98 HETEROCHROMATIN REPROGRAMMING IN MOUSE EARLY DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 149
Author(s):  
T. Wongtawan ◽  
J. Taylor ◽  
I. Wilmut ◽  
S. Pennings
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Es Cells ◽  
Cell Nuclei ◽  
Mouse Development ◽  
Fluorescent Intensity ◽  
Chromatin Architecture ◽  
Somatic Nuclear Transfer

Heterochromatin is essential for epigenetic gene silencing and nuclear chromatin architecture. Early mouse development is accompanied by dynamic epigenetic changes and heterochromatin restructuring in the cell nuclei of cleavage stage embryos. We have previously shown that disruption of heterochromatin markers such as DNA methylation following somatic nuclear transfer causes developmental failure (Beaujean N et al. 2004 Biol. Reprod. 71, 185–193). The aim of the present study was to investigate the transitions and maturation of heterochromatin during normal development to improve nuclear reprogramming technology and understand developmental abnormalities caused by epigenetic alterations in somatic nuclear transfer and assisted reproductive procedures. Mouse pre- and post-implantation embryos and ES cells from B6CBAF1 mice were used in experiments employing the following methodologies. Embryos and ES cells were stained by immunofluorescence to detect heterochromatin proteins and epigenetic markers. Images were captured using laser confocal microscopy. Relative quantification of fluorescent intensity was performed using Zeiss LSM-meta and WCIF-ImageJ software. mRNA of Histone methyltransferase (HMTase), suv39h, suv420h, ehmt, eset, and demethyltransferase (DHMTase), jmjd1a, jmjd2a, jmjd2c were quantified by real-time PCR. siRNA and specific inhibitors were used to study the function of suv39h, ehmt2, jmjd2c genes that may be responsible for heterochromatin reprogramming. Our results demonstrated that H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. The gene expression results showed the dynamic and temporal gene expression of HMTases and DHMTase during development. Inhibition of Ehmt2 and jmjd2c caused preimplantation developmental arrest. Furthermore, we found chromatin modification differences in the heterochromatin of ES cells, ICM and epiblast. We conclude that heterochromatin reprogramming might be essential for development because it may contribute to chromatin architecture, thus influencing gene expression. HMTases and DHTMases could be implicated in the mechanism of heterochromatin reprogramming. It is possible that Ehmt2 and jmjd2c play an important role in preimplantation development by modifying chromatin globally and at the local gene level. This work was supported by a The Royal Thai Studentship. We acknowledge the contributions of technical assistance from T. O’Connor, B. Wongtawan and P. Travers.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

scholarly journals Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

Endocrinology ◽  
2005 ◽  
Vol 146 (2) ◽  
pp. 776-783 ◽  
Author(s):  
Yan-Yun Liu ◽  
Gregory A. Brent
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Thyroid Hormone ◽  
Early Development ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Ec Cells

Abstract T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRα P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.

Gene Expression Patterns in Bovine In vitro-Produced and Nuclear Transfer-Derived Embryos and Their Implications for Early Development

2002 ◽  
Vol 4 (1) ◽  
pp. 29-38 ◽  
Author(s):  
H. Niemann ◽  
C. Wrenzycki ◽  
A. Lucas-Hahn ◽  
T. Brambrink ◽  
W.A. Kues ◽  
...  
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Patterns

scholarly journals 116 ACTIVE METHYLATION AND ACETYLATION OF HISTONE H3-K9 IN MOUSE EMBRYO WITH DIFFERENT PROPORTIONS OF MATERNAL AND PATERNAL GENOME

2005 ◽  
Vol 17 (2) ◽  
pp. 208
Author(s):  
V.T. Nguyen ◽  
S. Wakayama ◽  
H.-T. Bui ◽  
T. Wakayama
Keyword(s):  
Early Development ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Epigenetic Modification ◽  
Histone H3 ◽  
Paternal Genome ◽  
Somatic Nucleus ◽  
Histone H3 Methylation ◽  
Cell Embryo ◽  
Somatic Nuclear Transfer

Epigenetic modification of parental genomes plays a prominent role in regulating genome expression in the early development of embryos. In general, histone H3 of the paternal genome is demethylated at lysine 9 (H3-K9) during the first and second mitotic divisions in fertilized embryos, while the maternal genome is methylated. We investigated the effects of maternal genomes (Mgen) and paternal genomes (Pgen) on H3-K9 methylation and acetylation during the early development of murine embryos. Histone H3-K9 methylation and acetylation were detected by anti-trimethyl histone H3-K9 and anti-triacetyl histone H3-K9 antibodies. The following embryos were used in this study: (1) intracytoplasmic sperm injection (ICSI) embryos (50% Mgen, 50% Pgen); (2) parthenogenetic diploid embryos (100% Mgen, 0% Pgen); (3) somatic nuclear transfer embryos (50% Mgen, 50% Pgen from previous generation); (4) androgenetic diploid embryos (0% Mgen, 100% Pgen); and (5) haploidized somatic nucleus and sperm embryo (about 25% Mgen, about 75% Pgen). Each experiment was repeated five times to obtain more than 120 embryos per group. Our results show that: (1) in the ICSI embryo, histone H3 methylation occurs in Mgen but not in Pgen at the first and second mitotic divisions; (2) in the parthenogenetic embryo, histone H3 methylation occurs in both nuclei at the first and second mitotic divisions; (3) in the somatic nuclear transfer embryo, histone H3 is methylated in all of the nuclei at the first and second mitotic divisions; (4) in the androgenetic embryo, methylated H3-K9 is detected weakly in the heterochromatin enclosed around the nucleolus of the pronuclei of the one-cell embryo, and methylated in the entire nuclei of the two-cell embryo; and (5) in the haploidized somatic and sperm embryo, the pattern of histone H3-L9 methylation resembles that of the ICSI embryo. While histone H3-K9 acetylation occurs in both paternal and maternal genomes during interphase, even when the nuclear membrane is completely degraded and the chromosome is condensed, it disappears rapidly when the chromosome enters the real metaphase, and reappears at the early stage of pronuclear formation in all types of embryo. These results suggest that the absence of maternal genomes results in histone H3-K9 methylation in the paternal genomes during the first and second mitotic divisions of embryos in mice. In addition, histone H3-K9 acetylation is independent of the presence or absence of maternal or paternal genomes during pre-implantation development in mice. This study was supported by grants-in-aid for Creative Scientific Research (13GS0008) and a project for the realization of regenerative medicine (the research field for the technical development of stem cell manipulation) to T.W. from MEXT, Japan.

scholarly journals 26ABERRANT REPROGRAMMING OF IMPRINTED GENE EXPRESSION IN ENLARGED PLACENTAS OF MICE CLONED FROM ES CELLS TREATED WITH TSA OR 5AZAC

2004 ◽  
Vol 16 (2) ◽  
pp. 135
Author(s):  
S.G. Baqir ◽  
Q. Zhou ◽  
A. Jouneau ◽  
J.-P. Renard ◽  
D.H. Betts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Imprinted Genes ◽  
Imprinted Gene ◽  
Donor Cells

The success rate of producing cloned animals is very low, and in many cases is associated with the formation of enlarged placentas. Increasing evidence has pointed towards epigenetic deregulation of imprinted genes due to incomplete or abnormal resetting of DNA methylation and/or histone acetylation patterns during development. It has previously been shown that drugs that alter DNA methylation (5AzaC) and histone acetylation (TSA) over-express imprinted genes in mouse ES cells (Baqir and Smith, 2001, Theriogenology 55, 410). Our objective in this study was to determine whether nuclear transfer is able to reprogram imprinted gene expression patterns in the placenta of mice cloned from ES donor nuclei exposed to 5AzaC and TSA. ES donor cells were treated with either TSA or 5AzaC prior to injection into enucleated oocytes. Total RNA was extracted from placentas of day 14–15 fetus clones, and reversed transcribed; the expression pattern of imprinted genes (Ipl, Mash2, Igf2, H19, Igf2r, p57, Peg1), non-imprinted placental-specific genes (Esx1, Dlx3, Tpbp) and a housekeeping gene (Gapdh) was examined by Real Time PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to placentas of cloned fetus derived from non-treated donor cells. Data were analyzed by ANOVA and mean gene expression values were compared using the Tukey-Kramer test. Our results show that several imprinted genes (Mash2, H19, Ipl) and placenta-specific genes (Esx1 and Dlx1) were properly reprogrammed in non-enlarged (71mg) placentas of fetus clones derived from the TSA and 5AzaC treated ES donor cells. Although Gapdh expression did not differ among normal and enlarged 210mg) placenta groups, the expression level of Igf2 and Mash2 was higher in enlarged placentas from fetus clones produced from TSA-treated ES donor cells (4.6 and 3.5 fold) compared to non-enlarged placentas from non-treated ES cells (1 fold). Conversely, oversized placentas from cloned fetuses derived from TSA-treated donor ES cells under-expressed Peg1, H19 and Ipl (0.5, 0.2 and 0.2 fold, respectively) compared to control placentas (1 fold). In addition, enlarged placentas from the TSA- and 5AzaC-treated group displayed down-regulation of placenta specific genes Esx1 and Dlx3 and up-regulation of Tpbp, suggesting the presence of abnormal distribution of placental layers. These results indicate that while several imprinted and non-imprinted placenta specific genes were correctly expressed in normal size placentas of fetus clones derived from TSA and 5AzaC treated donor ES cells, enlarged placentas displayed aberrant gene expression patterns, suggesting that improper resetting of the epigenetic program after nuclear transfer is directly related to altered DNA methylation and histone acetylation patterns. Funded by NSERC & CIHR.

scholarly journals Epigenetic Reprogramming Induced Pluripotency

2011 ◽  
Vol 3 (2) ◽  
pp. 93
Author(s):  
Anna Meiliana ◽  
Andi Wijaya
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Ips Cells ◽  
Genetic Alterations ◽  
Epigenetic Reprogramming ◽  
Patient Specific

BACKGROUND: The ability to reprogram mature cells to an embryonic-like state by nuclear transfer or by inducing the expression of key transcription factors has provided us with critical opportunities to linearly map the epigenetic parameters that are essential for attaining pluripotency.CONTENT: Epigenetic reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible tissue, such as skin in the same individual. This has potential applications for cell replacement without the immunosuppression treatments commonly required when cells are transferred between genetically different individuals.SUMMARY: Reprogramming with transcription factors offers tremendous promise for the future development of patient-specific pluripotent cells and for studies of human disease. The identification of optimized protocols for the differentiation of iPS cells and ES cells into multiple functional cell types in vitro and their proper engraftment in vivo will be challenged in the coming years. Given that the first small molecule approaches aimed at activating pluripotency genes have already been devised and that murine iPS cells have recently been derived by using non-integrative transient expression strategies of the reprogramming factors, we expect that human iPS cells without permanent genetic alterations will soon be generated.KEYWORDS: epigenetics, reprogramming, pluripotency, stem cells, iPS cells, chromatin, DNA methylation

28 EFFECT OF TREATMENT OF OVINE OOCYTES WITH CAFFEINE ON GENE EXPRESSION IN NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 122 ◽  
Author(s):  
I. Choi ◽  
J.-H. Lee ◽  
K. Campbell
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Early Development ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Group Iv ◽  
Expression Levels ◽  
Number Of Genes

The efficiency of animal production by somatic cell nuclear transfer (SCNT) remains low and this has been linked to incomplete epigenetic reprogramming of the donor somatic cell nucleus. Previous studies have reported that embryos produced by SCNT exhibit abnormal expression patterns for a number of genes, including IL6, FGF4, FGFr2, Hsp, IF-tau, DNMT, and Mash2 (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040; Wrenzycki et al. 2001 Biol. Reprod. 65, 309-317). Recently, we demonstrated that treatment of telophase I or metaphase II ovine oocytes with 10 mM caffeine for 6 h increased the activities of both MPF and MAPK kinases. In NT embryos produced using caffeine treated oocytes as recipient cytoplasts, no increase in the frequency of development to the blastocyst stage was observed; however, blastocyst stage embryos had an increased cell number (Lee and Campbell 2005 Reprod. Fertil. Dev. 16, 125). The objective of the present study was to examine the effects of caffeine treatment on the expression levels of a number of genes involved in early development. Target genes were categorized into seven groups based on their function: (1) transcription factors (Oct-4, Sox-2), (2) growth factors (IGF-1, IGF-1r, IGF-2r, FGF-2, and FGF-4), (3) stress adaptation (Hsp70.1 and Hsp27), (4) metabolism (Glut-1, Glut-3, and Glut-4), (5) compaction/cavitation (DcII), (6) trophoblastic function (IF-tau), and (7) nuclear reprogramming factors (Hist4h4, H2A.Z, and Lmna). To determine the transcript levels semiquantitatively, different PCR cycle parameters were used (35 and 40 cycles). Expression levels were compared in blastocyst-stage embryos obtained from five groups produced as previously described (Lee and Campbell 2005; Maalouf et al. 2005 Reproduction 32, 49): (I) IVF embryos, (II) caffeine-treated IVF embryos, (III) parthenotes, (IV) SCNT embryos, and (V) caffeine treated SCNT embryos. No differences in overall expression patterns were observed among groups I, II, and III. In group IV non-treated SCNT-derived embryos, an aberrant gene expression pattern was found with respect to Oct-4 and genes regulated by Oct-4: H2A.Z, IF-tau, and FGF-4, Oct-4, H2A.Z, and FGF-4 were down-regulated and IF-tau was up-regulated. In contrast, the expression patterns of group V caffeine-treated SCNT embryos resembled those of control groups I, II, and III. In comparison to gene expression in group IV embryos, Oct-4, FGF-4, and H2A.Z were up-regulated but IF-tau was down-regulated. Previous studies have demonstrated that FGF-4 and H2A.Z play an important role in early development and implantation, whereas expression of IF-tau is related to embryo quality (Feldman et al. 1995 Science 267, 246-249; Fasst et al. 2001 Curr. Biol. 11, 1183-1187; Wrenzycki et al. 2001). Our results demonstrate that treatment of oocytes with caffeine prior to embryo reconstruction can alter the expression patterns of developmentally regulated genes in SCNT embryos to more closely resemble those of IVF controls.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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