Single-cell RNA sequencing of mitoticarrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

Background:Germ cell mitotic arrest is conserved in many vertebrates,including birds,although the time of entry or exit into quiescence phase differs.Mitotic arrest is essential for the normal differentiation of male germ cells into spermatogonia and accompanies epigenetic reprogramming and meiosis inhibition from embryonic development to post-hatch.However,mitotic arrest was not well studied in chickens because of the difficulty in obtaining pure germ cells from relevant developmental stage.Results:We performed single-cell RNA sequencing to investigate transcriptional dynamics of male germ cells during mitotic arrest in DAZL::GFP chickens.Using differentially expressed gene analysis and K-means clustering to analyze cells at different developmental stages(E12,E16,and hatch),we found that metabolic and signaling pathways were regulated,and that the epigenome was reprogrammed during mitotic arrest.In particular,we found that histone H3K9 and H3K14 acetylation(by HDAC2)and DNA demethylation(by DNMT3B and HELLS)led to a transcriptionally permissive chromatin state.Furthermore,we found that global DNA demethylation occurred gradually after the onset of mitotic arrest,indicating that the epigenetic-reprogramming schedule of the chicken genome differs from that of the mammalian genome.DNA hypomethylation persisted after hatching,and methylation was slowly re-established 3 weeks later.Conclusions:We found a unique epigenetic-reprogramming schedule of mitotic-arrested chicken prospermatogonia and prolonged hypomethylation after hatching.This will provide a foundation for understanding the process of germ-cell epigenetic regulation in several species for which this process is not clearly described.Our findings on the biological processes related to sex-specific differentiation of prospermatogonia could help studying germline development in vitro more elaborately.

Molecular and epigenetic pathogenesis of germ cell tumors

The development of germ cell tumors(GCTs)is a unique pathogenesis occurring at an early developmental stage during specification,migration or colonization of primordial germ cells(PGCs)in the genital ridge.Since driver mutations could not be identified so far,the involvement of the epigenetic machinery during the pathogenesis seems to play a crucial role.Currently,it is investigated whether epigenetic modifications occurring between the omnipotent two-cell stage and the pluripotent implanting PGCs might result in disturbances eventually leading to GCTs.Although progress in understanding epigenetic mechanisms during PGC development is ongoing,little is known about the complete picture of its involvement during GCT development and eventual classification into clinical subtypes.This review will shed light into the current knowledge of the complex epigenetic and molecular contribution during pathogenesis of GCTs by emphasizing on early developmental stages until arrival of late PGCs in the gonads.We questioned how misguided migrating and/or colonizing PGCs develop to either type Ⅰ or type Ⅱ GCTs.Additionally,we asked how pluripotency can be regulated during PGC development and which epigenetic changes contribute to GCT pathogenesis.We propose that SOX2 and SOX17 determine either embryonic stem cell-like(embryonal carcinoma)or PGC-like cell fate(seminoma).Finally,we suggest that factors secreted by the microenvironment,i.e.BMPs and BMP inhibiting molecules,dictate the fate decision of germ cell neoplasia in situ(into seminoma and embryonal carcinoma)and seminomas(into embryonal carcinoma or extraembryonic lineage),indicating an important role of the microenvironment on GCT plasticity.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes,followed by major epigenetic remodeling leading to the formation of a totipotent embryo.During the development of the pre-implantation embryo,precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality,but the underlying molecular mechanisms remain elusive.For the past few years,unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development,taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies.The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals,including DNA methylation,histone modifications,chromatin accessibility and 3D chromatin organization.

Nuclear TIGAR mediates an epigenetic and metabolic autoregulatory loop via NRF2 in cancer therapeutic resistance

Metabolic and epigenetic reprogramming play important roles in cancer therapeutic resistance.However,their interplays are poorly understood.We report here that elevated TIGAR(TP53-induced glycolysis and apoptosis regulator),an antioxidant and glucose metabolic regulator and a target of oncogenic histone methyltransferase NSD2(nuclear receptor binding SET domain protein 2),is mainly localized in the nucleus of therapeutic resistant tumor cells where it stimulates NSD2 expression and elevates global H3K36me2 mark.Mechanistically,TIGAR directly interacts with the antioxidant master regulator NRF2 and facilitates chromatin recruitment of NRF2,H3K4me3 methylase MLL1 and elongating Pol-II to stimulate the expression of both new(NSD2)and established(NQO1/2,PRDX1 and GSTM4)targets of NRF2,independent of its enzymatic activity.Nuclear TIGAR confers cancer cell resistance to chemotherapy and hormonal therapy in vitro and in tumors through effective maintenance of redox homeostasis.In addition,nuclear accumulation of TIGAR is positively associated with NSD2 expression in clinical tumors and strongly correlated with poor survival.These findings define a nuclear TIGAR-mediated epigenetic autoregulatory loop in redox rebalance for tumor therapeutic resistance.

HIF1α epigenetically repressed macrophages via CRISPR/Cas9-EZH2 system for enhanced cancer immunotherapy

Immune suppressive microenvironment in tumor emerges as the main obstacle for cancer immunotherapy.In this study,we identified that HIF1α was activated in the tumor associated macrophages and acted as an important factor for the immune suppressive microenvironment.Epigenetically silencing of Hif1αvia histone H3 methylation in the promoter region was achieved by CRISPR/dCas9-EZH2 system,in which histone H3 methylase EZH2 was recruited to the promoter region specifically.The Hif1αsilenced macrophage,namely HERM(Hif1αEpigenetically Repressed Macrophage)manifested as inheritable tumor suppressing phenotype.In the subcutaneous B16-F10 melanoma syngeneic model,intratumoral injection of HERMs reprogrammed the immune suppressive microenvironment to the active one,reducing tumor burden and prolonging overall survival.Additionally,HERMs therapy remarkably inhibited tumor angiogenesis.Together,our study has not only identified a promising cellular and molecular target for reverting immune suppressive microenvironment,but also provided a potent strategy for reprogramming tumor microenvironment via epigenetically reprogrammed macrophages.

In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases

How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers.Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases.Using primary human colon cancer cells engrafted in mice,we find that transient expression of OCT4,SOX2,KLF41/2 cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells.Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression.Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG,but enhanced NANOG is not sufficient to induce these phenotypes.Finally,reprogrammed tumors enhance GLI2,and we show that GLI2high and AXIN2low,which are markers of the metastatic transition of colon cancers,are prognostic of poor disease outcome in patients.We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors.

Epigenetic reprogramming: roads to pluripotency

Epigenetic reprogramming provides valuable resources for customized pluripotent stem cells generation,which are thought to be important bases of future regenerative medicine.Here we review the commonly used methods for epigenetic reprogramming:somatic cell nuclear transfer,cell fusion,cell extract treatment,inducing pluripotency by defined molecules,and briefly discuss their advantages and limitations.Finally we propose that mechanisms underlying epigenetic reprogramming and safety evaluation platform will be future research directions.

A REF6-dependent H3K27me3-depleted state facilitates gene activation during germination in Arabidopsis

Seed germination is a critical developmental switch from a quiescent state to active growth,which involves extensive changes in metabolism,gene expression,and cellular identity.However,our understanding of epigenetic and transcriptional reprogramming during this process is limited.The histone H3 lysine 27 trimethylation(H3K27me3)plays a key role in regulating gene repression and cell fate specification.Here,we profile H3K27me3 dynamics and dissect the function of H3K27 demethylation during germination in Arabidopsis.Our temporal genome-wide profiling of H3K27me3 and transcription reveals delayed H3K27me3reprogramming compared with transcriptomic changes during germination,with H3K27me3 changes mainly occurring when the embryo is entering into vegetative development.RELATIVE OF EARLY FLOWERING 6(REF6)-mediated H3K27 demethylation is necessary for robust germination but does not significantly contribute to H3K27me3 dynamics during germination,but rather stably establishes an H3K27me3-depleted state that facilitates the activation of hormone-related and expansin-coding genes important for germination.We also show that the REF6 chromatin occupancy is gradually established during germination to counteract increased Polycomb repressive complex 2(PRC2).Our study provides key insights into the H3K27me3 dynamics during germination and suggests the function of H3K27me3 in facilitating cell fate switch.Furthermore,we reveal the importance of H3K27 demethylation-established transcriptional competence in gene activation during germination and likely other developmental processes.

DNA methylation dynamics during germline development

DNA methylation plays essential homeostatic functions in eukaryotic genomes.In animals,DNA methylation is also developmentally regulated and,in turn,regulates development.In the past two decades,huge research effort has endorsed the understanding that DNA methylation plays a similar role in plant development,especially during sexual reproduction.The power of whole-genome sequencing and cell isolation techniques,as well as bioinformatics tools,have enabled recent studies to reveal dynamic changes in DNA methylation during germline development.Furthermore,the combination of these technological advances with genetics,developmental biology and cell biology tools has revealed functional methylation reprogramming events that control gene and transposon activities in flowering plant germlines.In this review,we discuss the major advances in our knowledge of DNA methylation dynamics during male and female germline development in flowering plants.

Toward pluripotency by reprogramming:mechanisms and application

The somatic epigenome can be reprogrammed to a pluri-potent state by a combination of transcription factors.Altering cell fate involves transcription factors coopera-tion,epigenetic reconfi guration,such as DNA methylation and histone modification,posttranscriptional regulation by microRNAs,and so on.Nevertheless,such reprogram-ming is inefficient.Evidence suggests that during the early stage of reprogramming,the process is stochastic,but by the late stage,it is deterministic.In addition to con-ventional reprogramming methods,dozens of small mol-ecules have been identifi ed that can functionally replace reprogramming factors and signifi cantly improve induced pluripotent stem cell(iPSC)reprogramming.Indeed,iPS cells have been created recently using chemical com-pounds only.iPSCs are thought to display subtle genetic and epigenetic variability;this variability is not random,but occurs at hotspots across the genome.Here we dis-cuss the progress and current perspectives in the fi eld.Research into the reprogramming process today will pave the way for great advances in regenerative medicine in the future.

Tgfbr2 inactivation facilitates cellular plasticity and development of Pten-null prostate cancer

Mutations in tumors can create a state of increased cellular plasticity that promotes resistance to treatment. Thus, there is an urgent need to develop novel strategies for identifying key factors that regulate cellular plasticity in order to combat resistance to chemotherapy and radiation treatment. Here we report that prostate epithelial cell reprogramming could be exploited to identify key factors required for promoting prostate cancer tumorigenesis and cellular plasticity. Deletion of phosphatase and tensin homolog （Pten） and transforming growth factor-beta receptor type 2 （Tgfbr2） may increase prostate epithelial cell reprogramming efficiency in vitro and cause rapid tumor development and early mortality in vivo. Tgfbr2 ablation abolished TGF-β signaling but increased the bone morphogenetic protein （BMP） signaling pathway through the negative regulator Tmeff1. Furthermore, increased BMP signaling promotes expression of the tumor marker genes ID1, Oct4, Nanog, and Sox2; ID1/STAT3/NANOG expression was inversely correlated with patient survival. Thus, our findings provide information about the molecular mechanisms by which BMP signaling pathways render stemness capacity to prostate tumor cells.

Chronic activation of MUC1-C in wound repair promotes progression to cancer stem cells

The mucin 1(MUC1)gene emerged in mammals to afford protection of barrier epithelial tissues from the external environment.MUC1 encodes a transmembrane C-terminal(MUC1-C)subunit that is activated by loss of homeostasis and induces inflammatory,proliferative,and remodeling pathways associated with wound repair.As a consequence,chronic activation of MUC1-C promotes lineage plasticity,epigenetic reprogramming,and carcinogenesis.In driving cancer progression,MUC1-C is imported into the nucleus,where it induces NF-κB inflammatory signaling and the epithelial-mesenchymal transition(EMT).MUC1-C represses gene expression by activating(i)DNA methyltransferase 1(DNMT1)and DNMT3b,(ii)Polycomb Repressive Complex 1(PRC1)and PRC2,and(iii)the nucleosome remodeling and deacetylase(NuRD)complex.PRC1/2-mediated gene repression is counteracted by the SWI/SNF chromatin remodeling complexes.MUC1-C activates the SWI/SNF BAF and PBAF complexes in cancer stem cell(CSC)models with the induction of genome-wide differentially accessible regions and expressed genes.MUC1-C regulates chromatin accessibility of enhancer-like signatures in association with the induction of the Yamanaka pluripotency factors and recruitment of JUN and BAF,which promote increases in histone activation marks and opening of chromatin.These and other findings described in this review have uncovered a pivotal role for MUC1-C in integrating lineage plasticity and epigenetic reprogramming,which are transient in wound repair and sustained in promoting CSC progression.