Studying human germline development presents many challenges. In particular, primordial germ cell (PGC) specification and PGC migration occur shortly after implantation when the developing embryo is largely inaccessible. Profiling of later stage gonadal PGCs has allowed comparison with mouse and other species, revealing not only many similarities but also notable differences. Recently, in vitro systems to generate functional PGCs from pluripotent stem cells have been established in mouse. These protocols are easily adapted to human, suggesting remarkable conservation in PGC specification mechanisms. Such in vitro systems may now provide an important tool in which to study early human germline development and disease. Here, we review the recent advances both in vivo and in vitro, and how these may influence the study of the human germline in the years to come.
Single-cell sequencing is a powerful methodology for revealing traits of individual cells in heterogeneous populations such as embryonic stem cell cultures or whole tissues. As cellular behaviors typically depend on coordinated expression of many genes and translated proteins, unbiased sequencing methods that can extract genome-wide profiles from single cells without prior knowledge about the initial cellular sample are needed. In recent years, single-cell RNA sequencing was used to identify complex cell populations, reconstruct developmental trajectories, and model transcriptional dynamics. As the DNA representation in a cell is much lower than that of RNA, single-cell DNA-based methods such as genomic sequencing, ChIP-seq, ATAC-seq, and methods for studying the 3-D architecture of genomes are limited by the low number of molecules that can be detected making them less sensitive and more challenging for computational analysis. In this chapter, we review current single-cell sequencing techniques, discuss the power and limitations of these methods, and discuss their implementation in the context of pluripotency and differentiation studies.
Here, we discuss how epigenetic processes, including posttranslational modifications of histones and posttranscriptional modifications of RNA, differ in pluripotent embryonic stem cells (ESCs) in comparison with their differentiated counterparts. Additionally, we discuss the specificity of DNA repair epigenetics in ESCs, in which the OCT4 protein play an important role, not only in pluripotency maintenance but also in the DNA damage response. Additionally, in ESC biology, the N6-methyladenosine (m6A) modification of RNA was found to be functionally essential. It is known that m6A RNAs regulate the balance between the self-renewal capability and differentiation properties of ESCs. This posttranscriptional modification, regulating gene expression and the DNA damage response, appears to also be crucial, especially during embryonic development.
Chromosomes must be tightly folded within cell nuclei but in a manner ensuring accessibility to the machinery regulating transcription, replication, and repair. Recent studies have identified the hierarchical organization principles of chromosome folding, which impact on transcriptional regulation of gene programs. Differentiation of pluripotent stem cells is characterized by a transition from generally open and transcriptionally permissive chromatin to a more polarized state, whereby lineage-specific genes are maintained in an "open" configuration and genes in other lineages are shut down within repressive, more compacted chromatin. Despite such an apparent difference between pluripotent and differentiated chromatin states, the underlying architectural organization principles are very similar. Here, we provide an overview on our understanding of how chromosome folding is linked to cell fate control via transcriptional regulation, focusing on the similarities and differences between pluripotent and somatic cells and special cases (e.g., mitosis) where chromatin folding appears very different.
During mammalian development the pluripotency appears in cells within the inner cell mass (ICM) of the blastocyst and disappears during gastrulation, corresponding to a 4- to 5-day period in mice and approximately 2 weeks in humans. During this period the pluripotent tissue, the epiblast, expands from about 10 homogeneous cells to several hundred. This rapid increase in cell number correlates with the transcriptional and epigenetic rewiring of pluripotent escapees that differentiate during gastrulation. The differentiation processes have long been attributed mainly as a coordinated response to extrinsic signaling cues. However, in the last years, the cell cycle has emerged as an intrinsic determinant that contributes to cell fate decision-making. In this review, we discuss the recent discoveries in this area and, in particular, how the cell cycle is regulated and impacts in the biology of the pluripotent cells.
Recent exome-sequencing studies on patients with (nonsyndromic) autism and their parents have identified the first tens of genes that, if mutated, likely contribute to the autism phenotype. Surprisingly, nearly half of these genes encode chromatin modifiers, both ATP-dependent chromatin remodelers and histone-modifying enzymes, many of which are highest expressed in neural stem cells or progenitors during human brain development. This review discusses these findings and potential explanations why this category of genes is overrepresented among autism-associated genes, whether their mutation may cause defects in neural stem cells/progenitors, as well as the autism-relevant pathways in which these chromatin modifiers may act. We will also discuss the relevance of chromatin modifiers for another common psychiatric disorder, schizophrenia, and for human intelligence in general.
Disruptions to transcriptional and epigenetic mechanisms are of fundamental importance in cancer. Elucidating the role of the various genes and pathways involved should reveal ways to limit tumor growth and lead to new therapeutic strategies. During malignant transformation, genetic and epigenetic disruptions accrue within the tissue-specific chromatin landscapes associated with the cell of origin. What emerges is a cellular state capable of unconstrained self-renewal and proliferation. In this article, we discuss the major transcriptional circuits and epigenetic mechanisms associated with the glioblastoma (GBM)—a disease driven by cells with neural stem cell-like characteristics. Transcriptional factor networks, chromatin regulation (histone modifications and chromatin remodeling) and DNA methylation, are all frequently altered and "lock" cells into a stem cell-like state. We discuss these molecular pathways in three distinct contexts: (i) evidence of ongoing "normal" epigenetic processes associated with differentiation hierarchies—as predicted by the "cancer stem cell" model, (ii) identification of site-specific and global epigenetic abnormalities in glioblastoma that may be reversible, and (iii) genetic disruptions of core epigenetic regulators. Most prominent are recurrent mutations within chromatin regulators (e.g., SWI/SNF), isocitrate dehydrogenase genes IDH1/2 and variant histone genes H3.3/H3F3A. These have been discovered as hallmarks of primary, secondary, and pediatric GBM, respectively. Our current understanding suggests that despite the diversity of genetic and epigenetic insults in GBM patients, a shared feature of all GBMs is selective pressures that enforce a neural stem/progenitor cell state with unconstrained self-renewal.
During early mammalian development a functional blastocyst is generated through complex mechanisms specifying two extraembryonic lineages, the trophectoderm and primitive endoderm, and a population of undifferentiated and pluripotent cells that will subsequently differentiate into the three germ layers. While the specification of these early cell identities by transcription factors is relatively well understood, how they are transiently maintained during cell division remains unknown. Indeed, transcription factors are often inactivated during mitosis, breaking the regulatory continuity among cellular generations. Recently, it has been shown that some transcription factors, including early developmental regulators, are capable of binding to a subset of their targets in mitotic cells. This opens the possibility that they act as mitotic bookmarking factors directly instructing gene regulation from mother to daughter cells. In this chapter, we discuss how early embryonic cell identities and their corresponding derived stem cells may be transiently maintained through mitotic bookmarking processes.
Epigenetic mechanisms, including posttranslational modifications of histones and methylation of DNA, control gene expression patterns in development and disease. Characteristically, embryonic stem cells (ESCs) have a unique "open" and hyperdynamic chromatin, a state that was suggested to be important for the maintenance of pluripotency. As cells progress toward a specific differentiation pathway and gain a specialized function, some parts of their chromatin rearrange into a more repressive form. Remarkably the somatic cell state, which is stable in vivo, can be reprogrammed into a pluripotent state by the ectopic expression of the pluripotency-related transcription factors OCT4, KLF4, SOX2, and cMYC (OSKM). Somatic cell reprogramming also takes place in oncogenic processes. Cancer and especially cancer stem cells (CSC) share several characteristics with pluripotent cells, including self-renewal and multipotency, maintaining, in some tumors, hierarchical organization. Thus elucidating the epigenetic mechanisms that regulate the transition into an undifferentiated state can be significant to cancer therapy and to regenerative medicine. This chapter covers chromatin biology in (cancer) stem cells.
Endogenous retroviruses (ERVs) represent abundant retrotransposon families that have coevolved in the mammalian genome for millions of years. Although long considered junk DNA, it is becoming increasingly evident that ERVs are likely to play important roles during normal development. Retroviral sequences have been coopted to create new regulatory elements, used by cells to rewire their gene expression patterns. On the other hand, ERVs are capable of retrotransposition into new genomic locations, thus carrying mutagenic potential. As a result, specialized host regulatory mechanisms have evolved to control ERV expression and activity. In this review, we discuss the exquisite regulation of ERVs in pluripotent cells and throughout early development in human and mouse.
Early embryonic development is characterized by a dynamic epigenome. Some of the most drastic changes occur as the mammalian embryo transitions from the free-floating, preimplantation blastocyst to the early embryo embedded in the uterus. Efforts to understand early embryonic molecular phenotypes have driven the derivation of mammalian embryonic stem cells that represent the pre- and postimplantation embryo, termed naïve and primed embryonic stem cells. Here, we describe in detail mouse and human embryonic stem cell derivation as it pertains to pre- and postimplantation development. We look at the epigenetic research conducted in these systems and what we have learned to date. We highlight whenever possible in vivo embryo studies as a comparison with the in vitro embryonic stem cell studies. Where data are lacking, we describe what is currently known. Altogether, we hope to focus attention on the research that has led to major discoveries in naïve and primed pluripotent stem cells.
Mass spectrometry (MS)-based analytical approaches to examine histone post-translational modifications (PTMs) are useful tools to understand epigenetic function. Histone H3K9 methylation plays an important role in chromatin remodeling, which is important in stem cell self-renewal and differentiation. With a simple, rapid, and accurate top-down strategy using matrix assisted laser desorption/ionization (MALDI)-in source decay (ISD), we have recently identified K9 PTMs on H3 variants (H3.1, H3.2, H3.3, and H3t) in the mouse testis. Mono-, di-, and tri-methylated K9 sites were identified on H3 variants separated using liquid chromatography and an ion-pairing reagent. These modifications were also observed in the testis-specific histone H3 using MALDI-ISD. Our findings demonstrate a novel top-down approach for characterizing PTMs on histone tails that will have applications in future research. Keywords: MALDI-ISD, Testis-specific H3 histone, K9 modifications
Mitochondrion, the major organelle involved in cell energy metabolism, plays important roles in many human diseases. Nowadays, its dysfunction during initation and progression of cancers is calling more and more attention. Since Otto Warburg brought out his hypothesis, mitochondrial DNA (mtDNA) may affect on cancer development by regulating oxidative phosphorylation, many important findings have been discovered. It is widely recognized that research the potential correlations between mitochondrion and mtDNA in cancers are of great importance. And the trends of change of mtDNA content are different in different cancers. Thus leaves us numerous works to do. Although some cancers have already been reported their relation with mtDNA alteration, different experimental methods or evidence may lead to different conclusions. This article presents a brief in summarizing the mitochondrion dysfunction in human disease and the alteration of mtDNA in cancers. Moreover, the recent highlighting achievements on colorectal cancer and mtDNA are also summarized to provide a discussion on possible pathological mechanism and clinical usage.
Male infertility is a complex multifactorial pathology that is correlated in some extent with semen parameters including sperm counts, motility, morphology, viability and nucleus integrity. The large debate surrounding global decline of semen parameters motivated numerous studies dealing with this important issue. The present article reviews different factors shown to impact on male fertility by affecting semen parameters, namely, epigenetics and sperm genome integrity.
Abstract Many epidemiological studies suggest that diet particulary rich in fruit and vegetables have cancer preventive properties. The beneficial effects of these diets are attributable, at least in part, to polyphenols that have emerged as very promising anticancer bioactive compounds. We report that polyphenolic extracts from the edible part of artichoke show a potential chemopreventive and anticancer properties on several cancer cell lines. High dose of extracts of artichoke (AEs) reduced cell viability and inhibited cell growth in a dose dependent-manner. In particular AEs triggered apoptosis and modulate other tumour related features such as migration and invasion in highly metastatic breast cancer cells. In addition, we provide evidences that low dose of AEs-treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. These findings demonstrated that high dose-AEs activate apoptotic machinery whereas low level treatment induce senescence in cancer cells. Since apoptosis and cellular pro-senescence are considered relevant anticancer therapeutic mechanisms, artichoke polyphenols extracts from the edible part, could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a non-conventional, adjuvant therapy.
A medical case was reported by Neha and Colleen of a 32-year-old female who had received Fecal microbial transplantation (FMT) as a consequent of recurring Clostridium difficile infection (CDI). Following this procedure, the recipient began to experience significant weight comorbid with constipation and dyspepsia episodes. The weight gain is unamendable by strict protein diet and exercise regimen, thus, leading the authors to hypothesize nutritional-neural-microbiota connection . This hypothesis is further reinforced as animals were transformed into obese phenotypes following fecal transplant from obese human .
In order to expand the genetic code to study metalloproteins and protein posttranslational modifications, we constructed several tyrosine and pyrrolysyl-tRNA synthetase libraries based on tyrosine and pyrrolysyl-tRNA synthetase/tRNA pairs to expand the genetic code, to screen unnatural amino acids with similar chemical structure to tyrosine or lysine. We had incorporated several unnatural amino acids into proteins, including metal-chelating, proton and electron transfer mediators, bioorthogonal reaction groups containing amino acids, and played a series of bio-orthogonal reactions in vivo and in vitro, such as copper click of azide and alkyne, copper free click of azide and cycloalkyne, photoclick of alkene and tetrazole, which laid the foundation for proteins specifically labeling, protein-protein interaction probing, and biological processes photo-regulation. Besides that, we applied this method to incorporate redox unnatural amino acids to improve the property of fluorescent proteins. We explored the activities of proteins and downstream signaling pathways by using sulfur or fluorine mimetics of acetylated lysine as probes of protein posttranslational modifications. We also directed Ne-Formyl-L-lysine into histones to study the impact of Ne-formylation of lysine naturally caused by oxidative damage in cells on histones.
Chemotherapy for acute myeloid leukemia (AML) principally induces intrinsic apoptosis in circulating leukemic blasts. Unfortunately, standard therapy is relatively ineffective in eliminating AML stem/progenitor cells that drive leukemogenesis. The inhibitors of apoptosis (IAP) protein family are critical regulators of cell survival. IAP proteins, in particular cIAP1 and cIAP2, have drawn great attention in recent years as targets for cancer therapy due to the development of SMAC mimetics. We discovered that the expressions of cIAP1, which primarily inhibits the extrinsic apoptosis pathway and a main target of SMAC mimetics, and caspase-8, the key caspase of the extrinsic apoptosis were significantly higher in AML stem/progenitor cells than in bulk AML cells. Conversely, the expression of SMAC, an endogenous antagonist of IAP proteins, was significantly lower by comparison. We investigated the therapeutic potential of targeting IAPs by SMAC mimetics in AML and reported that the novel SMAC mimetic birinapant effectively induced apoptosis in AML cells, including AML stem/progenitor cells. This effect was present even in leukemic cells co-cultured with bone marrow derived-mesenchymal stromal cells under hypoxic conditions representative of the bone marrow microenvironment. Furthermore, this anti-leukemia activity was enhanced in vitro and in vivo by combination with demethylating agents, which apparently modulated NFkB signaling and many key members of the extrinsic apoptosis pathway. The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress the extrinsic apoptosis. We have demonstrated that ARC is an effective negative prognostic factor for AML, is regulated by cIAP1/NIK signaling in AML cells, and can be inhibited by demethylating agents. Our findings suggest that the activation of the extrinsic pathway has the potential to eradicate AML stem/progenitor cells and that mechanistic combinations of SMAC mimetics with agents that modulate the extrinsic apoptosis pathway may benefit patients with AML. In fact, our preliminary results served as the catalyst for recently-initiated phase I/II trials of birinapant in combination with 5-azacytidine in pre-leukemia myelodysplastic syndrome and AML patients.
The genomes of herpesviruses and HIV become silent during latency through multiple chromatin silencing mechanisms including: histone deacetylation, repressive histone methylation, and DNA methylation. Reactivation of the latent virus requires removal of the chromatin silencing marks and their replacement by activating modifications such as histone acetylation and activating histone methylation. In a complementary mechanism, RNA Polymerase II (RNAP II) elongation is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. In resting T-cells latently infected by HIV, expression of P-TEFb is restricted. We found that a group of short chain fatty acids (SCFAs) produced by oral bacteria not only promote histone acetylation but also change the histone methylation dynamics by decreasing repressive histone methylation while increasing activating histone methylation. SCFAs also block DNA methylation and activate P-TEFb to enable elongation of stalled RNA polymerase II. Thus these molecules do not simply act as histone deacetylase (HDAC) inhibitors as previously claimed. Instead, they impact multiple complementary epigenetic regulatory mechanisms to promote highly efficient reactivation of latent viruses.
