State Of Embryonic Stem Cells Research Articles

Single-cell 3D genome structure reveals distinct human pluripotent states

BackgroundPluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive.ResultsBy single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes.ConclusionsOur data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Tbl1 promotes Wnt-β-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-β-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3β, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-β-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with β-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-β-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells.

Sox2 is known for its roles in maintaining the stem cell state of embryonic stem cells and neural stem cells. In particular, it has been shown to slow the proliferation of these cell types. It is also known for its effects as an activating transcription factor. Despite this, analysis of published studies shows that it represses as many genes as it activates. Here, we identify a new set of target genes that Sox2 represses in neural stem cells. These genes are associated with centrosomes, centromeres and other aspects of cell cycle control. In addition, we show that SUMOylation of Sox2 is necessary for the repression of these genes and for its repressive effects on cell proliferation. Together, these data suggest that SUMO-dependent repression of this group of target genes is responsible for the role of Sox2 in regulating the proliferation of neural stem cells.

Aneuploid embryonic stem cells drive teratoma metastasis

Aneuploidy, a deviation of the chromosome number from euploidy, is one of the hallmarks of cancer. High levels of aneuploidy are generally correlated with metastasis and poor prognosis in cancer patients. However, the causality of aneuploidy in cancer metastasis remains to be explored. Here we demonstrate that teratomas derived from aneuploid murine embryonic stem cells (ESCs), but not from isogenic diploid ESCs, disseminated to multiple organs, for which no additional copy number variations were required. Notably, no cancer driver gene mutations were identified in any metastases. Aneuploid circulating teratoma cells were successfully isolated from peripheral blood and showed high capacities for migration and organ colonization. Single-cell RNA sequencing of aneuploid primary teratomas and metastases identified a unique cell population with high stemness that was absent in diploid ESCs-derived teratomas. Further investigation revealed that aneuploid cells displayed decreased proteasome activity and overactivated endoplasmic reticulum (ER) stress during differentiation, thereby restricting the degradation of proteins produced from extra chromosomes in the ESC state and causing differentiation deficiencies. Noticeably, both proteasome activator Oleuropein and ER stress inhibitor 4-PBA can effectively inhibit aneuploid teratoma metastasis.

Heterogeneity and Differentiation Patterns of pDC Cells in pDC-AML: A Novel Perspective from Single-Cell RNA Sequencing

Background: Plasmacytoid dendritic cells (pDCs), being a primary source of natural type I interferon (IFN-I), have pivotal roles in orchestrating the immune response. Typically, only less than 1% pDCs can be detected in the total bone marrow or peripheral blood nuclear cells. However, the uncontrollable proliferation of transformed pDCs is a remarkable characteristic of blastic plasmacytoid dendritic cell neoplasm (BPDCN). Recently, an abnormal amplification of pDCs in myeloid neoplasms has also been reported in MDS/AML patients (pDC-AML/MDS). This abnormal increase in pDCs could potentially promote disease progression. In this study, we utilized single-cell RNA-sequencing (scRNA-seq) to examine the characteristics of pDCs in pDC-AML. Method: Our study employed a mixed retrospective and prospective design. 85 patients diagnosed with PDC-AML/MDS/BPDCN, including 1 pDC-MDS, 17 pDC-AML, 50 non-pDC-AML (AML patients without pDCs expansion), 17 BPDCN from Tongji Hospital of Huazhong University of Science and Technology in Wuhan, China, between July 2014 and June 2023. scRNA-seq using 10x Genomics platform were performed on bone marrow (BM) samples from 8 pDC-AML patients and 1 BPDCN patient. Furthermore, we expanded our dataset by integrating publicly available scRNA-seq data of BM samples from 5 BPDCN patients, 5 non-pDC-AML patients, and 5 healthy donors (HD). Result: The median pDCs proportion of pDC-AML/MDS was 8.0% (range 2.5-52.0%). As of June 2023, The median overall survival (OS) of patients with pDC-MDS/AML was 10.5 months shorter than non-pDC-AML (21.5 months; P＜0.05). In addition, the prognosis of pDC-AML/MDS patients can only be significantly improved after receiving allogenic hematopoietic stem cell transplantation, showing a similar OS to that of non-pDC-AML patients. To further clarified the role of pDCs in these patients, we performed scRNA-seq and acquired 126,693 cells across all samples (8 pDC-AML, 6 BPDCN, 5 non-pDC-AML and 5 HD), and identified 13 clusters based on their transcriptional profile in the overall UMAP (Figure 1 A). Firstly, our single-cell sequencing data revealed significant differences in the proportions of bone marrow pDCs across pDC-AML, HDs, non-pDC-AML, and BPDCN patients. Notably, pDC proportions were significantly elevated in pDC-AML and BPDCN patients. Further differential expression analysis revealed striking differences in the transcriptomes of pDCs across these four groups. When comparing pDC-AML to HDs, we identified 801 upregulated and 150 downregulated genes. Upon comparing pDC-AML to BPDCN, we found 1110 upregulated and 765 downregulated genes, and when comparing pDC-AML to non-pDC-AML, we observed 1674 upregulated and 945 downregulated genes. To explore the functional implications of these differentially expressed genes (DEGs), these findings were explored further through Gene Set Enrichment Analysis (GSEA), and indicated a heightened response to interferon signaling within the pDC cells of pDC-AML patients. Concurrently, we also discovered an increased expression of gene sets associated with tumorigenic pathways (such as KRAS, PTEN, p53, RAPA, etc.) and those related to embryonic stem cell states in pDCs of pDC-AML. Drawing on our findings and existing research, we hypothesize that interferon signaling might play a pivotal role in the pathogenesis of pDC-AML. This could be achieved through the enhancement of cell proliferation and self-renewal capabilities, thereby facilitating disease progression. This hypothesis is further supported by our pseudotime analysis. After conducting a more detailed subgroup classification of pDC-AML patients, we found a developmental trajectory linking AML_HSC and pDCs specific to pDC-AML patients. Notably, such a correlation was not observed in the non-pDC-AML patient group. Conclusions: Our study revealed heterogeneity and underscores the crucial role of pDCs in pDC-AML, as we found that these patients had worse OS. We observed upregulation of interferon signaling and tumorigenic pathways in these pDCs. Moreover, pseudotime analysis suggested a potential developmental relationship between pDCs and AML_HSCs. These findings provide invaluable insights for further investigations into pDCs biological properties in pDC-AML pathology and therapy design.

Molecular characterizations and functional roles of NANOG in early development of porcine embryos

Nanog homeobox (NANOG) is the gateway to the pluripotent ground state in mouse embryonic stem cells and early embryos. However, understanding of the molecular signatures and functional characteristics of porcine NANOG remains limited. In this study, we analyzed the gene structure and sequence characteristics of porcine NANOG and found that the porcine NANOG gene is localized on chromosome 5, while NANOG sequence on chromosome 1 is the processed pseudogene. We explored the expression pattern of NANOG in porcine early embryos by immunofluorescence staining and Realtime-PCR and RNA-seq, the results showed that transcription of porcine NANOG commences at the 4-cell stage, while expression of the NANOG protein is initially observed in the inner cell mass of blastocysts. Furthermore, we identified a NANOG splicing variant in porcine early embryos, which maintain the overall structure of the original NANOG mRNA, except for a deletion of 38 base pairs in the second exon. To further investigate the function of NANOG in early embryo development in pigs, we employed siRNA-mediated deletion of the two specific transcripts on porcine zygotes. The results showed that blastocyst rate was significantly reduced after NANOG deleting. A significant decrease in the expression of DNA methylation-related gene DNMT3B was also observed in D3 embryo from the NANOG deleting group. In conclusion, the porcine NANOG gene, accompanied by a single-exon processed pseudogene, exhibits two transcripts and plays a pivotal role in the development of early-stage embryos.

Zyxin inhibits the epithelial-mesenchymal transition process in gastric cancer by upregulating SIRT1.

Tumor development relies on the stemness of cancer stem cells, which is regulated by environmental cues. Previous studies have shown that zyxin can inhibit the expression of genes for embryonic stem cell status. In the present study, the expression levels of zyxin protein in cancer tissues and adjacent noncancerous tissues from 73 gastric cancer patients with different clinical stages were analyzed by Western blot. We showed that the relative expression levels of zyxin in gastric cancer tissues (cancer tissues/adjacent tissues) were significantly downregulated in advanced clinical stages. Overexpression of zyxin inhibited the stemness and epithelial-mesenchymal transition (EMT) processes in gastric cancer cells. Zyxin also inhibited the proliferation, migration, and invasion but increased the sensitivity of cancer cells to drugs. Overexpression of zyxin in MKN45 cells inhibited tumor growth in nude mice. We show that the interactions between zyxin and SIRT1 led to the upregulation of SIRT1, reduced acetylation levels of histone H3 K9 and K23, decreased transcription levels of SNAI 1/2, and inhibition of the EMT process. This study demonstrated that zyxin negatively regulates the progression of gastric cancer by inhibiting the stemness of cancer stem cells and EMT. Our findings shed new light on the pathogenesis of gastric cancer.

OCT4's role and mechanism underlying oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), a common malignancy of the head and neck, ranks sixth worldwide in terms of cancers with the most negative impact, owing to tumor relapse rates, cervical lymphnode metastasis, and the lack of an efficacious systemic therapy. Its prognosis is poor, and its mortality rate is high. Octamer-binding transcription factor 4 (OCT4) is a member of the Pit-Oct-Unc (POU) family and is a key reprogramming factor that produces a marked effect in preserving the pluripotency and self-renewal state of embryonic stem cells (ESCs). According to recent studies, OCT4 participates in retaining the survival of OSCC cancer stem cells (CSCs), which has far-reaching implications for the occurrence, recurrence, metastasis, and prognosis of oral carcinogenesis. Therefore, we summarize the structure, subtypes, and function of OCT4 as well as its role in the occurrence, progression, and prognosis of OSCC.

NELFA and BCL2 induce the 2C-like state in mouse embryonic stem cells in a chemically defined medium.

A minority of mouse embryonic stem cells (ESCs) display totipotent features resembling 2-cell stage embryos and are known as 2-cell-like (2C-like) cells. However, how ESCs transit into this 2C-like state remains largely unknown. Here, we report that the overexpression of negative elongation factor A (Nelfa), a maternally provided factor, enhances the conversion of ESCs into 2C-like cells in chemically defined conditions, while the deletion of endogenous Nelfa does not block this transition. We also demonstrate that Nelfa overexpression significantly enhances somatic cell reprogramming efficiency. Interestingly, we found that the co-overexpression of Nelfa and Bcl2 robustly activates the 2C-like state in ESCs and endows the cells with dual cell fate potential. We further demonstrate that Bcl2 overexpression upregulates endogenous Nelfa expression and can induce the 2C-like state in ESCs even in the absence of Nelfa. Our findings highlight the importance of BCL2 in the regulation of the 2C-like state and provide insights into the mechanism underlying the roles of Nelfa and Bcl2 in the establishment and regulation of the totipotent state in mouse ESCs.

The Trophoblast Compartment Helps Maintain Embryonic Pluripotency and Delays Differentiation towards Cardiomyocytes.

Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research.

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions.

Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naïve pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naïve state is impaired upon heme biosynthesis pathway blockade, linked in mESCs to the incapacity to activate MAPK- and TGFβ-dependent signaling pathways after succinate accumulation. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extracellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.

Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells

Embryonic stem cells (ESCs) are an attractive model to study the relationship between signaling and cell fates. Cultured mouse ESCs can exist in multiple states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. The signaling mechanisms regulating the totipotent state and coexistence of these states are poorly understood. Here we identify bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. However, we discover that BMP's role is constrained by the cross-activation of FGF, NODAL, and WNT pathways. We exploit this finding to enhance the proportion of totipotent cells by rationally inhibiting the cross-activated pathways. Single-cell mRNA sequencing reveals that induction of the totipotent state is accompanied by suppression of primed and primitive endoderm states. Furthermore, reprogrammed totipotent cells we generate in culture resemble totipotent cells of preimplantation embryo. Our findings reveal a BMP signaling mechanism regulating both the totipotent state and heterogeneity of ESCs.

A 1-kb human CDCA8 promoter directs the spermatogonia-specific luciferase expression in adult testis

Cell division cycle associated 8 (CDCA8) is a component of the chromosomal passenger complex and plays an essential role in mitosis, meiosis, cancer growth, and undifferentiated state of embryonic stem cells. However, its expression and role in adult tissues remain largely uncharacterized. Here, we studied the CDCA8 transcription in adult tissues by generating a transgenic mouse model, in which the luciferase was driven by a 1-kb human CDCA8 promoter. Our previous study showed that this 1-kb promoter was active enough to dictate reporter expression faithfully reflecting endogenous CDCA8 expression. Two founder mice carrying the transgene were identified. In vivo imaging and luciferase assays in tissue lysates revealed that CDCA8 promoter was highly activated and drove robust luciferase expression in testes. Subsequently, immunohistochemical and immunofluorescent staining showed that in adult transgenic testes, the expression of luciferase was restricted to a subset of spermatogonia that were located along the basement membrane and positive for the expression of GFRA1, a consensus marker for early undifferentiated spermatogonia. These findings for the first time indicate that the CDCA8 was transcriptionally activated in testis and thus may play a role in adult spermatogenesis. Moreover, the 1-kb CDCA8 promoter could be used for spermatogonia-specific gene expression in vivo and the transgenic lines constructed here could also be used for recovery of spermatogonia from adult testes.

Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast.

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.

Changes in the Expression of Pyruvate Dehydrogenase Kinase Isoenzymes During Early Differentiation of Mouse Embryonic Stem Cells

The embryonic stem cells (ESCs) are pluripotent, self-renewing cells that able to differentiate into any of the germ layers involved in embryogenesis. However, the molecular mechanisms that control ESC pluripotency and differentiation remain poorly understood. The family of Pyruvate dehydrogenase kinase (PDK1-4), inactivates the mitochondrial pyruvate dehydrogenase complex via phosphorylation, plays a crucial role in the control of glucose homeostasis. In the current study, gene expression levels of PDK isoenzymes were analyzed on undifferentiated mouse embryonic stem cells (mESCs) and compared to mESCs induced to differentiate by removal of leukemia inhibitory factor (LIF) for 1, 3 and 5 days. Besides, we performed gene expression analysis of several genes related to pluripotency and differentiation. In addition, we also determined glucose uptake rates by a colorimetric assay kit in early differentiated and undifferentiated mESCs. Differently expression level of PDK isoenzymes in pluripotent and LIF-depleted mESCs suggest that they may have roles in differentiation and pluripotency of ESCs. Furthermore, this study lays the foundation for detailed investigation of molecular mechanisms underlying the roles of PDKs in the pluripotency and transition to differentiated state of ESCs.

Single-cell mass cytometry analysis reveals stem cell heterogeneity.

Cellular heterogeneity is fundamental to both developmental differentiation and disease establishment. Recent advances in high-throughput single-cell technology have been rapidly revolutionizing the resolution of our understanding of development and disease. However, while the study of single-cell transcriptomes is easily accessible, the analysis of single-cell proteomes is still in its infancy. In this study, we describe simultaneous profiling of multiple regulatory proteins at a single-cell level using mass cytometry or cytometry by time of flight. We develop mass cytometry reagents to study key transcription factors, signaling proteins and chromatin modifiers that regulate mouse embryonic stem cells. Our data reveal that the protein level of stem cell regulators significantly varies and that cell signaling pathways are extensively cross-activated across defined culture conditions of embryonic stem cells. In addition, the mass cytometry data enabled us to identify distinct multiple cell states of embryonic stem cells and determine their variation across culture conditions. We discuss the mass cytometry method, our results of the multi-protein analysis in embryonic stem cells and potential future perspectives for single-cell protein analysis.

Abstract IA008: Chromatin regulation by reversible chromatin-associated RNA methylation

Abstract Over 150 types of post-transcriptional RNA modifications have been identified in all kingdoms of life. We have previously discovered two RNA demethylases, FTO and ALKBH5, which catalyze oxidative demethylation of the most prevalent modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). We have also characterized reader proteins that preferentially recognize methylated transcripts to facilitate either decay or translation of these target RNAs. Most recently we have discovered m6A methylation of chromatin-associated regulatory RNAs (carRNAs), which modulates chromatin state and transcription in mouse embryonic stem cells (mESCs). These findings suggest that reversible RNA modification could impact chromatin regulation analogous to the reversible DNA and histone chemical modifications. I will present recent mechanistic investigation of chromatin and transcription regulation through m6A methylation and demethylation, and impacts on mammalian development and cancer progression. Citation Format: Chuan He. Chromatin regulation by reversible chromatin-associated RNA methylation. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA008.

Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism.

Amino acids are crucial nutrients involved in several cellular and physiological processes, including fertilization and early embryo development. In particular, Leucine and Arginine have been shown to stimulate implantation, as lack of both in a blastocyst culture system is able to induce a dormant state in embryos. The aim of this work was to evaluate the effects of Leucine and Arginine withdrawal on pluripotent mouse embryonic stem cell status, notably, their growth, self-renewal, as well as glycolytic and oxidative metabolism. Our results show that the absence of both Leucine and Arginine does not affect mouse embryonic stem cell pluripotency, while reducing cell proliferation through cell-cycle arrest. Importantly, these effects are not related to Leukemia Inhibitory Factor (LIF) and are reversible when both amino acids are reconstituted in the culture media. Moreover, a lack of these amino acids is related to a reduction in glycolytic and oxidative metabolism and decreased protein translation in mouse embryonic stem cells (mESCs), while maintaining their pluripotent status.

Transcription factor antagonism regulates heterogeneity in embryonic stem cell states

Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.