scholarly journals Single-cell multiomic analysis of in vivo and in vitro matured human oocytes

2020 ◽  
Vol 35 (4) ◽  
pp. 886-900 ◽  
Author(s):  
Min Ye ◽  
Zhi-Yong Yang ◽  
Yu Zhang ◽  
Ya-Xin Xing ◽  
Qi-Gui Xie ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Methylation Level ◽  
Copy Number Variations ◽  
Cpg Methylation ◽  
Differentially Expressed ◽  
Sequencing Analysis ◽  
Human Oocytes

Abstract STUDY QUESTION Are there any differences between in vivo (IVV) and in vitro (IVT) matured metaphase II (MII) oocytes at the molecular level? SUMMARY ANSWER Between IVV and IVT oocytes, 507 differentially expressed genes (DEGs) were identified; the non-CpG methylomes were significantly different, but the CpG methylomes and genomic copy number variations (CNVs) were similar. WHAT IS KNOWN ALREADY A previous study using microarray and single-cell RNA-seq analysis revealed that numerous genes were differentially expressed between IVV and IVT oocytes. Independent studies of DNA methylation profiling in human oocytes have revealed negative correlations between gene transcription and the DNA methylation level at gene promoter regions. No study has compared global CpG or non-CpG methylation between these two groups of oocytes. Although a high level of aneuploidy has been reported in MII oocytes, no direct comparison of IVV and IVT oocytes based on single-cell sequencing data has been performed. STUDY DESIGN, SIZE, DURATION We collected eight IVV oocytes from six patients and seven IVT oocytes from seven patients and then analysed each oocyte using the previously established single-cell triple omics sequencing (scTrioseq) analysis to determine associations among the transcriptome, DNA methylome and chromosome ploidy in the oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS All IVV oocytes were donated by patients who received 150 IU gonadotropin per day from the third day of their menstrual cycle, followed by GnRH antagonist after 5 days of gonadotropin stimulation. All IVT oocytes were from immature oocytes which were donated by volunteers undergoing delivery by caesarean section then cultured in oocyte maturation medium containing 75 mIU/ml hMG for 24 to 48 h. Every single oocyte was analysed using the previously established single-cell multiomic sequencing analysis. MAIN RESULTS AND THE ROLE OF CHANCE There were 507 genes differentially expressed between the IVV (n = 8) and IVT (n = 7) oocytes, even though their global transcriptome profiles were similar. The enriched genes in IVV oocytes were related to the cell cycle process while those in IVT oocytes were related to mitochondrial respiration biogenesis. Although the global CpG methylation of the two groups of oocytes was similar, the non-CpG methylation level in IVV oocytes was higher than that in IVT oocytes. A high aneuploidy ratio was found in both groups, but the aneuploidy did not affect transcription according to the correlation analysis. LARGE-SCALE DATA N/A. LIMITATIONS AND REASONS FOR CAUTION Due to the difficulty in collecting MII oocytes, especially IVV matured oocytes, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS Our findings indicate that single-cell multiomic sequencing can be utilised to examine the similarity and differences between IVV and IVT matured MII oocytes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Ministry of Science and Technology of China, National Key R&D Program of China (No. 2017YFC1001601). The donated oocytes were collected by Shanghai Tenth People’s Hospital. The authors declare no competing interests.

scholarly journals Novel insights into the regulation of chemerin expression: role of acute-phase cytokines and DNA methylation

2019 ◽  
Author(s):  
Kamila Kwiecien ◽  
Piotr Brzoza ◽  
Pawel Majewski ◽  
Izabella Skulimowska ◽  
Kamil Bednarczyk ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Phase ◽  
Molecular Mechanisms ◽  
Retinoic Acid Receptor ◽  
Constitutive Expression ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
Pleiotropic Actions

AbstractChemerin is a chemoattractant protein with adipokine properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. It has gained more attention over the past few years due to its multilevel impact on metabolism and immune responses. The pleiotropic actions of chemerin include chemotaxis of dendritic cells, macrophages and natural killers (NK) subsets, bactericidal activity as well as regulation of adipogenesis and glucose metabolism. Therefore, reflecting the pleiotropic actions of chemerin, expression of RARRES2 is regulated by a variety of inflammatory and metabolic mediators. However, for most cell types, the molecular mechanisms controlling constitutive and regulated chemerin expression are poorly characterized. Here we show that RARRES2 mRNA levels in murine adipocytes are upregulated in vitro and in vivo by acute-phase cytokines, IL-1β and OSM. In contrast to adipocytes, these cytokines exerted a weak, if any, response in mouse hepatocytes, suggesting that the effect of IL-1β and OSM on chemerin expression is specific to fat tissue. Moreover, we show that DNA methylation controls the constitutive expression of chemerin. Bisulfite sequencing analysis showed low methylation levels within −735 to +258 bp of the murine RARRES2 gene promoter in unstimulated adipocytes and hepatocytes. In contrast to these cells, the RARRES2 promoter is highly methylated in B lymphocytes, cells that do not produce chemerin. Together, our findings reveal previously uncharacterized mediators and mechanisms controlling chemerin expression in various cells.

scholarly journals Genome-scale oscillations in DNA methylation during exit from pluripotency

2018 ◽  
Author(s):  
Steffen Rulands ◽  
Heather J Lee ◽  
Stephen J Clark ◽  
Christof Angermueller ◽  
Sébastien A Smallwood ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Epigenetic Mark ◽  
List Type ◽  
Dna Hypomethylation ◽  
Cell Fate Decisions ◽  
Oscillatory Dynamics ◽  
Genome Scale

SummaryPluripotency is accompanied by the erasure of parental epigenetic memory with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, coexpression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modelling, we show that this variability is associated with coherent, genome-scale, oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide fresh insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.HighlightsCo-expression of DNMT3s and TETs drive genome-scale oscillations of DNA methylationOscillation amplitude is greatest at a CpG density characteristic of enhancersCell synchronisation reveals oscillation period and link with primary transcriptsMultiomic single-cell profiling provides evidence for oscillatory dynamics in vivo

scholarly journals Progesterone-induced RNA Hand2os1 regulates decidualization in mice uteri

Reproduction ◽  
2020 ◽  
Vol 159 (3) ◽  
pp. 303-314
Author(s):  
Yanni Jia ◽  
Rui Cai ◽  
Tong Yu ◽  
Ruixue Zhang ◽  
Shouqin Liu ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Embryo Implantation ◽  
Pregnant Mouse ◽  
Differentially Expressed ◽  
Sequencing Analysis ◽  
Functional Roles ◽  
Decidual Cells ◽  
Functional Clustering Analysis

Decidualization is a critical process for successful embryo implantation and subsequent placenta formation. The characterization and physiological function of lncRNA during decidualization remain largely unknown. In the present study, we conducted RNA-sequencing analysis to compare gene expression between decidua of days 6 and 8, and normal pregnant endometrium (day 4). A total of 2332 high-confidence putative lncRNA transcripts were expressed. Functional clustering analysis of cis and trans lncRNA targets showed that differentially expressed lncRNAs may regulate multiple gene ontology terms and pathways that have important functions in decidualization. Subsequent analyses using qRT-PCR validated that eight of all lncRNAs were differentially regulated in mice uteri during decidualization, both in vivo and in vitro. Furthermore, we showed that differentially expressed lncRNA of Hand2os1 was specifically detected in stromal cells on days 2 to 5 of pregnancy and was strongly upregulated in decidual cells on days 6–8 of pregnancy. Similarly, Hand2os1 expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. In uterine stromal cells, P4 was able to significantly upregulate the expression of Hand2os1, but upregulation was impeded by RU486, whereas E2 appeared to have no regulating effect on Hand2os1 expression. Concurrently, Hand2os1 significantly promoted the decidual process in vitro and dramatically increased decidualization markers Prl8a2 and Prl3c1. Our results provide a valuable catalog for better understanding of the functional roles of lncRNAs in pregnant mouse uteri, as it relates to decidualization.

50 DNA METHYLATION PROFILES OF UPSTREAM ELEMENTS OF Oct-3/4 GENE IN IN VITRO FERTILIZATION (IVF) AND SOMATIC CELL NUCLEAR-TRANSFERRED (SCNT) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 143
Author(s):  
M. Kawasumi ◽  
Y. Unno ◽  
M. Nishiwaki ◽  
K. Matsumoto ◽  
M. Anzai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Methylation Level ◽  
Nuclear Transfer ◽  
Upstream Region ◽  
Sequencing Analysis ◽  
Vitro Fertilization ◽  
Cpg Sites

Cloning by adult somatic cell nuclear transfer (SCNT) has proven to be successful for the production of clones from many species (Keith 2004 Cytogenet. Genome Res. 105, 285). However, somatic cloning is currently highly inefficient. One of the reasons for this is that SCNT is believed to be associated with epigenetic errors including abnormal DNA methylation of the reconstructed embryo. The Oct-3/4 gene, a member of the POU transcription factor family, is expressed throughout the pre-implantation embryo. Abnormal expression of the Oct-3/4 gene in the nuclear-transferred embryo is either directly or indirectly caused by nuclear transfer and is suggested to be indicative of a general failure to reset the genetic program (Boiani et al. 2002 Genes Dev. 16, 1209). In this study, we investigated the DNA methylation profiles of the Oct-3/4 gene in the genome of SCNT embryos, using bisulfite sequencing analysis. Then, we observed the detailed subcellular localization of Oct-3/4 proteins in SCNT embryos using immunocytochemical (ICC) analysis. Nuclear transfer of cumulus cell nuclei was carried out as previously described (Wakayama et al. 1998 Nature 394, 369). After nuclear transfer, embryos were subsequently cultured in KSOM media to the morula and blastocyst stages. We compared the methylation profiles of 3 transcriptional control elements (distal enhancer, DE; proximal enhancer, PE; and promoter) of the upstream region of the Oct-3/4 gene with the genome of in vitro fertilization (IVF) and SCNT embryos. The methylation rate of CpG sites in the DE and promoter regions of both IVF and SCNT embryos was low at both the morula and the blastocyst stages. What's interesting is that there was a significant difference in the methylation level on CpG sites in the PE element between IVF and SCNT embryos. At the morula stage, the methylation level on CpG sites in the PE element was very low in the IVF embryo and moderately high in the SCNT embryo (0.9% and 26.3%). Conversely, at the blastocyst stage, CpG sites in the PE element showed high methylation in the IVF embryo and low methylation in the SCNT embryo (55.2% and 10.5%). CpG sites in the PE element were lightly methylated (3.0%) in the inner cell mass (ICM) of the IVF embryo. This means that the main portion of methylation in the IVF blastocyst embryo occurred at the trophectoderm (TE). On the other hand, in ICM of the SCNT embryo, the methylation level of each embryonic cell was almost the same in the whole blastocyst embryo (9.8% and 10.5%). As a result, it is highly possible that the CpG sites in the PE element of ICM were methylated as in the TE. ICC analysis revealed that some SCNT embryos showed aberrant Oct-3/4 expression in the TE. These results indicate that the methylation of CpG sites in the Oct-3/4 PE element may be related to expression of Oct-3/4 in the mouse IVF and SCNT embryos. These differences in methylation level between IVF and SCNT embryos were reflected as abnormal expressions of Oct-3/4 on SCNT embryos. This study was supported by the 21st COE Program of MEST. M.K. is a JSPS Research Fellow and supported by Grant-in Aid for Scientific Research (No. 1751132) of JSPS.

CBIO-14. SINGLE CELL RNA SEQUENCING ANALYSIS OF HUMAN GLIOBLASTOMA STEM-LIKE CELL CULTURES AND XENOGRAFT TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
Sonali Arora ◽  
Anca Mihalas ◽  
John Bassett ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Single Cell ◽  
Developmental Trajectories ◽  
Expression Patterns ◽  
Experimental Models ◽  
Tumor Formation ◽  
Sequencing Analysis ◽  
Rna Seq ◽  
Human Glioblastoma

Abstract Single cell RNA-seq (scRNA-seq) studies for glioma have yielded critical insight into intratumoral heterogeneity and developmental gene expression patterns for primary gliomas. One key conclusion from these studies is that each tumor represents a complex, yet maligned, neuro-developmental ecosystem, harboring diverse cell types, which presumably contribute to tumor growth and homeostasis in specific ways (e.g., vascular mimicry, immune evasion, recreating NSC niches, neural injury responses, etc.). Here, to better understand experimental models of human glioblastoma (GB), we performed single cell RNA-seq analysis of human GB stem-like cells (GSCs) of distinct tumor subtypes (mesenchymal and proneural) during their in vitro culture in serum-free conditions and also during tumor formation in immunocompromised mice. This analysis revealed surprising differences between in vitro and in vivo grown GSCs. Among our results, we find that in vivo mesenchymal GSCs are capable of transitioning to proneural-like states, while proneural GSCs are capable of transitioning to mesenchymal states. We characterize cycling cells based on expression of and G2/M and S phase makers, estimate RNA velocity, and examine different developmental trajectories arising in vitro and in vivo. We also compare and discuss different analysis pipelines for scRNA-seq data.

Single-Cell Analysis of Ploidy and Transcriptome Reveals Functional and Spatial Divergency in Murine Megakaryopoiesis

Blood ◽  
2021 ◽  
Author(s):  
Shu Sun ◽  
Chen Jin ◽  
Jia Si ◽  
Ying Lei ◽  
Kunying Chen ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Rapid Expansion ◽  
Cellular Heterogeneity ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Hsc Niche

Megakaryocytes (MKs), the platelet progenitor cell, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually-isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N), performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within three distinct subpopulations possessing gene signatures related to platelet-generation, HSC niche interaction, and inflammatory responses, respectively. In situ immunostaining of mouse BM demonstrated that platelet-generation and HSC-niche related MKs were physically in close proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under the blood shear forces, were predominantly formed on platelet-generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented approximately 5% of total MKs in the BM. These MKs could specifically respond to pathogen infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a pre-existing PU.1 and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance, as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable to engulf and digest bacteria and to stimulate T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

scholarly journals Alcohol promotes renal fibrosis by activating Nox2/4-mediated DNA methylation of Smad7

2020 ◽  
Vol 134 (2) ◽  
pp. 103-122 ◽  
Author(s):  
Qin Yang ◽  
Hai-Yong Chen ◽  
Jia-nan Wang ◽  
Huai-Qin Han ◽  
Ling Jiang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Methylation ◽  
Animal Model ◽  
Renal Fibrosis ◽  
Collagen I ◽  
Sequencing Analysis ◽  
Fibrotic Response ◽  
Kidney Fibrosis

Abstract Alcohol consumption causes renal injury and compromises kidney function. The underlying mechanism of the alcoholic kidney disease remains largely unknown. In the present study, an alcoholic renal fibrosis animal model was first employed which mice received liquid diet containing alcohol for 4 to 12 weeks. The Masson’s Trichrome staining analysis showed that kidney fibrosis increased at week 8 and 12 in the animal model that was further confirmed by albumin assay, Western blot, immunostaining and real-time PCR of fibrotic indexes (collagen I and α-SMA). In vitro analysis also confirmed that alcohol significantly induced fibrotic response (collagen I and α-SMA) in HK2 tubular epithelial cells. Importantly, both in vivo and in vitro studies showed alcohol treatments decreased Smad7 and activated Smad3. We further determined how the alcohol affected the balance of Smad7 (inhibitory Smad) and Smad3 (regulatory Smad). Genome-wide methylation sequencing showed an increased DNA methylation of many genes and bisulfite sequencing analysis showed an increased DNA methylation of Smad7 after alcohol ingestion. We also found DNA methylation of Smad7 was mediated by DNMT1 in ethyl alcohol (EtOH)-treated HK2 cells. Knockdown of Nox2 or Nox4 decreased DNMT1 and rebalanced Smad7/Smad3 axis, and thereby relieved EtOH-induced fibrotic response. The inhibition of reactive oxygen species by the intraperitoneal injection of apocynin attenuated renal fibrosis and restored renal function in the alcoholic mice. Collectively, we established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA methylation of Smad7. Suppression of Nox-mediated oxidative stress may be a potential therapy for long-term alcohol abuse-induced kidney fibrosis.

scholarly journals Inhibition of DNMT1 and ERRα crosstalk suppresses breast cancer via derepression of IRF4

Oncogene ◽  
2020 ◽  
Vol 39 (41) ◽  
pp. 6406-6420
Author(s):  
Mathieu Vernier ◽  
Shawn McGuirk ◽  
Catherine R. Dufour ◽  
Liangxinyi Wan ◽  
Etienne Audet-Walsh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Large Scale ◽  
Transcriptional Control ◽  
Critical Role ◽  
Promoter Hypermethylation ◽  
Sequencing Analysis

Abstract DNA methylation is implicated in the acquisition of malignant phenotypes, and the use of epigenetic modulating drugs is a promising anti-cancer therapeutic strategy. 5-aza-2’deoxycytidine (decitabine, 5-azadC) is an FDA-approved DNA methyltransferase (DNMT) inhibitor with proven effectiveness against hematological malignancies and more recently triple-negative breast cancer (BC). Herein, genetic or pharmacological studies uncovered a hitherto unknown feedforward molecular link between DNMT1 and the estrogen related receptor α (ERRα), a key transcriptional regulator of cellular metabolism. Mechanistically, DNMT1 promotes ERRα stability which in turn couples DNMT1 transcription with that of the methionine cycle and S-adenosylmethionine synthesis to drive DNA methylation. In vitro and in vivo investigation using a pre-clinical mouse model of BC demonstrated a clear therapeutic advantage for combined administration of the ERRα inhibitor C29 with 5-azadC. A large-scale bisulfite genomic sequencing analysis revealed specific methylation perturbations fostering the discovery that reversal of promoter hypermethylation and consequently derepression of the tumor suppressor gene, IRF4, is a factor underlying the observed BC suppressive effects. This work thus uncovers a critical role of ERRα in the crosstalk between transcriptional control of metabolism and epigenetics and illustrates the potential for targeting ERRα in combination with DNMT inhibitors for BC treatment and other epigenetics-driven malignancies.

Single-cell profiling of human subventricular zone progenitors identifies SFRP1 as a target for stimulating progenitor activation

2021 ◽  
Author(s):  
Vanessa Donega ◽  
Astrid van der Geest ◽  
Jacqueline Sluijs ◽  
Roland Van Dijk ◽  
Chi Chiu Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Subventricular Zone ◽  
Wnt Pathway ◽  
Oligodendrocyte Progenitor Cells ◽  
Quiescent State ◽  
Sequencing Analysis ◽  
Adult Human ◽  
Single Cell Profiling

Abstract Following the decline of neurogenesis at birth, progenitors of the subventricular zone (SVZ) remain mostly in a quiescent state in the adult human brain. The mechanisms that regulate this quiescent state are still unclear. Here, we isolated CD271+ progenitors from the aged human SVZ for single-cell RNA sequencing analysis. Our transcriptome data revealed the identity of progenitors of the aged human SVZ as late oligodendrocyte progenitor cells. We identified the Wnt pathway antagonist SFRP1 as a possible signal that promotes quiescence of progenitors from the aged human SVZ. Administration of WAY-316606, a small molecule that inhibits SFRP1 function, stimulates activation of neural stem cells both in vitro and in vivo under homeostatic conditions. Our data unravel a possible mechanism through which progenitors of the adult human SVZ are maintained in a quiescent state and a potential target for stimulating progenitors to re-activate.

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