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Epigenetic reprogramming in mouse primordial germ cells.

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.

Animals↗

An intelligent system for pacemaker reprogramming.

The process of reprogramming a cardiac pacemaker can be described in terms similar to those used for describing diagnostic problem solving. In this paper, the process of reprogramming a pacemaker is formalized as a special form of abductive diagnostic reasoning, where observable findings are interpreted with respect to results obtained from diagnostic tests. The dynamics of this process is cast as a diagnostic strategy, where information is gathered in a structured fashion. This abductive theory of pacemaker reprogramming has been used as the basis for an actual system that in its present form is capable of assisting cardiologists in dealing with problems in atrial sensing and pacing. The performance of the system has been evaluated using data from actual patients.

Algorithms↗

Reprogramming DNA methylation in the preimplantation stage: peeping with Dolly's eyes.

Oocyte cytoplasmic factors can reprogramme the sperm genome during fertilisation or the somatic cell genome during cloning. Diverse reprogramming machinery acts sequentially and interdependently on the imported genome to drive it to totipotency, but their three-dimensional interactions in the cytoplasm remain unknown. Aberrant epigenetic phenomena in early cloned embryos indicate that parts of the somatic cell genome are unyielding to reprogramming forces, owing to their 'knotty' epigenetic features. This fastidious nature of the donor genome might prevent completion of epigenetic reprogramming. It might also help to explain the chronic developmental defects seen in many cloned embryos.

Animals↗

Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer.

Epigenetic modifications of the genome play a significant role in the elaboration of the genetic code as established at fertilisation. These modifications affect early growth and development through their influence on gene expression especially on imprinted genes. Genome-wide epigenetic reprogramming in germ cells is essential in order to reset the parent-of-origin specific marking of imprinted genes, but may have a more general role in the restoration of totipotency in the early embryo. In a similar way, on somatic nuclear cloning, a differentiated cell must become 'reprogrammed' restoring totipotency in order to undergo development. Here we discuss the dynamic epigenetic reprogramming that takes place during normal development and highlight those areas with relevance to somatic nuclear cloning and the possibility of improving the efficiency of this process. We propose the concept of 'epigenetic checkpoints' for normal progression of development and the loss of totipotency.

Animals↗

Reprogramming of epigenetic inheritance by somatic cell nuclear transfer.

Somatic cloning by nuclear transfer returns a differentiated cell to a totipotent stage, a process termed nuclear reprogramming. During this de-differentiation process, genes inactivated during tissue differentiation are re-activated in a temporal and spatial special manner. It is believed that tissue differentiation occurs through epigenetic mechanisms, genetic inheritance that does not involve changes in DNA sequences. Developmental abnormalities and a high mortality rate in cloned offspring have frequently been observed and probably result from incomplete nuclear reprogramming. In this review, the reprogramming of two epigenetic mechanisms, imprinting and X chromosome inactivation, as well as recent attempts to modify pre-existing epigenetic marks in donor cells to improve nuclear transfer efficacy, are discussed.

Animals↗

[Reprogramming and epigenesis].

The fact that the nucleus of a differentiated somatic cell can be reprogrammed in order to sustain embryonic development is now well established. Experiments of somatic cell nuclear transfer (cloning) have proved that a foreign nucleus introduced into an enucleated oocyte can give rise to physiologically normal offsprings, with a normal lifespan. Such evidence of genome expression plasticity is also observed experimentally with heterokaryons, created by the fusion or the nuclear transfer between two somatic cells, where differentiated nuclei are able to express genes characteristic of the host cell. However, the epigenetic mechanisms that permit nuclear plasticity remain poorly understood. In this paper we present the main evidences showing important modifications of the large scale organisation of chromosomal domains and of the DNA methylation pattern upon nuclear transfer and during the first cleavages. These modifications of epigenetic marks, brought by an intimate contact between the chromatin and the recipient oocyte cytoplasmic factors, appear essential for further development. They are established over the first cell cycles of development. The onset of embryonic genome activation and the first cellular differentiation events that occur over the implantation period are two additional check-points of reprogramming that appear to be also highly dependent on epigenetic alterations. Beyond those stages, defective placental functions might be directly responsible for the fetal and postnatal physiopathologies frequently observed in cloned animals. No direct link between preimplantation reprogramming defaults, placental dysfunctions and low development to term has been established yet. The epigenetics studies which are now used to characterise loci specific and probably genotype dependent alterations in cloned animals of different species will provide invaluable help to define the role of epigenesis in the achievement of a developmental program.

Animals↗

Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning.

Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.

Animals↗

AML1-ETO hijacks a distal enhancer of NAT10 to reprogram glutathione metabolism and sustain leukemia stem cell stemness.

Chromosomal translocations produce oncogenic fusion proteins such as AML1-ETO, which predominantly occupy gene promoters to induce transcriptional reprogramming in leukemia stem cells (LSCs), consequently driving the pathogenesis of t(8;21) acute myeloid leukemia (AML). However, whether AML1-ETO is recruited to additional regulatory DNA elements to orchestrate oncogenic gene expression programs has not been fully addressed. Here, we define AML1-ETO and H3K27ac CUT&Tag landscapes in primary t(8;21) AML CD34+ cells and t(8;21) AML cell lines, revealing AML1-ETO binding at a distal enhancer of the RNA N4-acetylcytidine (ac4C) writer N-acetyltransferase 10 (NAT10), thereby driving its transcriptional activation. Genetic ablation or pharmacological inhibition of NAT10 restricted the survival and self-renewal of LSCs in primary t(8;21) AML CD34+ cells, as well as in a retroviral AML1-ETO9a-driven t(8;21) AML mouse model, establishing NAT10 as a potential therapeutic vulnerability. Mechanistically, NAT10 is recruited to glutathione S-transferase omega 2 (GSTO2) mRNA to catalyze ac4C modification, thereby enhancing transcript stability and reprogramming glutathione metabolism, as demonstrated by ac4C profiling, RNA immunoprecipitation (RIP), and dCas13b-NAT10-based analyses. Silencing of GSTO2 in primary t(8;21) AML CD34+ cells decreased intracellular reduced glutathione (GSH) levels and compromised LSC survival and self-renewal, whereas GSTO2 overexpression or GSH supplementation largely rescued LSC maintenance following NAT10 loss. Collectively, these findings enrich and extend the understanding of AML1-ETO regulatory programs by linking distal enhancer activity to a NAT10-GSTO2 ac4C-GSH axis that integrates epigenomic, posttranscriptional, and metabolic reprogramming to sustain LSC stemness, highlighting this circuit as a potential therapeutic vulnerability in t(8;21) AML.

Humans↗

Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle.

Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30-50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15-23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14-18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves ( approximately 20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal.

Animals↗

Preprogramming, programming and reprogramming of aimed hand movements as a function of age.

The present experiments were conducted to investigate the relationship between age and the response programming operations underlying the execution of a ballistic motor act. In an initial experiment, two separate age groups of female subjects (mean ages of 21.9 and 69.1 years) performed aimed-movements of the right hand and arm in one of two movement directions (left or right), under preprogramming, programming, and reprogramming conditions. These operations were examined by providing advance information about the direction of an impending movement and manipulating the degree of correspondence between the advance information and a subsequent reaction signal. The results indicated that subjects in the older age group reacted and moved more slowly than subjects in the younger age group, however, there was no interaction between age and the three response programming conditions. Such findings indicated that the basic operational characteristics of these processes remain unaffected with advancing chronological age. Also, irrespective of age and response programming condition, responses to the right were initiated faster than responses to the left. This difference was especially accentuated for reprogramming. A second experiment, using a new stimulus-response mapping, replicated the left-right difference in initiation time; this difference was reversed when the left hand was used to execute the designated movement, indicating that this finding is indeed a response programming phenomenon. Further discussion focused on the possible operations underlying reprogramming.

Journal Article↗

Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro.

Primary mouse brain cells were cultured with HPRT (hypoxanthine phosphoribosyl transferase)-deficient ES (embryonic stem) cells to see if the ES cells could provide cues sufficient to reprogram a pluripotential state. After 5 days of coculture, HPRT-deficient ES cells were killed by selection in HAT (hypoxanthine, aminopterin, thymidine) medium. We observed islands of HAT-resistant ES-like cells surrounded by differentiated cells. Cell lines generated from three such "islands" proved to be spontaneous, pluripotential ES-neural hybrids, and gave rise to a chimera following blastocyst injection. Re-expression of the ES-specific gene Foxd3 from somatic-derived chromosomes suggested that the somatic nucleus had been reprogrammed. Our results raise the intriguing possibility that ASCs shown to contribute to multiple tissues in blastocyst-injection studies may not contribute as a result of pluripotency. Instead contributions may arise from spontaneous fusion events in which phenotype is determined by either cytoplasmic dominance, nuclear reprogramming, or both.

Animals↗

Nuclear cloning, stem cells, and genomic reprogramming.

The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning).

Animals↗

Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer.

Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.

Animals↗

Nuclear reprogramming in nuclear transplant rabbit embryos.

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.

Animals↗

Reprogrammed macrophage tumor necrosis factor and interleukin-1 release with inflammatory pretreatment: differential regulation by endotoxin and zymosan.

OBJECTIVE: To determine whether different reprogrammed alterations in endotoxin (lipopolysaccharide, LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 (IL-1) release are seen following pretreatment with endotoxin (LPSp) or pretreatment with the particulate inflammatory stimulus zymosan. METHODS: Murine peritoneal macrophages (M phi) pretreated for 24 hours in vitro with medium, LPSp, zymosan, latex beads, or killed Escherichia coli. After 24 hours M phi were restimulated with medium, LPSa, zymosan, latex beads, or E. coli, M phi supernatant TNF and IL-1 were measured after 24 hours and mRNA levels determined after 6 hours with reverse-transcriptase polymerase chain reaction. RESULTS: Pretreatment with low dose LPSp markedly inhibited TNF release by both LPSa or zymosan, while pretreatment with zymosan increased LPSa-stimulated TNF release. Pretreatment with both LPSp and zymosan augmented LPSa and zymosan-stimulated IL-1. Zymosan pretreatment augmentation of TNF and IL-1 was accompanied by lower than basal levels of cytokine message. CONCLUSION: Reprogrammed macrophage TNF and IL-1 release was differentially regulated by distinct inflammatory stimuli. Understanding reprogrammed macrophage cytokine regulation may enable specific therapy to modify dysregulated cytokine release during sepsis and trauma.

Animals↗

Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells.

We have explored the use of embryonic stem cells as an alternative to oocytes for reprogramming human somatic nuclei. Human embryonic stem (hES) cells were fused with human fibroblasts, resulting in hybrid cells that maintain a stable tetraploid DNA content and have morphology, growth rate, and antigen expression patterns characteristic of hES cells. Differentiation of hybrid cells in vitro and in vivo yielded cell types from each embryonic germ layer. Analysis of genome-wide transcriptional activity, reporter gene activation, allele-specific gene expression, and DNA methylation showed that the somatic genome was reprogrammed to an embryonic state. These results establish that hES cells can reprogram the transcriptional state of somatic nuclei and provide a system for investigating the underlying mechanisms.

Adult↗

Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization.

Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization. Physiol Genomics 7: 135-148, 2001. First published September 21, 2001; 10.1152/physiolgenomics.00061.2001.-Human decidual fibroblasts undergo a differentiative commitment to the acquisition of endocrine, metabolic, and structural cell functions in a process known as decidualization. Decidualization is critical for embryo implantation and placental function. We characterized gene expression pattern kinetics during decidual fibroblast differentiation by microarray analysis. Of 6,918 genes analyzed, 121 genes were induced by more than twofold, 110 were downregulated, and 50 showed biphasic behavior. Dynamically regulated genes were could be fit into nine K-means algorithm-based kinetic pattern groups, and by biologic classification, into five categories: cell and tissue function, cell and tissue structure, regulation of gene expression, expressed sequence tag (EST), and "function unknown." Reprogramming of genes within specific functional groups and gene families was a prominent feature that consisted of simultaneous induction and downregulation of a set of genes with related function. We previously observed a conceptually similar process during fetal trophoblast differentiation, in which the same phenomena applied to different genes. Of the 569 dynamically regulated genes regulated by either model, only 81 of these were in common. These results suggest that reprogramming of gene expression within focused functional categories represents a fundamental aspect of cellular differentiation.

Cell Differentiation↗

Nuclear reprogramming in mammalian somatic cell nuclear cloning.

Nuclear cloning is still a developing technique used to create genetically identical animals by somatic cell nuclear transfer into unfertilized eggs. Despite an intensive effort in a number of laboratories, the success rate of obtaining viable offspring from this technique remains less than 5%. In the past few years many investigators reported the reprogramming of specific nuclear activities in cloned animals, such as genome-wide gene expression patterns, DNA methylation, genetic imprinting, histone modifications and telomere length regulation. The results highlight the tremendous difficulty the clones face to reprogram the original differentiation status of the donor nuclei. Nevertheless, nuclei prepared from terminally differentiated lymphocytes can overcome this barrier and produce apparently normal mice. Study of this striking nuclear reprogramming activity should significantly contribute to our understanding of cell differentiation in more physiological settings.

Cell Differentiation↗