Pigs are an important species in both agriculture and biomedical research. With the recent publication of the swine genome sequence, interest in a methylome map for the pig has grown. DNA methylation is the addition of a methyl group to a cytosine at CpG sites in the mammalian genome. Increased methylation at CpG islands, associated with genes, is correlated with decreased gene expression. DNA methylation is tissue specific and has been shown to be important in imprinting, development, cancer, and responsible for changes in gene expression. A methylation map of multiple tissues at various developmental stages as well as disease/physiological states would be beneficial in determining the importance of methylation patterns associated with these events, as well as establishing normal methylation patterns. In partnership with the Rural Development Administration of Korea and Jeju National University, we are developing a porcine epigenome reference map and identifying epigenome variations. During the first year of the grant, we performed RNAseq and reduced representation bisulfite sequencing (RRBS) on 10 tissue samples from the Duroc pig used for production of the swine genome sequence (Sscrofa10.2), as an adult time point for the DNA methylation map. In addition, we performed RNAseq and RRBS on hippocampus samples obtained from two studies assessing the effects of early life environmental insult on cognitive development. The first model is a piglet undergoing iron deficiency. Iron deficiency is the most prevalent micronutrient deficiency in the world and has been linked to cognitive impairments as well as attention deficit/hyperactivity disorder in human infants. Our collaborators have shown that neonatal piglets are a good model to study the mechanisms by which iron deficiency effects cognition (Rytych et al., 2012). The second model of environmental insult involves postnatal infection of piglets with porcine reproductive and respiratory syndrome virus (PRRSV) and is regarded as a model of viral pneumonia. Little is known about the effects of peripheral viral infection on brain function and development in the neonatal period, and again our collaborators have shown that neonatal piglets are a good model to study the mechanisms by which viral infection effects cognition (Elmore et al., 2014). During the second grant year, we determined the optimum alignment pipeline for the RRBS and RNAseq datasets, determined the level of additional sequencing required to reach our depth goals (50x for RNAseq, 20x for RRBS), resequenced the libraries and have made progress performing subsequent statistical analysis. In addition, we began analysis of a third model, our inducible transgenic p53/Kras Onco-pig model of cancer. Due to the urgent need for more relevant animal models of cancer, we developed an inducible transgenic porcine model. The transgene construct contains oncogenic KRASG12D and dominant-negative p53R167H, downstream of a LoxP-polyA (STOP)-LoxP sequence (LSL) and a CAG promoter. These mutations were used because both KRASG12D, associated with increased cell motility and invasiveness and p53 R175H, a necessary tumor suppressor, occur at a high frequency in many types of human cancer. This design enables both temporal and spatial control of transgene expression. Fibroblast cell lines and animals derived from the transgenic pigs can be transformed by Cre recombination. This triggers oncogene expression and induces the cells to become tumorigenic. Therefore, we are comparing the gene expression and methylation differences between the control and tumorigenic cells. We will also continue to develop a methylome map of tissues and developmental stages to enhance the published pig genome. It is our hope that the results from this research will allow for the increased understanding of the importance of DNA methylation patterns in development and disease.
