|Freking, Bradley - Brad|
|Nonneman, Danny - Dan|
Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2009
Publication Date: 7/1/2009
Citation: Bischoff, S.R., Tsai, S., Hardison, N., Motsinger-Reif, A.A., Freking, B.A., Nonneman, D.J., Rohrer, G.A., Piedrahita, J.A. 2009. Characterization of Conserved and Non-conserved Imprinted Genes in Swine. Biology of Reproduction. 81(5):906-920.
Interpretive Summary: Genomic imprinting results in the silencing of a subset of mammalian alleles due to parent-of-origin inheritance. In contrast to the majority of genes where expression is from both alleles, genomic imprinting leads to monoallelic expression from either the paternal or maternal strand. Human disorders such as cancer, autism, and bipolar disease have been associated with imprinted genes. Additionally, due to the nature of their expression patterns they play a critical role in placental and early embryonic development. It is interesting and evolutionarily important to note that the phenomenon of genomic imprinting and placentation have co-evolved in both placental animals and flowering plants. Currently, there are more than 83 known imprinted genes, and their imprinting status is mostly conserved between human and mouse. In order to increase our understanding of the role of imprinted genes in swine it is important that a comprehensive analysis of imprinted genes be carried out. Understanding and accounting for imprinting in marker assisted management schemes is important for swine production since typical pork production mating systems utilize different germplasm sources or breeds in specifically defined paternal and maternal roles; e.g., Landrace x Yorkshire F1 females mated to Duroc boars in terminal sire production systems. By exploiting the differences in transcript dosage between uniparental and biparental fetuses in four different tissues, we categorically summarized our results of imprinted genes that are conserved and discordant across mammalian clades. Discrepancies may shed light on variations on placental biology. Using Affymetrix Porcine GeneChip microarrays, four tissues of day 30 fetuses were profiled: brain, fibroblast, liver, and placenta. Evidence was generated that identified 25 known imprinted genes differentially expressed in at least one of the four tissue types. To extend the expression analysis results, we screened the imprinting status of additional candidate imprinted genes. Samples of brain, carcass, liver, and placenta were collected from day 30 pregnancies generated from reciprocal crosses of Meishan and occidental breeds, and polymorphisms were identified in several of the candidate genes. Imprinting status was confirmed for 11 of these genes in at least one tissue. We also evaluated genes whose imprinting status has been disputed in human or mouse models, and found nine of these genes not to be imprinted in swine. This manuscript represents the most comprehensive study of comparative mammalian imprinting in swine to date and forms the basis for future studies and their functional significance.
Technical Abstract: In order to increase our understanding of the role of imprinted genes in swine reproduction we used two complementary approaches, analysis of imprinting by pyrosequencing, and expression profiling of parthenogenetic fetuses, to carry out a comprehensive analysis of this gene family in swine. Using Affymetrix Porcine GeneChip microarrays and/or semi-quantitative PCR, four tissues of day 30 fetuses were profiled: brain, fibroblast, liver, and placenta. Twenty-five known imprinted genes were differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE and SLC38A4 tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing (QUASEP). Samples of brain, carcass, liver, and placenta were collected from day 30 pregnancies generated from reciprocal crosses of Meishan and occidental breeds, and SNPs identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also analyzed a subset of genes whose imprinting status has been reported in human or mouse models, and found no evidence of imprinting in ASB4, ASCL2, CALCR, CD81, COMMD1, DCN, DLX5, H13, and HTR2A. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date and will play a key role in unraveling their role in placental and fetal growth in swine.