A transcriptional landscape of 28 porcine tissues obtained by super deepSAGE sequencing.

BACKGROUND: Gene expression regulators identified in transcriptome profiling experiments may serve as ideal targets for genetic manipulations in farm animals. RESULTS: In this study, we developed a gene expression profile of 76,000+ unique transcripts for 224 porcine samples from 28 tissues collected from 32 animals using Super deepSAGE technology. Excellent sequencing depth was achieved for each multiplexed library, and replicated samples from the same tissues clustered together, demonstrating the high quality of Super deepSAGE data. Comparison with previous research indicated that our results not only have good reproducibility but also have greatly extended the coverage of the sample types as well as the number of genes. Clustering analysis revealed ten groups of genes showing distinct expression patterns among these samples. Our analysis of over-represented binding motifs identified 41 regulators, and we demonstrated a potential application of this dataset in infectious diseases and immune biology research by identifying an LPS-dependent transcription factor, runt-related transcription factor 1 (RUNX1), in peripheral blood mononuclear cells (PBMCs). The selected genes are specifically responsible for the transcription of toll-like receptor 2 (TLR2), lymphocyte-specific protein tyrosine kinase (LCK), and vav1 oncogene (VAV1), which belong to the T and B cell signaling pathways. CONCLUSIONS: The Super deepSAGE technology and tissue-differential expression profiles are valuable resources for investigating the porcine gene expression regulation. The identified RUNX1 target genes belong to the T and B cell signaling pathways, making them novel potential targets for the diagnosis and therapy of bacterial infections and other immune disorders.

Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos.

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50nM) for 1h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5nM 5-aza-dC for 1h and subsequently treated with 50nM TSA for another 1h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5nM 5-aza-dC and 50nM TSA for 1h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.

Epigenetic characterization of the PBEF and TIMP-2 genes in the developing placentae of normal mice.

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.