTR (untranslated region) and TSS annotations of C. albicans, which were retrieved from the yeast transcription start site database (YeasTSS) (http://www.yeastss.org/). This file was further used to create a TxDb object using the GenomicFeatures “makeTxDbFromGFF” function  from the edgeR analysis were further subjected to motif search analysis using the MEME suite . First, the previously identified C. albicans Cap1 regulon [2O2-treated samples (settings: –parse-genomic-coord). The enrichment of the identified Cap1 binding sites was re-formatted to the BED file format and further used to compute a coverage matrix with the “computeMatrix” function from deepTools2  accession number GSE73409) were used to compare ATAC-seq peaks with increased signal in H2O2-treated samples with genes upregulated in response to oxidative stress. MNase-seq data . After spheroplasting, cells were then subjected to sample preparation for ATAC-seq analysis. To correct for the known sequence bias of the Tn5 transposase [C. albicans was included in the workflow  and non-treated (YPD) cells, which was not apparent in gDNA libraries , we further observed an enrichment of nucleosome-free ATAC-seq reads adjacent to the TSS. Mononucleosomal ATAC-seq signals were enriched up- and downstream of the nucleosome-free peak, reflecting a well-positioned +1 nucleosome and a typical nucleosomal organization of canonical active gene promoters [C. albicans native chromatin share typical features of mammalian and other fungal (S. cerevisiae and Schizosaccharomyces pombe) ATAC-seq samples [C. albicans and their changes during stress response.Moreover, nucleosomal positions predicted by NucleoATAC correlated very well with published seq data   based on their differential read coverage profile using K-means clustering , we observed that transcription of the majority of cluster 1 genes was also upregulated in response to H2O2—321 genes mapped to cluster 1, of which 309 were also present in the RNA-seq dataset. Out of those 309 genes, 200 were transcriptionally upregulated upon H2O2 treatment . The genomic locations of the majority of called nucleosome-free ATAC-seq peaks were located within gene promoters, exons and 5′ untranslated regions (UTRs) A reflectOf note, substantial peak overlaps between these three genomic regions were observed, indicating peaks stretching from promoter regions into the 5′ UTRs and the first exon. Interestingly, a previous study observed ATAC-seq signals accumulating towards sub-telomeric regions . Thus, w2O2-treated and non-treated cells from all three biological replicates were then subjected to principal component analysis (PCA) based on the presence and absence of called peaks. Samples clustered according to their growth conditions, suggesting that the peak calling of the ATAC-seq workflow was able to identify alterations in chromatin accessibility in response to oxidative stress (2O2-treated cells and located within the 2 kb upstream region of the annotated gene, with 298 peaks detected upstream of genes with increased transcription after oxidative stress (CAT1, ICL1, OYE32, and TSA1, the latter encoding an antioxidative protein [2O2 treatment without alterations in gene expression. For example, gene C2_09880C, encoding for a putative DNA-binding factor, was among the top 20 genes with increased chromatin accessibility upon oxidative stress. However, no significant alteration in transcription was detected in the corresponding RNA-seq dataset (2O2 treatment (2O2 for a total of 20 min. In summary, these data demonstrate that ATAC-seq detects changes in chromatin accessibility linked to gene expression control during fungal stress adaptation. Moreover, it reflects highly dynamic transcriptional responses and could be used to predict novel regulators that are primed for further transcriptional alterations during environmental adaptation.Nucleosome-free ATAC-seq peaks in He stress . Notablye stress , PC1. Hee stress . Peaks we stress . These d protein  (Figure  dataset . Such careatment . These dPlasmodium falciparum during intra-erythrocytic development [C. albicans, such as morphological transitions, are under the control of multi-layer genetic regulatory networks engaging both transcription factors and chromatin modifiers [C. albicans. As mentioned briefly above, the Cap1 transcription factor is the key driver of transcriptional induction during the oxidative stress response [cis-acting sequence motif emerged from an earlier study [Despite assessing the chromatin architecture at different genomic features, ATAC-seq has been used to detect sequence motifs used by transcriptional regulators in ATAC-seq peak regions ,39. Thisodifiers ,84,85. Hresponse . Its ciser study  (Figure 2O2-treated cells, we observed that ATAC-seq read signals were indeed highly increased around the Cap1 motif (S. cerevisiae [When we analyzed the enrichment of published Cap1-binding sites in nucleosome-free ATAC-seq peaks in Hp1 motif B. Importrevisiae ,87. In arevisiae . These drevisiae ,89.C. albicans. ATAC-seq offers a new robust tool to capture temporal changes in chromatin landscapes that are tightly connected to transcript abundance during fungal adaptation to stress. In addition, novel regulators that are not subject to transcriptional control at the time of sample collection might be identified based on chromatin accessibility signatures. This might aid in capturing a broader picture of highly dynamic cellular adaptations where sample drawing is limited. Finally, the combination of ATAC-seq with additional datasets has the potential to predict yet-unknown regulatory sequences and transcription factor binding sites that dictate transcriptional reprogramming and, thus, may be crucial to fungal survival upon stress encounter. Finally, the present workflow might be applicable to in vivo ATAC-seq approaches, since we were already able to obtain meaningful ATAC-seq data from as little as 50,000 C. albicans spheroplasts. The re-isolation of comparable fungal cell numbers from host model systems, such as mice, is feasible and thus, ATAC-seq might facilitate studies that elucidate fungal responses and adaptation to host defense in a tissue-specific manner. Such data will not only help to better understand fungal tissue tropism but also offer to identify new fungal genes amenable to therapeutic intervention.In conclusion, we here present an integrative workflow for ATAC-seq in the human fungal pathogen


JARID2 expression itself is regulated. Recently a number of non-protein-coding RNAs or ncRNAs are shown to regulate transcription. An antisense ncRNA, JARID2-AS1, is expressed from the first intron of JARID2 isoform-1 but its role in regulation of JARID2 expression has not been investigated. The objective of this study was to explore the role of JARID2-AS1 in regulating JARID2 and consequently PRC2.JARID2 is a member of chromatin-modifying Polycomb Repressive Complex-2 or PRC2. It plays a role in recruiting PRC2 to developmental genes and regulating its activity. JARID2 along with PRC2 is indispensable for normal development. However, it remains unclear how We found that JARID2-AS1 is localised in the nucleus and shows anti-correlated expression pattern to that of JARID2 isoform-1 mRNA. More interestingly, data mining approach strongly indicates that JARID2-AS1 binds to PRC2. These are important observations that provide insights into transcriptional regulation of JARID2, especially because they indicate that JARID2-AS1 by interacting and probably recruiting PRC2 participates in an auto-regulatory loop that controls levels of JARID2. This holds importance in regulation of developmental and differentiation processes. However, to support this hypothesis, further in-depth studies are needed which can verify JARID2-AS1-PRC2 interactions. Polycomb group (PcG) proteins are highly conserved transcriptional repressive regulators required for maintenance of cell identity during normal metazoan development , 2. TheyIn recent years, more attention has been given to understanding molecular mechanisms of non-protein-coding RNAs (ncRNAs) as essential factors involved in modulating PRC2 function. Antisense ncRNAs are a subclass of ncRNAs that are produced from the DNA strand opposite to the sense strand encoding mRNA. A number of antisense ncRNAs are shown to regulate transcription of sense strand gene. They can overlap with promoters, exons, introns or transcriptional termination/start sites of sense transcripts and their regulatory effect is dependent on their position vis-à-vis their sense strand partner –26. MoreJARID2 gene located at human chromosome 6. However, its function in regulating JARID2 isoform-1 expression has not been explored. Here, we studied expression of JARID2-AS1 and propose an interesting possibility that JARID2 isoform-1 might auto-regulate its own transcription through the antisense RNA.An antisense ncRNA, JARID2-AS1, is expressed in antisense direction to HaCaT, a spontaneously immortalized human keratinocyte cell line, HEK293T, a human embryonic kidney cell, A549 cells, a human lung carcinoma epithelial cells, Hela cells, a human cervical cancer cells, HepG2, a Hepatocellular carcinoma and metastatic breast cancer MDA-MB231 were grown in Dulbecco’s modified eagle medium. Human chronic myelogenous leukemia, K562 was grown in RPMI containing 10% foetal bovine serum and 1% penicillin–streptomycin (10.000U/mL) in a 5% humidified CO2 incubator at 37 °C. HaCaT cells were grown as per previously published protocol [HaCaT cell line was kindly donated by Dr. Hotchin, University of Birmingham (UoB) . HEK293T7cells were lysed using cold RLN buffer . After incubating on ice for 10 min, the lysate was centrifuged at 3700 rpm for 5 min at 4 °C. The supernatant was transferred to a new tube and kept as the cytosolic fraction. The pellet (nuclear fraction) was washed with cold RLN buffer. RNA was extracted from both fractions using the RNeasy Mini Kit . The quality of fractionation was tested by standard agarose gel.To prepare nuclear and cytosolic fractions, 1x10Total RNA was extracted from cultured cells, treated with Amplification Grade DNase I (Sigma) to remove genomic DNA and quantified by NanoDrop. Complementary DNA (cDNA) was synthesised using Tetro cDNA synthesis kit (Bioline). For Reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA samples were run using forward and reverse primers  is encoded from the first intron between the transcription start sites of two isoforms of JARID2, isoform-1 and isoform-2 , Keratin-10 (KRT10) and Involucrin (IVL)  are localized in the nucleus, in accordance with their roles in regulation of transcription , 37. To Based on the anti-correlation in the expression pattern between JARID2-AS1 and JARID2 isoform-1 mRNA as well as the nuclear localization of JARID2-AS1, we hypothesize that JARID2-AS1 might be involved in regulation of JARID2 transcription. A number of antisense RNAs are shown to regulate the sense transcription by binding to PRC2 protein, EZH2 –42. To eIn this data, JARID2-AS1 is highly enriched in EZH2 pull-down compared to IgG control Fig. d. MultipJARID2 is an important transcriptional regulator that plays an essential role during early development. Without JARID2, embryonic stem cells cannot differentiate. Although it is very clear that JARID2 plays a crucial role in regulating transcription of differentiation genes , 29, 35,JARID2 locus (Fig. JARID2 gene promoter. A similar mechanism is observed in case of transcription regulation of lymphoid enhancer binding factor, LEF1, gene that is expressed during epithelial-to-mesenchymal transition. Like JARID2-AS1, antisense LEF1 ncRNA overlaps with the first intron of LEF1 transcript and is enriched in the nucleus. It has been reported that antisense LEF1 transcript regulates the expression of LEF1 in Cis through binding to the promoter of LEF1 and PRC2. This allows binding of PRC2 to the LEF1 promoter, leading to the blocking of LEF1 transcription [Antisense ncRNAs play a key role in regulating expression levels of coding genes through epigenetic silencing, transcription and mRNA stability via pairing with protein coding mRNAs, thus forming sense-antisense hybrids , 27, 49.cus Fig. a. Howevecus Fig. d. We alscus Fig. , 2. In acus Fig. a–c. Sevecription . HOXD-AScription .Studies have reported that a tripartite interaction between PRC2, RNA and other proteins like JARID2 are needed for proper localization of PRC2 on target genes  as well The strength of current study is that it examines a novel ncRNA overlapping PRC2 component JARID2 and presents an autoregulatory model which might be applicable to other PRC2 genes in addition to JARID2. However, a further careful scrutiny of this hypothesis is needed. It would be necessary to knockout JARID2-AS1 and study its effects on JARID2 expression. The interaction of JARID2-AS1 with EZH2 as well as with JARID2 needs further verification using RNA-pulldown approaches.Additional file 1: Table S1. Primers used for qRT-PCR.Additional file 2: Figure S1. Full gel pictures corresponding to main Fig.


CircRNAs are a class of single-stranded RNA molecules with a covalently closed loop structure and have been characterized by high stability, abundance, conservation, and display tissue/developmental stage-specific expression, furthermore, based on the abundance in distinct body fluids or exosomes, circRNAs present novel biomarkers and targets for the diagnosis and prognosis of cancers. Recently, the regulatory mechanisms of biogenesis and molecular functions, including miRNAs and RBPs sponge, translation as well as transcriptional and splicing regulation, have been gradually uncovered, although various aspects remained to be elucidated in combination with deep-sequence and bioinformatics. Accumulating studies have indicated that circRNAs are more enriched in neuronal tissues partly due to the abundance of specific genes promoting circularization, suggesting dysregulation of circRNAs is closely related to diseases of the nervous system, including glioma. In this review, we elaborate on the biogenesis, functions, databases as well as novel advances especially involved in the molecular pathways, highlight its great value as diagnostic or therapeutic targets in glioma. In the last several decades, scientific research for non-coding RNAs provided new insights for exploring comprehensive molecular mechanisms in glioma. Numerous studies demonstrated that non-coding RNAs play crucial roles in biological processes that regulated glioma initiation and progression . MoreoveCircRNAs are newly discovered to define as a type of non-coding RNA and highly conserved across multiple species, exhibiting tissue-specific, and development stage-dependent patterns –14. HoweCircRNAs are highly stable RNAs that are resistant to exonucleases (RNase R) and are mainly generated by pre-mRNA back-splicing, which connects a downstream splice donor site (5’splice site) to an upstream acceptor splice site (3’splice site) –23. MoreRNA pol II can affect pre-mRNA processing via changing the transcription elongation rate (TER) Fig. a, which RNA-binding protein (RBPs), acting as trans-acting activators or inhibitors, has experimentally proved that they have significant roles in the regulation of circRNA production. Li et al. used genome-wide siRNA screening and an efficient circRNA expression reporter to identify over 100 double-stranded RNA-binding proteins for circRNA formation. The immune factors NF90/NF110 promoted circRNA production by binding to IRAlus and stabilizing intronic RNA pairs in the nucleus . The RNASeveral splicing factors can also participate in circRNA formation regulation via indirectly or directly binding to specific RNA motifs. For example, Quaking (QKI), was proposed to bind to sites flanking introns and brought the circularized exons closer together, leading to circRNA upregulation during EMT. Insertion of QKI binding sites into linear RNA can induce exon circularization, revealing that circRNAs abundance is dependent on intronic QKI binding motifs . YY1/p65Multiple circRNAs have been verified to be expressed more abundantly independent of their linear counterparts and have a cell type and stage-specific manner , 46, 47,MiRNA-mediated dysregulation of mRNA and relevant signaling pathways are closely associated with progression and treatment resistance for cancer. CircRNAs can function as competing endogenous RNA (ceRNAs) or miRNA sponges to inhibit miRNA and therefore up-regulate the expression of target genes by MREs  Fig. 2)2). The vCiRNA and EIciRNAs mainly produced from processed intron lariats (ciRNAs) and back-splicing with retained introns respectively, and they are dominantly located in the nucleus and regulate gene expression at the transcriptional and post-transcriptional level , 29, howCi-ankrd52 and ci-SIRT7 were able to interact with elongating pol II complex and positively modulate the transcriptional activity of their parental genes . EIciRNAThe processing of circRNAs naturally affected alternative splicing of their linear cognates and competed with pre-mRNA splicing, suggesting that the negative relationship between circRNA and their linear isoforms , 56. ForCircRNAs could bind to the different proteins to form specific circRNA-protein complexes (circRNPs) that subsequently regulates the action of associated protein, the subcellular localization of proteins as well as the transcription of parental or related genes Fig. . The stuFurthermore, the most recent report from Barbagallo proposed that circSMARCA5, predicted to be enriched in the splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1) protein-binding sites, acted as sponge for SRSF1 in GBM , raisingThe majority of circRNAs produced from middle exons of pre-mRNA and mainly located in the cytoplasm, revealing a lack of evidence of encoding proteins . But, inCap-independent translation through IRESs was derived and enhanced in cancers, which was subjected to diverse stress such as hypoxia, nutrient deprivation, and genotoxic stress . BesidesBagchi et al. demonstrated that the protein from circ_12152, derived from human chromosome chr9: 87482157e87570432, participated in glioblastoma progression . MoreoveS. Tan et al. discovered that circRNA F-circEA, produced from the EML4-ALK fusion gene, acting as a novel liquid biopsy biomarker for non-small cell lung cancer . Dong etCircRNAs are more enriched in neuronal tissues and exhibit differential expression in different brain areas. This phenomenon might be attributed to the fact the abundance of protein-coding genes producing various circRNAs and splicing factors and RBPs regulating circRNA formation . CircRNAMounting evidence revealed that aberrant expression of circRNAs implicated in the development of glioma. By RNA sequence, Xu et al. screened for circRNAs with differential expression between three glioma tissue samples and three paired normal tissue samples in combination with five methods, CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2, and finally identified 12 common circRNAs . Zhang eoles in glioma development and progression  protein family, could regulate the expression of 19 circRNAs via binding to introns flanking the splicing . SpecifiThe identification and functional researches of circRNAs suggest that circRNAs serve as oncogenes or tumor-suppressors governing downstream target genes and have significant clinical applications. In addition to unique covalently circular structure compared with other ncRNAs, there are several other unique characteristics. Specifically, 1) the structural character without 5’caps and 3’poly(A) tails makes circRNAs resistant to exonuclease, and the half-life is longer than 48 h  2) The sTo date, the association between circRNA expression in tissues or peptides/proteins encoding by circRNA and clinical parameters have been largely reported. Circ_0034642, circ_0074362, cir-ITCH, circHIPK3 and circCPA4 linked to clinical severity and poor prognosis in patients with glioma , 114. WaExtracellular vesicles  play critical roles in intercellular communication by transporting molecules into the surroundings, thereby altering or reprogramming tumor microenvironment. They contain carriers of various types of molecules such as proteins/peptides, mRNAs, non-coding RNAs and DNA, which determine their roles in tumor progression, metabolic regulation, immune modulation, angiogenesis, therapy resistance . What isCurrently, miRNA medicated TMZ sensitivity through regulating specific target genes have been widely verified. For example, miR-181b regulated the chemosensitivity of glioma to temozolomide by targeting Bcl-2 and EGFR , 124. AlPD-1/PD-L1 checkpoint blockade represents a promising target strategy for several types of tumours, including glioma. The report from Chen indicated that PD-L1 modulated immune cell infiltration in glioma microenvironment (TME) and served as signaling protein control multiple pathways including STAT3, PI3K/AKT/mTOR, Ras/ERKsignaling pathways . HoweverHigh-throughput RNA-seq and microarray analysis facilitate circRNAs detection. The bioinformatics community rapidly developed the process circRNA exploration that is from custom scripts working on mapped reads to the production of complete workflows . The curExperimental validation is fundamental to further study circRNAs function following computational prediction. qRT-PCR based on divergent primers, which flanking the BSJ sites, was employed to confirm that circular form was amplified in complementary DNA (cDNA) but not genomic DNA (gDNA) whereas sanger sequencing identified the junction sites. Following that, RNase R is a 3′ to 5′ exoribonuclease that degrades nearly all linear RNA but does not digest circular RNA and is used to further verify circRNAs . FinallyKnockdown and overexpression of genes are similarly applied to research of circRNAs function. SiRNA or shRNA complementary to the cognate linear RNA through back-splicing sites have potential influence on expression of linear RNA, demonstrating that strict control siRNA half-sequence (∼10 nt) replacement should be carried out . TargetiIn order to facilitate the analysis of circRNA research, several databases have been developed to provide tremendous valuable information. Circ2Traits is categorized based on disease-related SNPs, AGO interaction site and relative miRNAs to highlight potential relationships between circRNAs, miRNAs, and diseases . CircBasWith the prompt advance of deep-sequence and operational formulas, the mystery of circRNA is gradually uncovered. It is generally recognized that circRNAs currently play vital roles in various physiological and pathophysiological processes linked to the central nervous system. CircRNAs are more enriched in brain and often derived from genes for neuronal and synaptic function whereas less abundance in gliomas, it seems that identifying upregulated circRNAs may have more important clinical value, which is consistent with present researches. However, the regulatory mechanisms of circRNAs biogenesis are complicated. We suppose that high-confidence detection with low false-positive rate and high specificity through improving algorithms may exhibit profound significance into interpreting specific function and circRNA regulatory network in progression of glioma. Besides, it is stunning that circRNAs with translational potential and clinical relationships are prominently found in GBM, to a large extent, due to widely circRNA formation with IRSE by MBL or other regulatory proteins such as enriched splicing factors that are highly enriched in brain, although the precise and specific mechanism is unclear. More critical, peptide and protein encoded by circRNAs provide a considerable number of opportunities for GBM treatment responses, prognosis evaluation, and small-molecule peptide drug developments based on high specificity and activity, low immunogenicity and less cytotoxicity. Moreover, nanoparticle-based strategies are sufficient to help the delivery of specific compounds to brain tumors across the blood-brain barrier. Therefore, targeted circRNAs and relevant peptides combined with nanoparticle-based cerebral drug-delivery systems will represent a significant new perspective for further treatment of glioma.Currently, specific expression and functional studies have advocated that circRNAs are strictly related to the WHO grade and prognosis of gliomas patients, suggesting its potential diagnostic value. However, these studies dominantly focus on minimal clinical-pathological samples; there are no relevant reports from body fluids, especially blood and CSF. The particular traits containing more stability, long half-life as well as abundance in exosomes tend to deduce that Exo-circRNA from blood and CSF of GBM patients provides excellent insights into non-invasively diagnosis and distinguish different gliomas subtypes, as well as early treatment assessment with high sensitivity combined with magnetic resonance imaging or other traditional biomarkers. Therefore, the expression profile analysis and relevant roles research from body fluids are urgent to further explore molecular pathology of glioma medicated by circRNA.5C), pseudouridine, m7G, N1-methyladenosine, attracted increasing attention. Considerable evidence verified that RNA epigenetic modifications play a vital role in CNS diseases, including glioma. It is demonstrated that m6A modification induced highly cell-specific expression, and m6A circRNAs have long single exons [Epigenetics is involved in numerous critical natural processes and plays significant roles in the occurrence and progression of cancers. A variety of circRNAs have been identified to modulate the epigenetic alterations, such as DNA methylation and histone modifications. For instance, circFECR1 could recruit TET1 DNA demethylase to the FLI1 promoter and subsequently led to CpG DNA demethylation . Howeverle exons . Park etle exons . Also, ale exons . All datle exons , 155. Mele exons . Despitele exons . HoweverThe previous revealing mechanisms implicated in the tumorigenesis of gliomas majorly were paid more attention to the miRNA sponge, protein/peptides encoding associated with signaling pathways, EMT transformation. Undoubtedly, multiple circRNAs are located in the nuclear; accordingly, other potential functions, including regulating gene transcription, protein interactions or sponges, remain to be deeply addressed. Based on high heterogeneity of gliomas, circRNAs with specificity under discrete molecular subtypes are imperative to further discovered. Additionally, more precise methods changing circRNA expression levels but not affecting their residing genes also remain to be clarified.In conclusion, there is accumulating evidence regarding the crucial role of circRNAs in the initiation and progression of glioma, but circRNAs research is still in its infancy. We believe, accompanied by the development of research strategies and sequencing technologies, further elucidation for circRNAs implicated in the tumorigenesis of gliomas will eventually accelerate the clinical application of circRNAs in the diagnosis, treatment, prognosis evaluation.


Streptococcus mutans to persist in a variety of adverse environments and to emerge as a numerically dominant member of stable oral biofilm communities are essential elements for its cariogenicity. The S. mutans Cid/Lrg system has been studied as a key player in the integration of complex environmental signals into regulatory networks that modulate virulence and cell homeostasis. Cid/Lrg has also been shown to be closely associated with metabolic pathways of this organism, due to distinct patterns of cid and lrg expression in response to growth phase and glucose/oxygen levels. In this study, a comparison of cid and lrg promoter regions with conserved CodY (a regulator which responds to starvation stress)‐binding motifs revealed the presence of a potential CodY‐binding site, which is arranged similarly in both cid and lrg promoters. Electrophoretic mobility shift assays (EMSAs) and promoter reporter assays demonstrated that expression of the cid and lrg operons is directly mediated by the global transcriptional regulator CodY. DNase I footprinting analyses confirmed the predicted binding sequences for CodY in both the cid and the lrg promoter regions. Overexpression of CodY had no obvious effect on lrgAB expression, but deficiency of CodY still affected lrgAB expression in a lytST‐overexpressing strain, suggesting that CodY is required for the full regulation of lrgAB by LytST. We also demonstrated that both CodY and CcpA are involved in regulating pyruvate flux and utilization. Collectively, these data show that CodY directly regulates cid and lrg expression, and together with CcpA (previously shown to directly regulate cid and lrg promoters) contributes to coordinating pyruvate uptake and utilization in response to both the external environment and the cellular metabolic status.The ability of  cid and lrg operons, key players in the integration of complex environmental signals into regulatory networks that modulate virulence and cell homeostasis in Streptococcus mutans. It also improves our understanding of the functional links between stress response and cell death/lysis process.This article demonstrates the involvement of CodY as a direct regulator of  The potential binding sequences in each promoter region were also identified by DNase I footprinting analyses. We also show that lack of CodY influences pyruvate flux. The data presented clearly indicate that the balanced regulation between cid and lrg in response to carbohydrate availability and nutritional status of the organism is coordinated by the activities of CodY, together with CcpA, previously shown to directly regulate cid and lrg.CcpA often works together with CodY  to sense changes in nutrient availability and coordinates, directly and indirectly, the expression of hundreds of genes involved in carbon and nitrogen metabolism of gram‐positive bacteria  medium  or chemically defined medium FMC  or β‐fluoropyruvic acid sodium salt monohydrate , as necessary. Antibiotics were used to supplement growth media in the following concentrations: kanamycin (1 mg/ml), spectinomycin (1 mg/ml), and tetracycline (10 μg/ml). For growth measurements, fresh medium was inoculated with 1:100 dilutions of overnight cultures of S. mutans. The optical density at 600 nm (OD600) was measured at 37°C at 30‐min intervals using a Bioscreen C growth curve analysis system. At least three independent experiments, each in quadruplicate, were performed. A representative result is presented in each corresponding figure.2.2codY was constructed as previously described  was replaced by a fragment (ΩKm‐Pldh) containing a polar kanamycin resistance gene (ΩKm) and an ldh promoter region (Pldh). For this, two ~0.5‐kb fragments flanking the −35 and −10 sequences of the codY promoter were PCR‐amplified, ligated into the ΩKm‐Pldh cassette, and transformed into S. mutans. The lytST‐overexpressing strain (SAB163) was recently constructed  and its flanking arms (~0.5‐kb) in the ΔcodY strain by PCR and transformed into SAB163. Transformants were selected on BHI agar containing kanamycin (for a strain constitutively expressing codY) and tetracycline (for the deletion of codY in SAB163), and double‐crossover recombination into each gene was confirmed by PCR and sequencing to ensure that no mutations were introduced into flanking genes. The codY‐overexpressing (SAB399) and codY‐deficient strains in the lytST overexpression background (SAB392) were then each transformed by the PlrgA‐gfp construct for conducting the GFP promoter reporter assays described below.A strain constitutively expressing 2.3S. mutans strains harboring PlrgA‐gfp or PcidA‐gfp promoter fusion constructs  and incubated at 37°C. The optical density at 600 nm (OD600) and green fluorescence  were monitored at 30‐min intervals with a Synergy 2 multimode microplate reader (BioTek) controlled by Gen5 software. Fluorescence intensity was calculated by subtracting the fluorescence of wild‐type harboring plasmid without the reporter gene fusion from fluorescence readings of the S. mutans strains harboring the PlrgA‐gfp or PcidA‐gfp gene fusion.For measurement of GFP fluorescence of 2.4cid or lrg were amplified by primers labeled on their 5′ end with biotinylated nucleotides. To delete each predicted cre site(s) or CodY‐box in cid or lrg promoter region, biotinylated DNA probes were amplified with primers with the desired base deletions in each promoter sequence. Four fmol of biotin‐labeled promoter regions of cid and lrg was incubated in combination with the increasing amount (0 and 25 pmol) of purified, recombinant His6‐tagged CodY protein , 1 mM EDTA, 1 μg poly (dI‐dC), 1 μg bovine serum albumin (BSA), and 10% glycerol. The binding reaction, sample separation in a polyacrylamide gel, and DNA signal detection were performed as described previously  FMC medium. Time‐course measurements of extracellular pyruvate during growth were measured as described previously  CO2 atmosphere. To measure the growth‐dependent expression of cidA and lrgA genes, cells were harvested in early‐exponential (EE) and early‐stationary (ES) growth phases. Extraction of RNA, qPCR, and data analysis was performed as described elsewhere , and data were presented as the relative copy number of mRNA (copies/μl). Statistical analyses were performed on data generated from n = 3 independent experiments using an unpaired t test.To measure the expression of genes using qPCR, 33.1lrgAB expression is induced in stationary phase and its expression is strongly repressed in the presence of excess glucose  activity was dramatically repressed at stationary phase in the low‐glucose culture of the wild‐type strain , and observed a similar pattern in lrgAB and cidAB expression . In the cid promoter region, we discovered a potential CodY‐binding site (AAcTTTCTGAAAAgT) with two mismatches to the consensus sequence, AATTTTCWGAAAATT  was recognized, which contains 5 mismatches to the consensus sequence  were performed with increasing amounts of purified His‐CodY and biotin‐labeled cid or lrg promoter regions. The CodY protein bound to both promoter regions of cid  promoter occur more effectively in the presence of BCAAs and implicate CodY as another major regulator of lrg and cid expression, together with CcpA, recently shown to directly bind to the cid and lrg promoter regions  were conducted using a PCR‐amplified 6‐carboxyfluorescein (6‐FAM)‐labeled DNA probe of the cid promoter region (201‐bp) bound to CodY under the same conditions as used for EMSAs. Sites protected from DNase I digestion by DNA binding by CodY were visualized as regions lacking discernable peaks compared to a control reaction mixture containing BSA . Reaction mixtures containing CodY yielded only a single region of protection spanning from −77 to −54, with respect to the ATG start site of the cidA gene. This protected region corresponded to the predicted cid‐cre2 (nt −73/−59) and CodY‐binding sites (nt −68/−54)  was deleted, the probe was less bound by CodY, resulting to more smears on the gel , mapped to the positions −114 to −101 with respect to the ATG start site of the lrgA gene, but mismatched to the consensus sequence (AATTTTCWGAAAATT) , extended from −70 to −55 with respect to the ATG start site of the gene included a 15‐bp CodY motif  with five mismatches to the consensus. Since two potential CodY‐binding sites (lrg‐CodY1 and lrg‐CodY2) were identified in the lrg promoter region  Figure , showingl Figure , lane 6.l Figure , lane 9,l Figure . The datt Figure . The upsn Figure , we testre2 nt −7/−59 and 3.4lrgAB by lack of CodY affects the capacity of S. mutans to take up pyruvate. For this, we monitored the level of extracellular pyruvate during growth in a low‐glucose FMC medium using a Pyruvate Assay Kit  of 3‐fluoropyruvate (3FP), previously shown to compete with pyruvate and inhibit cell growth by binding to PDH complex in the cell , constitutively expressing codY from ldh promoter in the wild‐type background, as previously described  in a wild‐type background led to a dramatic increase of stationary phase lrgAB expression , followed by transformation with the PlrgA‐gfp reporter construct, and monitored the expression of lrg in low‐glucose cultures. As shown in Figure lrgAB in the lytST overexpression strain, an expression pattern similar to that observed in the ΔcodY strain  in the lrg promoter  partially overlaps a predicted LytT‐binding motif in the lrg promoter. However, the finding that the overexpression of codY had no evident effect on the stationary phase expression of lrgAB suggests that the abundance of CodY alone does not affect the accessibility of LytT to the promoter region. Nevertheless, based on the EMSA and DNases I footprinting data, it is likely that CodY may interact with LytT on the lrg promoter. Notably, unlike the codY gene, the lytT gene was >14‐fold downregulated at early‐exponential phase, compared to that of late‐exponential growth phase  without a further requirement for GTP, suggesting that BCAAs are predominant co‐effectors in mediating an interaction between CodY and lrg (or cid), and the cid and lrg operons respond to changes in amino acid availability. Therefore, it is possible that CodY loses its repressing activity as a result of the decrease in the intracellular levels of BCAAs and/or GTP that occurs at the onset of stationary phase, or by interacting with LytT. The levels of BCAAs may reflect the metabolic/nutritional status of cell, allowing CodY to coordinate a variety of genes that are induced when S. mutans cells make the transition from rapid exponential growth to stationary phase. However, it does not appear that CodY competes with LytT for the potential binding site on the lrgAB promoter, because (a) lack of CodY does not release the repression of lrgAB during exponential growth and (b) it still markedly reduces the stationary phase expression of lrgAB in the strain overexpressing lytST, showing dramatic elevation of lrgAB expression at stationary phase. Thus, the access of LytT to the lrgAB promoter or its full function as an activator may require CodY at stationary phase. To address this idea, we are currently investigating the potential interaction between CodY and LytT in the regulation of lrgAB. Given that CodY is a major repressor of stationary phase gene expression during exponential phase, and deficiency of CodY renders the cell more sensitive to 3FP during growth, it is also possible that CodY may be involved in both carbohydrate and pyruvate metabolism, related to cell growth. In fact, the S. mutans ΔcodY strain grows slowly  in the lrg promoter, indicating that activation of lrg by LytT is influenced by both CcpA and CodY. In our previous EMSAs, it is quite apparent that CcpA has a higher binding affinity to the cid and particularly lrg promoter regions than CodY, unless the level of BCAAs is increased in the culture  and the CodY‐binding sites further support the idea that CcpA and CodY work together in regulating both cid and lrg expression, and changes in glycolytic metabolite and amino acid pools act as signals that alter the balance between the effects these two regulatory proteins have on cid and lrg expression. This current study offers an intriguing potential insight toward understanding metabolic linkages with cid and lrg operons.More interestingly, the predicted LytT‐binding motif, partially overlapping with the cid and lrg expression ; formal analysis (supporting); funding acquisition (lead); investigation (supporting); methodology (lead); supervision (lead); validation (supporting); writing‐original draft (lead); writing‐review and editing (lead). Hey‐Min Kim: Formal analysis (lead); investigation (supporting); methodology (supporting); visualization (lead); writing‐original draft (supporting); writing–review and editing (supporting). Shailja Desai: Formal analysis (supporting); investigation (supporting); methodology (supporting); visualization (supporting). Kamal Deep: Formal analysis (supporting); investigation (supporting); methodology (supporting); visualization (supporting); writing–review and editing (supporting). Kelly C Rice: Conceptualization (supporting); funding acquisition (supporting); writing‐original draft (supporting); writing–review and editing (supporting).None required.


During the period of oocyte growth, chromatin undergoes global rearrangements at both morphological and molecular levels. An intriguing feature of oogenesis in some mammalian species is the formation of a heterochromatin ring-shaped structure, called the karyosphere or surrounded “nucleolus”, which is associated with the periphery of the nucleolus-like bodies (NLBs). Morphologically similar heterochromatin structures also form around the nucleolus-precursor bodies (NPBs) in zygotes and persist for several first cleavage divisions in blastomeres. Despite recent progress in our understanding the regulation of gene silencing/expression during early mammalian development, as well as the molecular mechanisms that underlie chromatin condensation and heterochromatin structure, the biological significance of the karyosphere and its counterparts in early embryos is still elusive. We pay attention to both the changes of heterochromatin morphology and to the molecular mechanisms that can affect the configuration and functional activity of chromatin. We briefly discuss how DNA methylation, post-translational histone modifications, alternative histone variants, and some chromatin-associated non-histone proteins may be involved in the formation of peculiar heterochromatin structures intimately associated with NLBs and NPBs, the unique nuclear bodies of oocytes and early embryos. The development of germ cells, the formation of a zygote and the subsequent cleavage of the embryo are amazing phenomena in nature. The period of oocyte growth at the diplotene stage of the meiotic prophase, the integration of parental genomes after fertilization, which gives rise to a new organism, and the zygotic genome activation (ZGA) at the species-specific stage of early embryonic development are all accompanied by dramatic structural and functional rearrangements of chromatin, both at the morphological and molecular levels.The morphodynamics and specificity of chromatin rearrangements observed in developing oocytes of several laboratory and farm animals are described in detail . Rapid development of modern methods especially using improved techniques of sequencing and chromatin mapping ,7,8,9,10The aim of our review is a brief analysis of modern data that can be interpreted in the context of the peculiar chromatin morphology, which had being described by classical morphologists in oocytes and embryos for many decades. We discuss the morphological and molecular features of chromatin changes during mammalian oogenesis and embryogenesis, focusing on the characteristic ring structures of heterochromatin, intimately associated with unique nuclear bodies known as the nucleolus-like bodies  or the nucleolus precursor bodies  that are observed in several species, including mouse and human. Both NLBs and NPBs are sometimes referred to as atypical nucleoli (NCLs) .Oogenesis and embryogenesis are a continuous process, during which the transformations of oocyte chromatin predetermine the outcome of further development after fertilization. Here, we tried to group data on the dynamics of chromatin configuration and its regulatory mechanisms according to the main stages of development, but not according to molecular processes as is typical for reviews on this topic. We found this approach more appropriate to describe the molecular mechanisms that can determine the morphodynamics of heterochromatin, the distribution pattern of which is specific for a certain stage of pro-embryonic and early embryonic development.The specific morphological features of chromatin configurations in growing and fully-grown oocytes had been discussed elsewhere for several mammalian species including mouse, rat, human, monkey, pig, horse, cattle, buffalo, goat, sheep, rabbit, dog, cat, and ferret ,4,12. InIn spite of the significant differences in the nomenclature proposed for different stages of chromatin rearrangements in the GV oocytes of various animals  and NSN (non-surrounded “nucleolus”) . In the The morphology of a heterochromatin mass (karyosphere) in SN or SN-related oocytes is distinct in different mammals. The differences mainly concern the karyosphere compactness and NLB size .The gradual formation of NSN and SN configurations in mouse oocytes directly correlates with a decrease in the transcriptional activity ,38. The The NSN configuration of chromatin anyhow precedes the SN one. An opposite situation, when SN oocytes might acquire an NSN-like chromatin configuration before the GVBD has not yet been described, with except of a recent study  proposedA striking feature of mammalian oogenesis is that both NSN- and SN-type oocytes can be isolated from the follicles of the same stage, but with a gradual increase in the percentage of SN oocytes in more advanced follicles. For example, the percentage of SN-type oocytes reached 86% in fully-grown antral follicles of rhesus monkeys .However, the SN-type oocytes are observed only in those follicles that have reached a certain stage of folliculogenesis. For example, all mouse GV oocytes from pre-antral follicles exhibit the NSN configuration of chromatin ,30, whilThe ratio of NSN- and SN-type oocytes in the same size follicles may depend on the age of the female. For example, GV oocytes of young (two-month old) normally fertile mice might be identified as “canonical” NSN- or SN-type oocytes. In contrast, the oocytes of 11-month-old low-fertility mice had a chromatin configuration that can be referred to as “neither NSN nor SN” (nNSN–nSN) . The nNSThere are some differences between murine SN and NSN oocytes in terms of the GVBD dynamics and metaphase plate (MI) formation: the GVBD occurs approximately 17 min later in NSN than in SN oocytes, and MI also appears approximately 40 min later . More im2+ oscillations and nuclear accumulation of PLC-β1—an important cofactor in the intracellular transduction of many signals—characterize only SN human oocytes with a fully-developed compact karyosphere.Ferret oocytes of the SN type were also shown to be more competent for proper development: approximately 75% of ferret SN oocytes can reach MII, while 72.6% of NSN oocytes stop their development at the GV stage before the onset of meiotic divisions . FinallyCombelles et al.  have disFurther evidence that SN oocytes exhibit a higher competence to develop came recently from a study on pig oocytes . As mentThe formation of the SN chromatin configuration in murine GV oocytes is accompanied by a redistribution of centromeric and pericentromeric heterochromatin ,66,67,68A powerful tool in studies of heterochromatin structure in mammalian oocytes and zygotes is the so-called “enucleolation”—microsurgical removing of NLBs from mouse GV oocytes and NPBs from the pronuclei (PNs) of zygotes followed by NLB/NPB transfer . With thA marker of chromocenters is HP1—the main structural protein of heterochromatin . Two HP1It is not surprising that the distribution of chromatin remodeling proteins associated with the pericentromeric heterochromatin changes significantly during the NSN–SN transition. For example, ATRX—α-thalassemia/mental retardation X-linked protein, an ATP-dependent helicase, essential for heterochromatin formation and maintenance during meiosis—is predominantly associated with the NLB-surrounding heterochromatin ,77. ATRXSince NLBs are a nucleolar derivative, it is not surprising that rDNA is part of the NLB-associated heterochromatin. The rDNA-containing regions assemble together in NSN oocytes to form several highly condensed foci at the NLB periphery ,66. OtheThe total number of rDNA-positive zones decreases during NSN–SN transition, but their association to MaSat sequences, in contrast, increases at this time. The vast majority of rDNA signals detecting in SN oocytes are associated with MaSat signals. However, a number of those MaSat signals that are not associated to rDNA, as well as the total number of MaSat signals, also increase during the NSN–SN transition. This allows suggesting that the pericentromeric regions, largely presented by MaSat signals, undergo a decondensation to wrap the NLB, while rDNA sequences become more condensed .Thus, drastic chromatin transformations during mammalian oocyte development involve different functional areas of chromatin, including pericentomeric, centromeric and rDNA-containing regions. As a result, a complex heterochromatin compartment (karyosphere) forms around the NLBs. Numerous data on the large-scale chromatin rearrangements during late mammalian oogenesis allows one to conclude that the NLB-associated heterochromatin formed during the NSN–SN transition is indeed a dynamic hub of many activities. The formation of this unique heterochromatin compartment is necessary for the successful completion of meiosis and subsequent early embryonic development.The acquisition of a specific morphological configuration by heterochromatin is also characteristic of mouse zygotes, in which the parental genomes are initially separated and exist in two haploid pronuclei (PNs): paternal (pPN) and maternal (mPN). Within the first hours after fertilization, before the formation of the PNs, maternal chromatin looks like a round or elongated mass localized near the polar body. Paternal chromatin retains an oblong shape during this period, resembling the sperm head . The nucThe main tendency to the formation of a common chromatin landscape in zygotes is the gradual appearance of condensed chromatin regions against the background of more diffuse chromatin, initially dispersed throughout the PN. The NPBs play a role in the formation of the specific regions of heterochromatin and represent the centers around which these regions concentrate. Again, as a result, ring-shaped zones intensely stained with DAPI appear at the NPB periphery in both PNs and persist for several cleavage divisions in the blastomere nucleus . StudiesAs in fully-grown oocytes where the NLB-associated heterochromatin is enriched with DNA tandem repeats, including centromeric DNA sequences , the perAt the beginning of the second cell cycle, pericentromeric heterochromatin retains in a close association with the periphery of NPBs, forming partial “rims” or, rarely, spherical zones corresponding to the pro-chromocenters. By the end of the second cell cycle, the ratio of these zones changes: the spherical zones become more numerous, and the number of NBPs surrounded by the pericentromeric “rim” decreases. In turn, the regions of centromeric heterochromatin are always associated with both zones of pericentromeric chromatin .At the late two-cell stage of mouse development, DNA tandem repeats begin to regroup into chromocenters ,90  appear at different developmental stages: HP1α is not detected until the four-cell stage, HP1β appears already in both PNs at the G1 stage, and HP1γ is detected after the stage S/G2 . The locInterestingly, HP1β exists in the pPN of mouse zygotes despite the absence of its main binding target—H3K9me3 . Hence, Generally, the morphological pictures that reflect the changes of heterochromatin configuration in early embryogenesis resemble those in oogenesis at first glance. The unique nuclear bodies—NPBs and NLBs—serve as a platform around which chromatin condenses to form a peculiar heterochromatin ring containing the areas of centromeres and pericentromeres.In the oogenesis and early development of mammals, the functional activity of chromatin is largely regulated by the unique epigenetic landscape created by post-replication DNA modifications, post-translational modifications of DNA-associated proteins and ATP-dependent nucleosome remodeling.One of the main epigenetic mechanisms in the cell is DNA methylation, which changes the binding sites of a number of transcription factors, thereby modulating transcription . SeveralOther essential epigenetic marks that play a key role in chromatin remodeling in oocytes and embryos are post-translational modifications of core histones ,107,108.In addition, oocytes and early embryos are characterized by a specific pattern of alternative histone variants, providing another layer of chromatin control. Non-canonical histone variants can replace canonical histones, thereby potentially switching the state of chromatin. Replacing the canonical histones with their variants can erase or alter the pattern of histone post-translational modifications  to suppoThe epigenetic transitions including DNA methylation in mammalian oogenesis and pre-implantation development are well explored, especially in mouse and human . At the The level of global DNA methylation changes significantly during oogenesis. In oocytes from primordial follicles at the beginning of folliculogenesis, DNA is practically unmethylated compared to GV oocytes present in antral follicles, which demonstrate approximately 40% global DNA methylation , with a At the beginning stages of follicle development, de novo DNA methylation requires a permissive histone modification state. For example, histone H3 must be trimethylated at lysine 36 (H3K36me3), but should not be di- or trimethylated at lysine 4 (H3K4me2/me3) in areas destined for DNA methylation ,114. In If DNA methylation reprogramming is highly similar between mouse and human ,117, theGrowing mouse oocytes are characterized by numerous post-translational modifications of histones in certain lysine residues. The epigenetic reprogramming of chromatin mostly involves methylated H3  and acetylated H3 and H4  ,113, witHdac1 and Hdac2 genes encoding histone deacetylases results in follicle developmental arrest at the secondary follicle stage [Besides, histone deacetylation plays a role in the regulation of the spatial organization and functional status of oocyte heterochromatin . In micele stage . Applicale stage . Experimle stage .Cxxc1 knockout mice, but did not affect the chromatin configuration [The predictable reorganization of heterochromatin during oogenesis is impaired under experimental conditions when the specific landscape of histone modifications is disrupted, e.g., after oocyte-specific depletion of mammalian histone methyltransferase G9a , which leads to a decrease in H3K9me2 level and to an impaired SN chromatin structure . On the guration . HoweverThe distortion of normal NSN–SN transformation was described in aging oocytes, which coincides with the changes of histone methylation . AccordiThe distribution patterns of five histone modifications  were studied during the NSN–SN transition of mouse oocytes with the use of high resolution confocal microcopy and 3D-FISH in 3D-preserved nuclei . SignifiEzhip knockout mice resulted in a global increase in H3K27me2/3 deposition in the late stages of oocyte maturation [It has been documented that the NLB-associated heterochromatin “ring” is marked by H3K4me3 and H4K5ac ,68,74, wturation . The altturation , it is nturation —another turation . HoweverIn human SN-type oocytes, a highly condensed chromatin, which is organized into a compact transcriptionally inert karyosphere closely associated with the NLB ,22,45, bThe karyosphere in mammalian oocytes is a vivid example of a situation when the unpretentious logic “on/off” is too simple to describe the histone code. A class of developmental regulator genes that carry both activating H3K4me3 and repressive H3K27me3 marks have been identified and referred to as “bivalent genes” . They arSeveral highly sensitive variants of ChIP–seq allowed revealing non-canonical (nc) forms of H3K4me3 and H3K27me3 in mouse oocytes ,6,7,10. Pcbp1 knockdown mice.Growing oocytes are highly enriched in the poly(rC)-binding protein 1 , which plays an important role in controlling gene expression as a transcriptional regulator . ExperimDppa3-null mouse oocytes, the NSN–SN transition was significantly impaired and transcriptional repression was incomplete.In addition, the developmental pluripotency-associated protein 3  was shown to facilitate transcriptional repression and the subsequent NSN–SN transition in mouse oogenesis . In DppaChromatin rearrangement during karyosphere formation (the NSN–SN transition) is also regulated by the mitogen-activated proteinkinase (MAPK), as shown in studies of pig oocytes . In thisThere is no doubt that the NSN–SN transition is a highly complicated attribute of mammalian oogenesis, which involves many molecular and physiological events. Some of them closely related with the morphological reorganizations of oocyte chromatin are summarized in The pronounced changes in chromatin of maternal and paternal origin occur after fertilization. They are necessary for the integration of parental genomes and for the oocyte-to-embryo transition. Noticeably, there is a clear asymmetry between the pPN and mPN at the zygote stage in many respects.There are several forms of methylated DNA nucleotides including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)—a modification of 5mC generated by oxidation. The paternal genome is initially characterized by an extremely high degree of methylation. For example, about 80–90% of CpG dinucleotides are methylated in spermatozoa ,135,136.Two mechanisms are involved in DNA demethylation during pre-implantation development of mammals: the replication-dependent passive loss of methylation and the active process mediated by methylcytosine dioxygenase TET3 . AccordiThe level of paternal DNA demethylation after fertilization was previously thought to be significantly higher than that of maternal DNA. This conclusion came from immunofluorescent staining studies, in which anti-5mC signal was not detected in the pPN in the end of the first cell cycle but retained in the mPN . HoweverIn mouse zygotes, the NPB-associated regions of heterochromatin contain 5mC and its intermediate modification 5hmC. Moreover, both 5mC and 5hmC are detected simultaneously in both mPNs and pPNs, with no reciprocal change in the levels of these marks . Direct The epigenetic asymmetry between the pPN and mPN in mammalian zygotes can also be monitored in respect to the distribution patterns of post-translational histone modifications. In mouse, di- and trimethylated H3K9 modifications are revealed in the mPN, but not the pPN, at stage PN0, despite that monomethylated H3K9me1 and H3K27me1 are revealed in the pPN at this stage . This paThere are some species-specific features of the deposition of post-translational histone modifications in mammalian zygotes. A clear asymmetry between the pPN and mPN in relation to the content of H3K27me3 was described in the pig ,154,155 Remarkably, the early development is characterized by different dynamics of H3K4me3 and H3K27me3 in human and mouse. A special “priming” form of H3K4me3, which nonetheless are to be distinguished from non-canonical H3K4me3 in mouse, appears in four-cell human embryos, prior to ZGA, in CpG-rich promoters and distal regulatory elements of the genome. H3K27me3 is vice versa depleted after fertilization and is re-established yet afterwards the eight-cell stage. In the mouse, ncH3K4me3 and ncH3K27me3—the non-canonical forms of H3K4me3 and H3K27me3—are present in the GV oocytes and inherited after fertilization. Then, ncH3K4me3 is reprogrammed to the canonical H3K4me3 form upon ZGA, while ncH3K27me3 retains even in blastocysts and resets to the canonical H3K27me3 form in post-implantation embryos only .It can be also mentioned that the NPB-associated heterochromatin rings, at least in the mouse, contain the marks of both transcriptionally inert and transcriptionally active chromatin. For example, H3K9me3 and H4K5ac—the representative marks of “repressed” and “active” chromatin, respectively— are detected in these areas  Figure , indicatThe appearance of the histone variant H3.3 in zygotes is closely related to the replacement of sperm protamines with histones in the pPN. In mice, protamines are removed from sperm chromatin within 30 min after gamete fusion and completely disappear after 50 min . This prOct4 pluripotency marker gene and global de novo transcription from the paternal genome, which is crucial for early embryonic development.It was shown that H3.3 plays a critical role in the regulation of pPN formation in zygotes and significantly affects the development of mouse embryos ,163,164.Oct4. Injection of exogenous H3.3 mRNA into oocytes rescued the compromised reprogramming for developmental potentials [An important role of H3.3 as a key maternal factor for oocyte reprogramming was confirmed in somatic cell nuclear transfer (SCNT) embryos . It was tentials .In normal mouse development, H3.3 begins to be detected in the pPN earlier than in the mPN ,161. ThiDppa3-null mouse embryos, which are characterized by a violated formation of chromocenters. This indicates that DPPA3/STELLA is a participant of chromatin reorganization in early mouse embryos, apparently by controlling the expression of DAXX [It was shown that the histone variant H3.3 and especially H3.3K27 is required to establish the specific heterochromatin structure in early mouse embryos . Indeed, of DAXX .The H2A variants play a key role in the regulation of chromatin activity ,170. MacAlthough the canonical H2A and its variants, including H2A.X, H2A.Z, and macroH2A, are deposited in the nucleus of immature and fully-grown oocytes, as well as on the condensed chromosomes after the GVBD, only H2A.X is abundant in the PNs of mouse zygotes after fertilization, in contrast to the low abundance of canonical H2A and the absence of H2A.Z and macroH2A . The decParticularly, maternal macroH2A is lost from the zygote at the PN2 stage, reappears in embryos only after the eight-cell stage, and then persists in morulae and blastocysts, where it is revealed in the nucleus of cells both of the trophectoderm and the internal cell mass . These dH1foo recovered the ability of embryonic stem cells to differentiate [H1foo gene led to chromatin condensation and increased deposition of H3.1 and H3.2 at the periphery of zygotic NPBs [H1foo is an oocyte-specific variant of the linker histone H1, which is expressed in GV oocytes and persists until the late two-cell embryonic stage . H1foo irentiate . H1foo, rentiate . The exprentiate . H1foo itic NPBs .The nucleus of mammalian oocytes and pre-implantation embryos has a unique organization. However, oocytes and zygotes are the cells significantly different in nature: the oocyte is a highly specialized cell, while the zygote is a totipotent cell. Nevertheless, the specific heterochromatin rings form in both late oocytes and zygotes soon after fertilization, contouring the specific nucleolus-related bodies—NLBs and NPBs .Here, we briefly analyzed key epigenetic factors that could determine the morphodynamics of most prominent DAPI-positive structures—the karyosphere and the karyosphere-like rings—in mammalian oocytes, zygotes, and early embryos. In particular, we have discussed how DNA methylation, post-translational histone modifications, alternative histone variants, and some chromatin-associated non-histone proteins may be involved in the formation of these unique heterochromatin structures.Despite a major advance in our understanding of how differential gene expression is regulated during early mammalian development, a complete picture of the molecular interactions that underlie chromatin reorganization in oogenesis and embryogenesis has not yet been created. Most of the data on the molecular mechanisms of heterochromatin regulation were obtained in studies on a limited number of mammalian species, mainly in the mouse and human. However, both the morphology of heterochromatin and the dynamics of its transformations have species-specific features in oocytes and embryos. Therefore, only the expansion of the spectrum of objects used for molecular analysis can reveal the universal principles of chromatin reorganizations, which are crucial for the onset of the development of a new organism.Peculiar karyosphere-like structures are indeed a good example of the plasticity and complexity of heterochromatin. To date, some findings force to revise the classical definitions of euchromatin and heterochromatin proposed in the end of 1920s by Heitz (see  and refeOne of the intriguing and practically unexplored problems is the deciphering the mechanisms of interactions between heterochromatin and NLBs/NPBs . The modFurther studies in all these and related fields will probably help us to identify the driving forces that determine the morphological singularity of the nucleus during late oogenesis and early embryonic development.


Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of  The paternal genome in mice undergoes widespread DNA methylation loss post-fertilization. Here, the authors apply allele-specific analysis of WGBS data to show that a number of genomic regions are simultaneously de novo methylated on the paternal genome dependent on maternal DNMT3A activity, which induces transcriptional silencing of this allele in the early embryo. As DNAme is largely maintained throughout spermatogenesis, the genomes of mature spermatozoa harbour characteristically high levels of DNAme2. Following fertilization, the paternal genome undergoes another profound change in chromatin state, including replacement of protamines with histones and a global reduction in DNAme before the first S-phase4. Subsequently, DNAme levels on both parental genomes are progressively reduced with each DNA replication cycle5. While passive demethylation in the early embryo is likely explained by cytoplasmic sequestration of UHRF1, a cofactor of the maintenance DNA methyltransferase DNMT18, the mechanism of active demethylation remains controversial. Indeed, although TET3-mediated oxidation followed by base excision repair has been implicated in this process, a TET-independent mechanism is also clearly involved15. Regardless, whole-genome bisulfite sequencing (WGBS) analyses reveal that DNAme levels on both parental genomes reach a low point in inner cell mass (ICM) cells of embryonic day 3.5 (E3.5) mouse blastocysts, followed by widespread de novo DNAme during post-implantation development18. This wave of genome-wide demethylation followed by remethylation is conserved in human embryonic development, albeit with slower kinetics21. Notably, disruption of the machinery required for the establishment or maintenance of DNAme result in infertility and/or embryonic lethality in mice, revealing the importance of DNAme homeostasis in early mammalian development27.Male germ cell development in mammals is characterized by widespread de novo DNA methylation (DNAme) and compaction of DNA via histone-to-protamine exchange29, are an exception to the widespread DNAme observed in spermatozoa. Indeed, the vast majority of CGIs are hypomethylated in mature male and female germ cells, as well as in early embryonic development35. In addition, CGIs retain nucleosomes in spermatozoa, foregoing protamine exchange32. These retained nucleosomes include canonical H3 or its variant H3.3, which are enriched for di- and/or tri-methylation on lysine 4 (H3K4me2/3)37. Although the extent of histone post-translational modification (PTM) maintenance following fertilization is controversial39, the retention and/or rapid deposition of H3K4 methylation at CGI promoters may protect these regions against de novo DNAme40 in the developing male germline as well as the early embryo. Indeed, most CGI promoters are enriched for H3K4me3 and remain hypomethylated on both parental genomes throughout early embryonic development and in adult tissues41. H3K4me2/3 also likely facilitate the initiation of transcription from the paternal genome during zygotic gene activation36, which marks the transition between the oocyte and embryonic transcriptional programmes20. As in sperm, CGIs in oocytes are generally hypomethylated and harbour nucleosomes enriched for H3K4me338 and/or H3K27me343. However, a subset of CGIs are de novo methylated in growing oocytes by DNMT3A, which is highly expressed in oocytes45. Paradoxically, despite widespread DNAme loss from the paternal genome in mouse zygotes, maternal DNMT3A is also clearly detected in the paternal pronucleus at this stage27 and ongoing de novo DNAme is required for maintaining DNAme at the paternally methylated imprinted H19 locus in early embryos46. Direct evidence that the zygotic paternal genome is subject to de novo DNAme was provided in a recent study employing immunofluorescence (IF) and ultrasensitive liquid chromatography/mass spectrometry14. However, the genomic regions subject to such paternal DNAme acquisition (PMA) in the early embryo and the relevance of this phenomenon to transcriptional regulation of the paternal genome has not been systematically addressed.CpG islands (CGIs), CpG-rich regions that often overlap genic promoters16 and identify specific genomic regions, including CGI promoters, that show bona fide PMA. Corroborating these findings, we observe PMA of an overlapping set of CGI promoters in androgenetic but not in parthenogenetic blastocysts. Allele-specific analysis of ChIP-seq data from 2C embryos reveal that PMA is accompanied by loss of H3K4me3 over the same regions, indicating that such DNAme may inhibit transcription from the paternal allele in early embryonic development. Indeed, we show that PMA is lost in the absence of maternal DNMT3A and a subset of hypomethylated genes are concomitantly upregulated specifically from the paternal allele in the 4C embryo. Taken together, these experiments reveal that, beyond its role in maternal imprinting, DNMT3A methylates a subset of genes on the paternal genome by the 2C stage, inhibiting their expression in preimplantation development.To determine which loci gain DNAme following fertilization, here we carry out an allele-specific analysis of WGBS data from 2-cell (2C) F1 hybrid embryos47. We then applied our recently developed allele-specific pipeline for Methylomic and Epigenomic Analysis (MEA)48 to WGBS data generated from 2C (55× coverage) as well as 4C, ICM, E6.5 and E7.5 F1 (C57BL/6J × DBA/2J) hybrid embryos16. This integrated analysis yielded female/maternal and male/paternal DNAme profiles through fertilization and early mouse development  are enriched over annotated genic transcription start sites (TSSs) , spermatozoa and oocytes29. As expected, DNAme levels and CpG density are anti-correlated , as described for the human genomeT-test p = 0.46, Fig. T-test p = 2.15E−5). Though we cannot discriminate between active demethylation of H3K4 and histone H3 turnover, these results reveal that de novo DNAme of CGIs on the paternal genome is generally accompanied by a reduction of H3K4me3. For example, Tuba3a, Gdap2 and Bpi gain DNAme across 28, 58 and 7 CpGs proximal to their TSSs (+/−300 bp), respectively, coincident with loss of H3K4me3 on the paternal allele  androgenetic blastocysts, which contain only paternally inherited genomes, and conducted WGBS. Notably, androgenetic blastocysts have lower overall DNAme levels relative to the paternal allele of normal ICM cells  shows a ≥20% DNAme gain in these cells relative to germinal vesicle (GV) oocytes. In contrast, 28 CGI promoters show such a gain in androgenetic blastocysts relative to sperm, confirming that the paternal genome is the preferred target for such post-fertilization de novo DNAme  do not harbour a genetic variant and could therefore not be assessed in the F1 hybrid dataset, six, including Tuba3a, Bpi, Them7, Shisa7, Syn3 and A230077H06Rik, were also identified as PMA genes in our allele-specific analysis of F1 hybrid embryos  showed a gain in DNAme of 9–25%, below our original threshold of >30% but still higher than the genomic average of CGI promoters (1.7%+/−1.1). These results indicate that post-fertilization de novo DNAme of the paternal genome likely extends beyond the regions for which we have allele-specific data and that this phenomenon occurs independent of DBA/2J-specific variants. Thus, PMA is a bona fide parent-of-origin effect at these loci.Given that androgenetic and parthenogenetic blastocysts are uniparental, we were able to extend our parental genome-specific DNAme analysis from 18 to 90% of all annotated autosomal CGI promoters. As expected, maternally methylated imprinted gene CGI promoters, such as sts Fig. . Howeveryos Fig.  and four50 and MEME51. However, we did not detect any common DNA motif at regions that undergo PMA, including the subset that overlap CGI promoters. Therefore, we focussed on the relationship between histone PTMs and paternal DNAme acquisition and/or subsequent DNAme maintenance at these sites. We analyzed published H3K4me236, H3K4me338, H3K9me317, H3K27me342 and H3K36me339 ChIP-seq data from sperm, oocytes, zygote (1C), 2C and ICM cells using ChromHMM52. No single histone PTM or combination thereof in sperm predicted PMA at CGI promoters versus those that remain hypomethylated  are transcribed from the paternal allele in the early embryo  datasets from multiple stages in spermatogenesis as well as F1 hybrid preimplantation embryosTuba3a and Gdap2, are transcribed at this stage  C57BL/6J females with wild-type (WT) DBA/2J males and performed WGBS on F1 hybrids at the early-mid 2C stage  of PMA genes consistently showing relatively high DNAme on the paternal allele  matKO embryos  in the zygote, it is possible that this mark promotes DNAme. As KRAB-ZFPs have been shown to promote H3K9me3 deposition and potentially de novo DNAme at gDMRsDnmt3a maternal KO embryos die during post-implantation development at around E9.5–10.558. While it is tempting to speculate that aberrant expression of PMA genes from the paternal allele plays a role, our temporal analysis reveals that this is a transient phenomenon, with mRNA levels of these genes indistinguishable from wild type by the blastocyst stage. Alternatively, aberrant expression of imprinted genes as a consequence of failure of de novo DNAme in the oocyte may be responsible for such embryonic lethality74. Of note, several maternally methylated imprinted genes are already aberrantly expressed from the hypomethylated maternal allele in Dnmt3a matKO ICM cells (~E3.5).In summary, this study reveals that maternal DNMT3A is required for de novo DNAme of specific regions on the paternal genome by the 2C stage, and in turn silencing of a subset of methylated genes on the paternal allele in early mouse embryonic development. Whether maternal DNMT3A acts on the paternal genome following fertilization in other mammals, including humans, and what effect such DNAme has on transcriptional regulation of target genes remains to be determined.All animal experiments were performed under the ethical guidelines of Kyushu University and Tokyo University of Agriculture.All mice were at least 10-week old at the time of each experiment. All mice were maintained at 22 °C ambient temperature, 55% humidity and 12 h/12 h dark/light cycle.75. In brief, oocyte and spermatozoa were isolated from B6D2F1/Jcl and C57BL/6NJcl mice , respectively. Enucleated oocytes were in vitro fertilized and zygotes with two male pronuclei were cultured for 4 days in KSOM medium at 37 °C and 5% CO276. Five blastocyst pools were collected in duplicate for WGBS library construction.Diploid androgenotes were prepared as previously describedDnmt3a KO oocytes were generated using Dnmt3a2lox and Zp3-cre C57BL/6J mice as described previously77. Superovulation was induced using 7.5U PMSG/hCG and MII oocytes were collected from oviducts. Dnmt3a2lox;Zp3-cre MII oocytes were artificially inseminated with DBA/2J spermatozoa. Cumulus cells were removed using hyaluronidase after insemination and embryos were cultured in KSOM at 37 °C and 5% CO2. Early-mid 2C embryos were collected at 22 h (WGBS) and 24 h (RNA-seq), 4C embryos at 36 h and blastocysts at 96 h. Zona pellucida and polar bodies of 2C embryos were removed (WGBS). ICM cells were purified by immunodissection using anti-mouse IgG  and guinea pig complement 78. Genotyping was performed by PCR using primers for Dnmt3a (CTGTGGCATCTCAGGGTGATGAGCA and GCAAACAGACCCAACATGGAACCCT) and the Zp3-cre transgene (GCAGAACCTGAAGATGTTCGCGAT and AGGTATCTCTGACCAGAGTCATCC).27. Micrographs of all five zygotes analyzed are included in Source Data.C57BL6J females were mated with JF1 males and IF was performed on one-cell zygotes. DNMT3A was detected using the IMGENEX IMG-268 antibody (0.5 μg/ml), and DNA was counterstained using propidium iodide (2 μg/ml), as described previously47. DNA from 20–30 pooled 2C embryos per replicate was purified and spiked with 1% unmethylated lambda phage DNA, and WGBS libraries were generated by PBAT with 4 cycles of library amplification61. All WGBS libraries had >99% bisulfite conversion rates. Total RNA was extracted from 20–40 pooled 2C embryos, 5–10 4C embryos and 2–7 blastocysts per replicate using Trizol reagent. Strand-specific RNA-seq libraries were generated using NEBNext: rRNA Depletion Kit, RNA First-Strand Synthesis Module, Ultra Directional RNA Second Strand Synthesis Module, and Ultra II DNA Library Prep Kit. Libraries were sequenced on HiSeq 1500 or HiSeq 2500 (WGBS: HCS v2.2.68 and RTA v1.18.66.3)79. See Supplementary Data Lysates of androgenetic blastocysts were spiked with 0.1 ng lambda phage DNA and subjected to WGBS library construction according to the PBAT protocol for single-read sequencing80 and processed for total and allele-specific alignments using MEA v1.048 using default parameters  and the mm10 reference genome. 4 bases were removed from the 5′ end of PBAT sequences. All publicly available NGS data  and Ensembl Regulatory features (release 81) annotations were used to identify PMA regions that overlapped TSSs, gene bodies and enhancers  were scored, with the exception of allele-specific alignments of WGBS datasets generated in this study, for which a ≥1× allele-specific read coverage cutoff was used to score methylation status. DNAme levels were calculated over CGI promoters using Bedops v2.4.27ers Fig. . Overlap52 was employed to define distinct chromatin states  on the basis of filtered BAM files (BinarizeBam) using default parameters. CGI annotations  were downloaded from the UCSC Table Browser (last updated 2012-02-09).Raw sequencing reads were reprocessed as described above into total and allele-specific genomic tracks. RPKM values were calculated over TSSs (+/−300 bp) using VisRseq on the basis of normalized genomic tracks. ChromHMM v1.12https://software.broadinstitute.org/morpheus) on the basis of log2 transformed values. For genome browser visualization, biological replicates were merged and total  and allele-specific genomic tracks were organized into UCSC Track Hubs as described previously48. Total, paternal and maternal-specific changes in gene expression were calculated using DESeq2 v1.26.0 with default parameters (FDR = 10%)83. Genes with a ≥2-fold change in expression and a Benjamini-Hochberg adjusted p-value ≤ 0.1 were considered differentially expressed. Transcription initiation sites for Dnmt3a matKO and wild-type embryos were determined using StringTie v1.3.584 on the basis of total  aligned reads.Raw sequencing reads were reprocessed as described above into total and allele-specific genomic tracks. Gene expression (RPKM) values over genic exons were calculated using VisRseq (NCBI Refseq). Paternal RPKM values for each gene showing PMA was then plotted on a parallel-coordinate plot using VisRseq. Oocyte CGI promoter expression (RPKM) was calculated over TSSs +/−300bp and normalized to total aligned reads. Correlograms were generated using Morpheus , where 50% of the data are distributed (the box), and whiskers denote the values lying within 1.5 times the interquartile range.Two-tailed Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary