96 AN IMMUNOHISTOCHEMICAL STUDY ON MARKERS OF PLURIPOTENCY AND DNA METHYLATION IN THE DEVELOPING PORCINE GERM LINE

2009 ◽  
Vol 21 (1) ◽  
pp. 148
Author(s):  
M. Vejlsted ◽  
S. M. W. Hyldig ◽  
P. Maddox-Hyttel
Keyword(s):  
Dna Methylation ◽  
Germ Cells ◽  
Germ Line ◽  
Immunohistochemical Study ◽  
Methylation Status ◽  
Dna Demethylation ◽  
Santa Cruz ◽  
The Third ◽  
Pluripotency Markers ◽  
Gestational Week

Reprogramming of the germ line genome is a fundamental molecular process involving DNA demethylation. This has been demonstrated in the mouse (Seki Y et al. 2005 Dev. Biol. 278, 440–458), but has not yet been studied in the pig. From a large collection of porcine embryos/fetuses 2 to 7 weeks post-insemination (p.i.), a total of 35 randomly selected specimens from the end of the second (n = 10), third (n = 10), fourth (n = 5), and seventh (n = 10) gestational week were selected for an immunohistochemical study on pluripotency markers and DNA methylation in the developing germ line. Intact embryos and isolated developing gonads were paraffin embedded, sectioned (5 to 15 μm) and evaluated for the expression of markers of pluripotency [OCT4 (sc-8628, Santa Cruz Biotech., Santa Cruz, CA), Nanog (500-P236, PeproTech EC, Rocky Hill, NJ), and SOX2 (MAB2018, R&D Systems, Wiesbaden, Germany)], DNA methylation [5-methyl cytidine (ab10805, Abcam, Cambridge, MA)], and meiosis [SCP-3 (generous gift from C. Heyting)]. Heat-induced epitope retrieval (HIER) in an alkaline (pH 8.2) EDTA buffer (Shi SR et al. 2001 J. Histochem. Cytochem. 49, 931–937) and confocal laser scanning microscopy allowed for the evaluation of germ cells co-expressing OCT4 and 5-methyl cytidine. The expression of Nanog and SOX2 was found to be better visualized using HIER in an acidic (pH 6.0) citrate buffer. Isolated and clustered primordial germ cells (PGC) were identified by OCT4 labeling early during gastrulation in embryos around 2 weeks of age p.i. The amount of methylated DNA in PGC appeared similar to that in the nuclei of neighboring somatic cells at this stage. During colonization of the genital ridges, in embryos at the end of the third gestational week, this global DNA methylation status seemed to markedly decrease in PGC, remaining low in the gonadal maturing germ cells. Around onset of meiosis, in fetuses at the seventh gestational week, germ cells in 3 out of 5 female specimens studied had ceased to express markers of pluripotency. In contrast, such markers appeared to be retained in germ cells of male siblings. In conclusion, expression of pluripotency markers during porcine germ line development appears similar to what has been described in the mouse with expression ceasing at the beginning of meiosis in the female but not in the male fetus. Further, the timing of germ line DNA demethylation appears similar between the 2 species. In the mouse, PGC entering the genital ridges soon initiate meiosis, whereas in the pig, these events are separated by a 3-week period. The connection between porcine germ line pluripotency and DNA methylation status during the third to fourth week of development p.i. is presently being thoroughly investigated.

scholarly journals Title Page Title: The role of DNA methylation in the accumulation of iridoid glycosides in Rehmannia glutinosa

2021 ◽  
Author(s):  
Tianyu Dong ◽  
Xiaoyan Wei ◽  
Qianting Qi ◽  
Peilei Chen ◽  
Yanqing Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Secondary Metabolites ◽  
Iridoid Glycosides ◽  
Methylation Status ◽  
Plant Secondary Metabolites ◽  
Dna Demethylation ◽  
Terpene Biosynthesis ◽  
The Relationship

Abstract Background: Epigenetic regulation plays a significant role in the accumulation of plant secondary metabolites. The terpenoids are the most abundant in the secondary metabolites of plants, iridoid glycosides belong to monoterpenoids which is one of the main medicinal components of R.glutinosa. At present, study on iridoid glycosides mainly focuses on its pharmacology, accumulation and distribution, while the mechanism of its biosynthesis and the relationship between DNA methylation and plant terpene biosynthesis are seldom reports. Results: The research showed that the expression of DXS, DXR, 10HGO, G10H, GPPS and accumulation of iridoid glycosides increased at first and then decreased with the maturity of R.glutinosa, and under different concentrations of 5-azaC, the expression of DXS, DXR, 10HGO, G10H, GPPS and the accumulation of total iridoid glycosides were promoted, the promotion effect of low concentration (15μM-50μM) was more significant, the content of genomic DNA 5mC decreased significantly, the DNA methylation status of R.glutinosa genomes was also changed. DNA demethylation promoted gene expression and increased the accumulation of iridoid glycosides, but excessive demethylation inhibited gene expression and decreased the accumulation of iridoid glycosides. Conclusion: The analysis of DNA methylation, gene expression, and accumulation of iridoid glycoside provides insights into accumulation of terpenoids in R.glutinosa and lays a foundation for future studies on the effects of epigenetics on the synthesis of secondary metabolites.

scholarly journals The H19 Differentially Methylated Region Marks the Parental Origin of a Heterologous Locus without Gametic DNA Methylation

2004 ◽  
Vol 24 (9) ◽  
pp. 3588-3595 ◽  
Author(s):  
Kye-Yoon Park ◽  
Elizabeth A. Sellars ◽  
Alexander Grinberg ◽  
Sing-Ping Huang ◽  
Karl Pfeifer
Keyword(s):  
Dna Methylation ◽  
Mouse Chromosome ◽  
Germ Line ◽  
Transcriptional Silencing ◽  
Chromosome 7 ◽  
Differentially Methylated Region ◽  
Parental Origin ◽  
Imprinted Genes ◽  
Chromosome 5 ◽  
The Third

ABSTRACT Igf2 and H19 are coordinately regulated imprinted genes physically linked on the distal end of mouse chromosome 7. Genetic analyses demonstrate that the differentially methylated region (DMR) upstream of the H19 gene is necessary for three distinct functions: transcriptional insulation of the maternal Igf2 allele, transcriptional silencing of paternal H19 allele, and marking of the parental origin of the two chromosomes. To test the sufficiency of the DMR for the third function, we inserted DMR at two heterologous positions in the genome, downstream of H19 and at the alpha-fetoprotein locus on chromosome 5. Our results demonstrate that the DMR alone is sufficient to act as a mark of parental origin. Moreover, this activity is not dependent on germ line differences in DMR methylation. Thus, the DMR can mark its parental origin by a mechanism independent of its own DNA methylation.

Effects of ten–eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells

2019 ◽  
Vol 31 (3) ◽  
pp. 509 ◽  
Author(s):  
Minli Yu ◽  
Dongfeng Li ◽  
Wanyan Cao ◽  
Xiaolu Chen ◽  
Wenxing Du
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Germ Cells ◽  
Dna Methyltransferase ◽  
Primordial Germ Cells ◽  
Dna Demethylation ◽  
Caveolin 1 ◽  
Expression Of Genes ◽  
Deleted In Azoospermia

Ten–eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5–E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

DNA Demethylation By Activation-Induced Cytidine Deaminase in B Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3549-3549
Author(s):  
Yang Xi ◽  
Velizar Shivarov ◽  
Gur Yaari ◽  
Steven Kleinstein ◽  
Matthew P. Strout
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Repair ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Methylation Status ◽  
B Cell Lymphoma ◽  
Dna Demethylation ◽  
Cpg Sites ◽  
Average Beta

Abstract DNA methylation and demethylation at cytosine residues are epigenetic modifications that regulate gene expression associated with early cell development, somatic cell differentiation, cellular reprogramming and malignant transformation. While the process of DNA methylation and maintenance by DNA methyltransferases is well described, the nature of DNA demethylation remains poorly understood. The current model of DNA demethylation proposes modification of 5-methylcytosine followed by DNA repair-dependent cytosine substitution. Although there is debate on the extent of its involvement in DNA demethylation, activation-induced cytidine deaminase (AID) has recently emerged as an enzyme that is capable of deaminating 5-methylcytosine to thymine, creating a T:G mismatch which can be repaired back to cytosine through DNA repair pathways. AID is expressed at low levels in many tissue types but is most highly expressed in germinal center B cells where it deaminates cytidine to uracil during somatic hypermutation and class switch recombination of the immunoglobulin genes. In addition to this critical role in immune diversification, aberrant targeting of AID contributes to oncogenic point mutations and chromosome translocations associated with B cell malignancies. Thus, to explore a role for AID in DNA demethylation in B cell lymphoma, we performed genome-wide methylation profiling in BL2 and AID-deficient (AID-/-) BL2 cell lines (Burkitt lymphoma that can be induced to express high levels of AID). Using Illumina’s Infinium II DNA Methylation assay combined with the Infinium Human Methylation 450 Bead Chip, we analyzed over 450,000 methylation (CpG) sites at single nucleotide resolution in each line. BL2 AID-/- cells had a median average beta value (ratio of the methylated probe intensity to overall intensity) of 0.76 compared with 0.73 in AID-expressing BL2 cells (P < 0.00001), indicating a significant reduction in global methylation in the presence of AID. Using a delta average beta value of ≥ 0.3 (high stringency cut-off whereby a difference of 0.3 or more defines a CpG site as hypomethylated), we identified 5883 CpG sites in 3347 genes that were hypomethylated in BL2 versus BL2 AID-/- cells. Using the Illumina HumanHT-12 v4 Expression BeadChip and Genome Studio software, we then integrated gene expression and methylation profiles from both lines to generate a list of genes that met the following criteria: 1) contained at least 4 methylation sites within the first 1500 bases downstream of the primary transcriptional start site (TSS 1500; AID is most active in this region during somatic hypermutation); 2) average beta value increased by >0.1 in the TSS 1500 region in BL2 compared with BL2 AID-/- cells; and 3) down-regulated by >50% in BL2 compared with BL2 AID-/- cells. This analysis identified 31 candidate genes targeted for AID-dependent demethylation with consequent changes in gene expression. Interestingly, 15 of these genes have been reported to be bound by AID in association with stalled RNA polymerase II in activated mouse B cells. After validating methylation status in a subset of genes (APOBEC3B, BIN1, DEM1, GRN, GNPDA1) through bisulfite sequencing, we selected DEM1 for further analysis. DEM1 encodes an exonuclease involved in DNA repair and contains 16 CpG sites within its TSS1500, with only one site >50% methylated in BL2 cells compared with 8 of 16 in BL2 AID-/- cells. To assess a direct role for AID in DEM1 methylation status, a retroviral construct (AIDΔL189-L198ER) driving tamoxifen-inducible expression of a C-terminal deletion mutant of AID (increases time spent in the nucleus) was introduced into BL2 AID-/- cells. BL2, BL2 AID-/-, and BL2 AIDΔL189-L198ER cells were cultured continuously for 21 days in the presence of tamoxifen, 100 nM. Bisulfite sequencing of DEM1 TSS 1500 did not demonstrate any significant changes in methylation at day 7. However, at day 21, 13 of the 16 DEM1 TSS 1500 methylation sites in BL2 AIDΔL189-L198ER cells were found to have an increase in the ratio of unmethylated to methylated clones ~10-25% above that of BL2 AID-/- cells. By qPCR, this correlated with a 1.75-fold increase in DEM1 gene expression to levels that were equivalent to that seen in BL2 cells (P = 0.003). Although further investigations are needed, this data supports the notion that AID is able to regulate target gene expression in B cell malignancy through active DNA demethylation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transposable elements resistant to epigenetic resetting in the human germline are epigenetic hotspots for development and disease

2020 ◽  
Author(s):  
Sabine Dietmann ◽  
Michael J Keogh ◽  
Walfred Tang ◽  
Erna Magnusdottir ◽  
Toshihiro Kobayashi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cerebral Cortex ◽  
Transposable Elements ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Dna Demethylation ◽  
Hair Color ◽  
Epigenetic Changes ◽  
Early Human ◽  
Development And Evolution

ABSTRACTDespite the extensive erasure of DNA methylation in the early human germline, nearly eight percent of CpGs are resistant to the epigenetic resetting in the acutely hypomethylated primordial germ cells (week 7-9 hPGCs). Whether this occurs stochastically or represents relatively conserved layer of epigenetic information is unclear. Here we show that several predominantly hominoid-specific families of transposable elements (TEs) consistently resist DNA demethylation (henceforth called hPGC-methylated TEs or ‘escapees’) during the epigenetic resetting of hPGCs. Some of them undergo subsequent dynamic epigenetic changes during embryonic development. Our analysis of the fetal cerebral cortex also revealed multiple classes of young hPGC-methylated TEs within putative and established enhancers. Remarkably, specific hPGC-methylated TE subfamilies were associated with a multitude of adaptive human traits, including hair color and intelligence, and diseases including schizophrenia and Alzheimer’s disease. We postulate that hPGC-methylated TEs represent potentially heritable information within the germline with a role in human development and evolution.

scholarly journals Azacitidine Blocks GATA-1-Mediated Repression of the PU.1 Gene in Human Leukemic Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5220-5220
Author(s):  
Pavel Burda ◽  
Jarmila Vargova ◽  
Nikola Curik ◽  
John Strouboulis ◽  
Giorgio Lucio Papadopoulos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Regulatory Element ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Leukemic Cells ◽  
Epigenetic Therapy ◽  
Myeloid Differentiation

Abstract Introduction: GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. Expression of PU.1 is controlled by several transcription factors including PU.1 itself by binding to the distal URE enhancer (upstream regulatory element) whose deletion leads to acute myeloid leukemia (AML) (Rosenbauer F et al. 2004). Co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents efficient differentiation regulated by these transcription factors. Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000), however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. We have recently found that MDS/AML erythroblasts display repressive histone modifications and DNA methylation status of PU.1 gene that respond to 5-azacitidine (AZA) leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). We hypothesize that l eukemia blockade during early erythroid differentiation includes direct GATA-1-mediated inhibition of the PU.1 gene. Results: We herein document the GATA-1 mediated repression of the PU.1 gene in human EL cell lines (OCI-M2 and K562) together with the recruitment of DNA methyl transferase I (DNMT1) to the URE known to guide most of the PU.1 gene transcription. Repression of the PU.1 gene involves both DNA methylation at the URE and methylation/deacetylation of the histone H3 lysine-K9 residue and methylation of H3K27 at additional DNA elements and the PU.1 promoter. Inhibition of GATA-1 by siRNA as well as the AZA treatment in AML-EL led to the significant DNA-demethylation of the URE thorough the mechanism of DNMT1 depletion leading to upregulation of the PU.1 expression. Conclusions: Our data indicate that GATA-1 binds to the PU.1 gene at the URE and initiate events leading to the PU.1 gene repression in human ELs. The mechanism includes repressive epigenetic remodeling of the URE that is important for the PU.1 downregulation and leukemogenesis and that is also simultaneously sensitive to the DNA demethylation treatment with AZA. The GATA-1-mediated inhibition likely contributes to the PU.1 downregulation during progenitor cell differentiation that could be employed during leukemogenesis. Importantly, we also observed important differences between murine and human ELs and found that repression of the PU.1 gene in human ELs can become reverted by the epigenetic therapy with AZA. Our work also suggests that hypomethylating therapy using DNA methylation inhibitors in MDS/AML may become potentially effective in MDS/EL patients. We think that during early erythroid differentiation the GATA-1 binds and represses the PU.1 gene, however this is not fully completed in EL and therefore the erythroid as well as myeloid differentiation are blocked. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. Disclosures Off Label Use: Azacitidine, DNA demethylation agens tested in vitro in AML/MDS treatment. Stopka:Celgene: Research Funding.

Dynamic Changes Of DNA Methylation and a Functional Role For TET2 DNA Dioxygenase In Human Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3415-3415
Author(s):  
Jie Li ◽  
Papoin Julien ◽  
Chao An ◽  
Jingpin Hu ◽  
Ari Melnick ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Functional Role ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Parent Cell ◽  
Hematopoietic Stem ◽  
Mature Erythrocyte ◽  
Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. This process contains eight distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. Unlike most cell types, an important feature of erythropoiesis is that following each of the three or four mitoses that occur during terminal erythroid differentiation, the daughter cells are distinctly different from the parent cell from which they are derived. Thus, erythropoiesis is a complex process that requires tight regulation. The most extensively studied regulators of erythroid differentiation include the EPO/EPOR system and two major transcription factors, GATA1 and KLF1. In contrast to the well-established roles of growth factors, cytokines and transcription factors in regulating erythropoiesis, the regulation of erythropoiesis by other mechanisms is much less understood. In the present study, we explore the changes in DNA methylation during human terminal erythroid differentiation and DNA methylation/demethylation in human erythropoiesis. The methylation status of DNA influences many biologic processes. It has been recently reported that global demethylation occurs during both murine and human erythropoiesis. However, the dynamics of DNA methylation changes, the underlying molecular mechanism(s), and the function of DNA demethylation in erythropoiesis are not clear. To address these issues, we performed next-generation bisulfite sequencing on highly purified human erythroblasts at distinct differentiation stages. We show that while there is a global hypomethylation as terminal erythropoiesis proceeds, stage-specific analysis revealed that a significant proportion of differential methylation includes gains of methylation. Moreover, genes that presented with DNA methylation changes could be categorized into 3 groups based on the dynamics of their methylation changes. As Ten-eleven-translocation proteins (TETs) have been implicated in DNA demethylation by converting 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), we attempted to explore the role of TETs in DNA demethylation and terminal erythroid differentiation. We show that 5hmc is progressively increased during human terminal erythroid differentiation. Importantly, knockdown of TET2 by shRNA in human CD34+ cells impaired the production of 5hmc as well as terminal erythroid differentiation. Our findings demonstrate the complexity of DNA methylation dynamics and identify a functional role for TET2 in human erythroid differentiation. These findings provide new and novel insights into the mechanistic understanding of normal and disordered erythropoiesis. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, we suggest that these finding also provide novel insights into these diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Two types of pole cells are present in the Drosophila embryo, one with and one without splicing activity for the third P-element intron

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 885-893 ◽  
Author(s):  
S. Kobayashi ◽  
T. Kitamura ◽  
H. Sasaki ◽  
M. Okada
Keyword(s):  
Germ Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Egg Laying ◽  
P Element ◽  
Drosophila Embryo ◽  
The Third ◽  
Pole Cells ◽  
Almost All ◽  
Second Peak

In Drosophila, it has been postulated that the third intron of the P-element is spliced only in germ-line cells. To test whether this postulate is applicable to pole cells, the progenitor cells of germ line, we carried out a histochemical assay to detect the splicing activity in embryos. The splicing activity was detected in pole cells and primordial germ cells. The activity increased to reach a maximum at 5–6 hours AEL (after egg laying), then decreased to an undetectable level by 8–9 hours AEL. The splicing activity showed a small second peak at 12–15 hours AEL. It was rather unexpected that not all pole cells were capable of splicing the third intron. Almost all pole cells that had the splicing activity at 5–6 hours AEL penetrated the embryonic gonads and differentiated into primordial germ cells. Our findings suggest that pole cells are selected to penetrate the gonads while they are migrating from the proctodeal cavity to the gonads. Furthermore, these results suggest that the machinery to splice the P-element is active in some pole cells, and that this activity is used for processing transcripts of genes that play important roles in the differentiation of pole cells into primordial germ cells.

scholarly journals TDG regulates cell cycle progression in human neural progenitors

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 497
Author(s):  
Igal Germanguz ◽  
Jenny C. Park ◽  
Jessica Cinkornpumin ◽  
Aryeh Solomon ◽  
Minori Ohashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Methylation Status ◽  
Human Pluripotent Stem Cells ◽  
Dna Demethylation ◽  
Neural Progenitors ◽  
Specific Cell ◽  
Active Demethylation

Background: As cells divide, they must both replicate their DNA and generate a new set of histone proteins.  The newly synthesized daughter strands and histones are unmodified, and must therefore be covalently modified to allow for transmission of important epigenetic marks to daughter cells.  Human pluripotent stem cells (hPSCs) display a unique cell cycle profile, and control of the cell cycle is known to be critical for their proper differentiation and survival.  A major unresolved question is how hPSCs regulate their DNA methylation status through the cell cycle, namely how passive and active demethylation work to maintain a stable genome. Thymine-DNA glycosylase (TDG), an embryonic essential gene, has been recently implicated as a major enzyme involved in demethylation. Methods: We use human pluripotent stem cells and their derivatives to investigate the role of TDG in differentiation and proliferation.  To perform loss of function of TDG, RNA Interference was used.  To study the cell cyle, we engineered human pluripotent stem cells to express the FUCCI tool which marks cells at various stages of the cell cycle with distinct patterns of fluorescent proteins.  We also used cell cycle profiling by FACS, and DNA methylation analysis to probe a connection between DNA demethylation and cell cycle. Results: Here we present data showing that TDG regulates cell cycle dynamics in human neural progenitors (NPCs) derived from hPSCs, leading to changes in  cell cycle related gene expression and neural differentiation capacity.  These data show that loss of TDG function can block differentiation by driving proliferation of neural progenitors.  We also identify specific cell cycle related genes whose expression changes upon loss of TDG expression. Conclusions: These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.

42 DEVELOPMENTAL POTENTIAL AND REPROGRAMMING EFFICIENCY OF BOVINE EMBRYOS CLONED WITH ADULT FIBROBLASTS TREATED BY A DEMETHYLATING AGENT, S-ADENOSYL-HOMOCYSTEINE

2006 ◽  
Vol 18 (2) ◽  
pp. 130 ◽  
Author(s):  
B.-G. Jeon ◽  
S. D. Perrault ◽  
G.-J. Rho ◽  
D. H. Betts ◽  
W. A. King
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Somatic Cells ◽  
Telomerase Activity ◽  
Dna Demethylation ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Reprogramming Efficiency ◽  
Low Efficiency

Animal cloning by somatic cell nuclear transfer (SCNT) has been successfully applied to several species although with low efficiency and often associated with severe abnormalities. These poor outcomes are thought to be a consequence of aberrant DNA methylation patterns that result from incomplete epigenetic reprogramming of the transplanted nucleus into recipient oocytes. Telomerase, an enzyme not expressed in most somatic cells, should be expressed in cloned embryos. Therefore its activity has been used as an index of reprogramming in SCNT embryos. The objective of this study was to investigate the DNA methylation status of donor fibroblasts treated with a non-cytotoxic transmethylation inhibitor, S-adenosyl homocysteine (SAH), and to assess the relative telomerase activity (RTA) and developmental potential of SCNT embryos derived from such cells. Adult ear skin fibroblasts were cultured in DMEM supplemented with 0, 0.5, 1.0, or 2.0 mM SAH for 144 h by daily media change prior to nuclear transfer. The SAH-treated fibroblasts were immunostained with a fluorescein isothiocyanate (FITC) conjugated 5-methylcytosine antibody and the relative fluorescence intensity (RFI) was analyzed using a fluorescence microscope equipped with an Openlab" program (Improvision, Coventry, UK). RTA was measured in Day 8 SCNT blastocysts using the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP). Fibroblasts treated with 0.5, 1.0, and 2.0 mM SAH showed lower levels of DNA methylation compared to nontreated controls, and the values did not differ among the treatment groups. Cleavage rates did not differ between the SCNT embryos derived from 0.5 mM SAH-treated cells and nontreated control cells (92.3% vs. 91.3%, respectively). However, the rates of blastocyst development and hatching were significantly (P < 0.05) higher in SCNT embryos derived from 0.5 mM SAH treated donor cells compared to controls (60.0 and 40.0% vs. 34.3 and 26.4%, respectively). Moreover, RTA of the 0.5 mM SAH SCNT embryos was significantly (P < 0.05) increased (1.5-fold) in relation to controls. S-adenosyl homocysteine treatment induces global DNA demethylation in donor fibroblasts and enhances the blastocyst frequencies for bovine SCNT embryos that also exhibit greater telomerase activity levels. These results suggest that use of hypomethylated donor somatic cells increases the developmental potential for SCNT embryos by enhancing the nuclear reprogramming efficiency. This work was funded by NSERC, OMAF, OCAG, and CRC.

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