Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

The developmental programme of embryogenesis is controlled by both genetic and epigenetic mechanisms. An emerging theme from recent studies is that the regulation of higher-order chromatin structures by DNA methylation and histone modification is crucial for genome reprogramming during early embryogenesis and gametogenesis, and for tissue-specific gene expression and global gene silencing. Disruptions to chromatin modification can lead to the dysregulation of developmental processes, such as X-chromosome inactivation and genomic imprinting, and to various diseases. Understanding the process of epigenetic reprogramming in development is important for studies of cloning and the clinical application of stem-cell therapy.

Chromatin modification and epigenetic reprogramming in mammalian development

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP:

Functional genomics reveals a bmp driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming (oral presentation)

Cloning of mammals by nuclear transfer (NT) results in gestational or neonatal failure with at most a few percent of manipulated embryos resulting in live births. Many of those that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Cloned embryos derived from donors, such as embryonic stem cells, that may require little or no reprogramming of early developmental genes develop substantially better beyond implantation than NT clones derived from somatic cells. Although recent experiments have demonstrated normal reprogramming of telomere length and X chromosome inactivation, epigenetic information established during gametogenesis, such as gametic imprints, cannot be restored after nuclear transfer. Survival of cloned animals to birth and beyond, despite substantial transcriptional dysregulation, is consistent with mammalian development being rather tolerant to epigenetic abnormalities, with lethality resulting only beyond a threshold of faulty gene reprogramming encompassing multiple loci.

https://science.sciencemag.org/content/sci/293/5532/1093.full.pdf

Nuclear Cloning and Epigenetic Reprogramming of the Genome

Phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/AKT/mTOR signalling cascade, regulating cell proliferation, survival and metabolism. Although these functions are well-defined in the context of tumorigenesis, recent studies - in particular those using pluripotent stem cells - have highlighted the importance of this pathway to development and cellular differentiation. Here, we review the recent in vitro and in vivo evidence for the role PI3K/AKT/mTOR signalling plays in the control of pluripotency and differentiation, with a particular focus on the molecular mechanisms underlying these functions.

Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination.

Development to blastocyst following nuclear transfer is dependent on the donor cell’s ability to reprogram its genome to that of a zygote. This reprogramming step is inefficient and may be dependent on a number of factors, including chromatin organization. Trichostatin A (TSA; 0‐5 mM), a histone deacetylase inhibitor, was used to increase histone acetylation and 5-aza-29deoxycytidine (5-aza-dC; 0‐5 mM), a DNA methyl-transferase inhibitor, was used to decrease methylation of chromatin in donor cells in an attempt to improve their reprogrammability. Adult fibroblast cells treated with 1.25 or 5 mM TSA had elevated histone H3 acetylation compared to untreated controls. Cells treated with 0.3 mM 5-aza-dC had decreased methylation compared to untreated controls. Both drugs at 0.08 mM caused morphological changes of the donor cells. Development to blastocysts by embryos cloned from donor cells after 0.08 or 0.3 mM 5-aza-dC treatments was lower than in embryos cloned from untreated control cells (9.7% and 4.2%, respectively, vs. 25.1%), whereas 0.08 mM TSA treatment of donor cells increased blastocyst development compared to controls (35.1% vs. 25.1%). These results indicate that partial erasure of preexisting epigenetic marks of donor cells improves subsequent in vitro development of cloned embryos. assisted reproductive technology, embryo

https://bioone.org/journals/biology-of-reproduction/volume-69/issue-3/biolreprod.103.017954/Epigenetic-Characteristics-and-Development-of-Embryos-Cloned-from-Donor-Cells/10.1095/biolreprod.103.017954.pdf

Epigenetic Characteristics and Development of Embryos Cloned from Donor Cells Treated by Trichostatin A or 5-aza-2′-deoxycytidine

Success of cloning using adult somatic cells has been reported in sheep1, mice2,3 and cattle4,5,6,7. The report that `Dolly'8 the sheep, the first clone from an adult mammal, inherited shortened telomeres from her cell donor and that her telomeres were further shortened by the brief culture of donor cells has raised serious scientific and public concerns about the `genetic age' and potential developmental problems of cloned animals. This observation was challenged by a recent report9 that showed calves cloned from fetal cells have longer telomeres than their age-matched controls. The question remains whether Dolly's short telomeres were an exception or a general fact, which would differ from the telomeres of fetal-derived clones.

Normal telomere lengths found in cloned cattle.

Differentiated somatic cells and embryos cloned from somatic cells by nuclear transfer (NT) have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells before NT by treating them with chemicals such as the DNA methyl-transferase inhibitor (5-aza-29-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Previously, high levels of this reagent were used to treat donor cells, and decreased development of cloned embryos was observed. In this study, we tested a lower range (0.005 to 0.08 mM) of this drug and used cell cycle distribution changes as an indicator of changes in the characteristics of donor cells. We found that at 0.01 mM 5-aza-dC induced changes in the cycle stage distribution of donor cells, increased the fusion rate of NT embryos, and had no deleterious effect on the percentage of blastocyst development. Levels of 5-aza-dC greater than 0.01 mM significantly decreased embryo development. Embryos cloned from donor cells treated with a low dose of 5-aza-dC had higher levels of DNA methylation than embryos produced by in vitro fertilization, but they also had higher levels of histone acetylation. Although 5-aza-dC at 0.04 mM or higher reduced DNA methylation and histone acetylation levels to those of in vitro-fertilized embryos, development to blastocyst was reduced, suggesting that this concentration of the drug was detrimental. In summary, 5-aza-dC at 0.01 mM altered donor cell characteristics while showing no deleterious effects on embryos cloned from treated cells. assisted reproductive technology

Methylation and Acetylation Characteristics of Cloned Bovine Embryos from Donor Cells Treated with 5-aza-2′-Deoxycytidine

During mammalian pre-implantation embryonic development dramatic and orchestrated changes occur in gene transcription. The identification of the complete changes has not been possible until the development of the Next Generation Sequencing Technology. Here we report comprehensive transcriptome dynamics of single matured bovine oocytes and pre-implantation embryos developed in vivo. Surprisingly, more than half of the estimated 22,000 bovine genes, 11,488 to 12,729 involved in more than 100 pathways, is expressed in oocytes and early embryos. Despite the similarity in the total numbers of genes expressed across stages, the nature of the expressed genes is dramatically different. A total of 2,845 genes were differentially expressed among different stages, of which the largest change was observed between the 4- and 8-cell stages, demonstrating that the bovine embryonic genome is activated at this transition. Additionally, 774 genes were identified as only expressed/highly enriched in particular stages of development, suggesting their stage-specific roles in embryogenesis. Using weighted gene co-expression network analysis, we found 12 stage-specific modules of co-expressed genes that can be used to represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the bovine expressed gene networks. Their vast association with other embryonic genes suggests that they may have important regulatory roles in embryo development; yet, the majority of the hub genes are relatively unknown/under-studied in embryos. We also conducted the first comparison of embryonic expression profiles across three mammalian species, human, mouse and bovine, for which RNA-seq data are available. We found that the three species share more maternally deposited genes than embryonic genome activated genes. More importantly, there are more similarities in embryonic transcriptomes between bovine and humans than between humans and mice, demonstrating that bovine embryos are better models for human embryonic development. This study provides a comprehensive examination of gene activities in bovine embryos and identified little-known potential master regulators of pre-implantation development.

/pdf/transcriptional-profiles-of-bovine-in-vivo-pre-implantation-edm8u8xq1m.pdf

Transcriptional profiles of bovine in vivo pre-implantation development.

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5–15) in G0/G1- and G2/M-stage cumulus an...

https://bioone.org/journals/Biology-of-Reproduction/volume-69/issue-5/biolreprod.103.019950/Epigenetic-Characteristics-of-Bovine-Donor-Cells-for-Nuclear-Transfer/10.1095/biolreprod.103.019950.pdf

Xiuchun Cindy Tian

Papers

Epigenetic Characteristics and Development of Embryos Cloned from Donor Cells Treated by Trichostatin A or 5-aza-2′-deoxycytidine

Normal telomere lengths found in cloned cattle.

Methylation and Acetylation Characteristics of Cloned Bovine Embryos from Donor Cells Treated with 5-aza-2′-Deoxycytidine

Transcriptional profiles of bovine in vivo pre-implantation development.

Epigenetic Characteristics of Bovine Donor Cells for Nuclear Transfer: Levels of Histone Acetylation