Methylation patterns in 5' terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rexl and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rexl and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rexl respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion,the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bo-vine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.     

Methylation patterns in 5＇ terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rex1 and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rex1 respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion, the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bovine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.     

The regulatory role of histone deacetylase inhibitors in Fgf4 expression is dependent on the differentiation state of pluripotent stem cells

The identity of embryonic stem cells (ESCs) is controlled by a set of pluripotency genes, including Oct4, Sox2, Nanog, and Fgf4. How their expression is repressed during differentiation and reactivated during reprogramming is largely unknown. Here, using mouse ESCs as well as F9 and P19 cells (mouse embryonal carcinoma cell lines, P19 being considered further differentiated than F9 cells) as models, we found that HDAC inhibitors elevated Fgf4 expression in P19 cells, but reduced it in F9 cells. We also observed that HDAC inhibitors enhanced the expression of Fgf4 and a subset of pluripotency genes in differentiated ESCs, but reduced their expression in undifferentiated and less differentiated ESCs. Mechanistically, we observed more HDAC1 recruitment and a weaker association of histone 4 lysine 5 acetylation at the Fgf4 enhancer in P19 cells compared to F9 cells. Additionally, we demonstrated the interaction between Sox2 and HDAC1 both in vitro and in vivo, implicating a possible role for Sox2 in the recruitment of HDAC1 to the Fgf4 enhancer. We also found that Nanog bound to the Fgf4 enhancer, and this binding was stronger in F9 cells, indicating the involvement of Nanog in the regulation of Fgf4 expression in undifferentiated and less differentiated pluripotent stem cells. This study uncovers an important role of HDAC1 and histone modifications in the repression of Fgf4 and perhaps other pluripotency genes during ESC differentiation. Our results also suggest that HDAC inhibitors may promote reprogramming partially through activating pluripotency genes at some intermediate stages.     

FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na?ve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.     

Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation

We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on approximately 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression.     

Efficient derivation of human embryonic stem cell lines from discarded embryos through increases in the concentration of basic fibroblast growth factor

We describe the derivation and characterization of three novel human embryonic stem (hES) cell lines (YT1, YT2, YT3). One hES line (YT1) was obtained from six discarded blastocysts in a culture medium supplemented with 12 ng/ml basic fibroblast growth factor (bFGF), and two lines (YT2,YT3)were obtained from three discarded blastocysts in the same medium but supplemented with 16 ng/ml bFGF. These cell lines were derived by partial or whole embryo culture followed by further expansion after manual dissection of the passaged cells. These cells were passaged continuously for more than 6 or 8 months and possessed all of the typical features of pluripotent hES cell lines, such as typical morphological characteristics and the expression of hES-specific markers (TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, alkaline phosphatase, Oct4, Nanog) and pluripotency-related genes (Oct4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex1). The lines maintained normal karyotypes after long-term cultivation. The karyotype of YT1 and YT3 was 46,XX, and that of YT2 was 46, XY. Pluripotency was confirmed by in vitro and in vivo differentiation, and genetic identity was demonstrated by DNA fingerprinting.Our results indicate that higher concentrations of bFGF at the early culture stage support efficient the hES cell derivation.     

不同培养条件对鸡胚胎生殖细胞Oct4启动子DNA甲基化的影响

旨在在体外通过胚胎生殖细胞(EGC)和细胞外基质共同构建一个EGC的小生境(niche)模型,通过比较Oct4DNA甲基化的变化,研究niche中特有的表观遗传效应对胚胎生殖细胞自我更新的影响.构建EGC细胞标准培养方案(对照组)和改良培养方案(试验组),采用甲基化特异性PCR( MS-PCR)技术、RT-PCR技术,对比分析两种培养方案中Oct4启动子的甲基化状态,判断基因表达与其CpG岛甲基化的关系,分析DNA甲基化模式与EGC细胞自我更新的关系.结果显示,试验组可扩增出Oct4的非甲基化扩增产物,而对照组为其甲基化扩增产物,试验组Oct4表达水平明显高于对照组.试验组EGC生长状态明显好于对照组.试验表明,在改良培养条件下,Oct4基因启动子DNA甲基化程度较低,且与基因表达水平呈负相关,更有利于维持胚胎生殖细胞的自我更新状态.     

Identification and characterization of subpopulations in undifferentiated ES cell culture

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells. The Rex1(-)/Oct3/4(+) and Rex1(+)/Oct3/4(+) populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells have characteristics similar to those of ICM and early-PrE cells, Rex1(+)/Oct3/4(+) cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1(-)/Oct3/4(+) cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.     

Oct4 and nanog directly regulate dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreased?proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.