Yong-Kook Kang

Limited demethylation leaves mosaic‐type methylation states in cloned bovine pre‐implantation embryos

Cloning by nuclear transfer (NT) has been riddled with difficulties: most clones die before birth and survivors frequently display growth abnormalities. The cross‐species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the fidelity of epigenetic reprogramming of the donor genome. Here, we found that single‐copy sequences, unlike satellite sequences, are demethylated in pre‐implantation NT embryos. The differential demethylation pattern between genomic sequences was confirmed by analyzing single blastocysts. It suggests selective demethylation of other developmentally important genes in NT embryos. We also observed a reverse relationship between methylation levels and inner cell mass versus trophectoderm (ICM/TE) ratios, which was found to be a result of another type of differential demethylation occurring in NT blastocysts where unequal methylation was maintained between ICM and TE regions. TE‐localized methylation aberrancy suggests a widespread gene dysregulation in an extra‐embryonic region, thereby resulting in placental dysfunction familiar to cloned fetuses/animals. These differential demethylations among genomic sequences and between differently allocated cells produce varied overall, but specified, methylation patterns, demonstrating that epigenetic reprogramming occurs in a limited fashion in NT embryos.

Aberrant Allocations of Inner Cell Mass and Trophectoderm Cells in Bovine Nuclear Transfer Blastocysts

Abstract Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20–40%, III: 40–60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.

Inheritable histone H4 acetylation of somatic chromatins in cloned embryos

A viable cloned animal indicates that epigenetic status of the differentiated cell nucleus is reprogrammed to an embryonic totipotent state. However, molecular events regarding epigenetic reprogramming of the somatic chromatin are poorly understood. Here we provide new insight that somatic chromatins are refractory to reprogramming of histone acetylation during early development. A low level of acetylated histone H4-lysine 5 (AcH4K5) of the somatic chromatin was sustained at the pronuclear stage. Unlike in vitro fertilized (IVF) embryos, the AcH4K5 level remarkably reduced at the 8-cell stage in cloned bovine embryos. The AcH4K5 status of somatic chromatins transmitted to cloned and even recloned embryos. Differences of AcH4K5 signal intensity were more distinguishable in the metaphase chromosomes between IVF and cloned embryos. Two imprinted genes, Ndn and Xist, were aberrantly expressed in cloned embryos as compared with IVF embryos, which is partly associated with the AcH4K5 signal intensity. Our findings suggest that abnormal epigenetic reprogramming in cloned embryos may be because of a memory mechanism, the epigenetic status itself of somatic chromatins.

Nuclear reprogramming of cloned embryos produced in vitro.

Despite the fact that cloned animals derived from somatic cells have been successfully generated in a variety of mammalian species, there are still many unsolved problems with current cloning technology. Somatic cell nuclear transfer has shown several developmental aberrancies, including a high rate of abortion during early gestation and increased perinatal death. One cause of these developmental failures of cloned embryos may reside in the epigenetic reprogramming of somatic donor genome. In mammals, DNA methylation is an essential process in the regulation of transcription during embryonic development and is generally associated with gene silencing. A genome-wide demethylation may be a prerequisite for the formation of pluripotent stem cells that are important for later development. We analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant methylation profiles of cloned bovine embryos were observed in various genomic regions, except in single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in vitro or in vivo. These results suggest that the developmental failures of cloned embryos may be due to incomplete epigenetic reprogramming of donor genomic DNA. We expect that advances in understanding the molecular events for reprogramming of donor genome will contribute to clarify the developmental defects of cloned embryos.

In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer

Abstract This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 ± 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 ± 2.3%) (P < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 ± 2.9, 6.6 ± 2.3, and 8.1 ± 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 ± 10.4 > 24.6 ± 10.1 > 16.5 ± 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P < 0.05). Nuclear-transferred embryos (9.4 ± 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 ± 1.9%) or parthenogenetic embryos (22.4 ± 7.2%) (P < 0.01). The mean cell number (28.9 ± 11.4) of NT-derived blastocysts was smaller than that (38.6 ± 10.4) of IVF-derived blastocysts (P < 0.05) and was similar to that (29.9 ± 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.

Loss of the polycomb protein Mel-18 enhances the epithelial–mesenchymal transition by ZEB1 and ZEB2 expression through the downregulation of miR-205 in breast cancer

The epithelial–mesenchymal transition (EMT) is the pivotal mechanism underlying the initiation of cancer invasion and metastasis. Although Mel-18 has been implicated in several biological processes in cancer, its function in the EMT of human cancers has not yet been studied. Here, we demonstrate that Mel-18 negatively regulates the EMT by epigenetically modulating miR-205. We identified miR-205 as a novel target of Mel-18 using a microRNA microarray analysis and found that Mel-18 increased miR-205 transcription by the inhibition of DNA methyltransferase-mediated DNA methylation of the miR-205 promoter, thereby downregulating its target genes, ZEB1 and ZEB2. Furthermore, the loss of Mel-18 promoted ZEB1- and ZEB2-mediated downregulation of E-cadherin transcription and also enhanced the expression of mesenchymal markers, leading to increased migration and invasion in MCF-7 cells. In MDA-MB-231 cells, Mel-18 overexpression restored E-cadherin expression, resulting in reduced migration and invasion. These effects were reversed by miR-205 overexpression or inhibition. A tumor xenograft with Mel-18 knockdown MCF-7 cells consistently showed increased ZEB1 and ZEB2 expression and decreased E-cadherin expression. Taken together, these results suggest that Mel-18 functions as a tumor suppressor by its novel negative control of the EMT, achieved through regulating the expression of miR-205 and its target genes, ZEB1 and ZEB2.

Developmental potential and transgene expression of porcine nuclear transfer embryos using somatic cells

We examined whether porcine nuclear transfer (NT) embryos carrying somatic cells have a developmental potential and NT embryos carrying transformed fibroblasts express transgenes in the preimplantation stages. In Experiment 1, different activation methods were applied to NT embryos and the development rates were examined. Relative to A23187 only or A23187/6‐DMAP, electrical pulse made a significant increase in both cleavage rate (58.1 ± 13.9 or 60.7 ± 6.3 vs. 74.9 ± 7.5%) and development rate of NT embryos to the blastocyst stage (2.2 ± 2.8 or 2.2 ± 1.5 vs. 11.0 ± 4.1%). In Experiment 2, in vitro developmental competence of NT embryos was investigated. The developmental rate to the blastocyst stage of NT embryos (9.9 ± 2.4% for cumulus cells and 9.8 ± 1.6% for fibroblast cells) was significantly lower than that (22.9 ± 3.5%) of IVF‐derived embryos (P < 0.01). NT blastocysts derived from either cumulus (28.9 ± 11.4, n = 26) or fibroblast cells (30.2 ± 9.9, n = 27) showed smaller mean nuclei numbers than IVF‐derived blastocysts (38.6 ±  10.4, n = 62) (P < 0.05). In Experiment 3, nuclear transfer of porcine fibroblasts expressing the GFP (green fluorescent protein) gene resulted in green blastocysts without losing developmental potential. These results suggest that porcine embryos reconstructed by somatic cell nuclear transfer are capable of developing to preimplantation stage. We conclude that somatic cells expressing exogenous genes can be used as nuclei donors in the production of NT‐mediated transgenic pig. Mol. Reprod. Dev. 58:15–21, 2001.

Influence of oocyte nuclei on demethylation of donor genome in cloned bovine embryos

We recently demonstrated that satellite regions exhibit an aberrant DNA methylation in cloned bovine embryos. Here, we examined, using bisulfite‐sequencing technology, whether the inefficient demethylation of cloned donor genomes could be rescued by the presence of oocytic nuclei. Both AciI digestion and sequencing analyses showed that satellite sequence was demethylated more efficiently in cloned tetraploid blastocysts than in diploid clones. When methyl‐CpG density (the number of methyl‐CpG sites per string) was scored, a significant decrease was observed in tetraploids (P<0.001). These results suggest that unknown mechanisms provided by oocytic nuclei could assist the demethylation of satellite sequences in tetraploid clones.

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

BackgroundStudies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.ResultsFrom hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.ConclusionOur results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.

Dual functions of histone-lysine N-methyltransferase Setdb1 protein at promyelocytic leukemia-nuclear body (PML-NB): maintaining PML-NB structure and regulating the expression of its associated genes.

Background: Setdb1 regulates gene expression with various transcription factors. Results: Setdb1 is a constitutive member of PML-NB and suppresses Id2 expression. Conclusion: Setdb1 maintains PML-NB structure and concurrently controls PML-NB-associated genes. Significance: This provides the mechanism of Setdb1 being involved in PML-mediated transcriptional regulation. Setdb1/Eset is a histone H3 lysine 9 (H3K9)-specific methyltransferase that associates with various transcription factors to regulate gene expression via chromatin remodeling. Here, we report that Setdb1 associates with promyelocytic leukemia (Pml) protein from the early stage of mouse development and is a constitutive member of promyelocytic leukemia (PML)-nuclear bodies (PML-NBs) that have been linked to many cellular processes such as apoptosis, DNA damage responses, and transcriptional regulation. Arsenic treatment, which induces Pml degradation, caused Setdb1 signals to disappear. Setdb1 knockdown resulted in dismantlement of PML-NBs. Immunoprecipitation results demonstrated physical interactions between Setdb1 and Pml. Chromatin immunoprecipitation revealed that, within the frame of PML-NBs, Setdb1 binds the promoter of Id2 and suppresses its expression through installing H3K9 methylation. Our findings suggest that Setdb1 performs dual, but inseparable, functions at PML-NBs to maintain the structural integrity of PML-NBs and to control PML-NB-associated genes transcriptionally.