Imprinted Gene Expression and Phenotype of Bovine Concepti with Bos taurus and Bos indicus Genetics

Abstract

Epigenetic parent-of-origin effects contribute significantly to phenotypic variation in animals. Imprinted genes, which show differential allelic expression in a parent of-origin dependent manner, are critical regulators of prenatal development. Altered epigenetic modifications of imprinted genes in response to maternal environmental stimuli are believed to impact on prenatal development with long-term consequences for postnatal phenotype, a process which is known as fetal programming. Most studies in the area of fetal programming have focused on the epigenetic link between intrauterine environment and fetal phenotype, and genetic programming of variation in pre- and postnatal growth traits remains largely unexplored. We hypothesised that heterosis, i.e., the superiority of F1 hybrids compared to their parents, in growth traits is programmed prenatally through changes in expression patterns of imprinted genes. The purpose of this thesis was (i) to perform a comparative in silico analysis of promoter-specific transcripts and splice variants of the imprinted IGF2 and IGF2R genes as well as their imprinted regulatory non-coding RNAs, H19 and AIRN, (ii) to analyse expression patterns of all identified transcripts in bovine pre- and postnatal tissues, and (iii) to investigate fetal genetic effects on gene expression and their association with heterotic phenotype. To this end, a bovine model was employed using two genetically and phenotypically distinct subspecies of domesticated cattle, Bos taurus and Bos indicus, and their reciprocal crosses. Real time quantitative PCR was used to quantify expression of IGF2 promoterspecific transcripts, IGF2R, H19 and AIRN, in liver, brain, heart, cotyledon, skeletal muscle, kidney, lung and testis at three developmental stages, Day-48 embryo, Day-153 fetus and 12- month juvenile. Heterotic effects on transcript abundances and expression patterns of imprinted genes and their correlations with fetal body weight and weights of fetal tissues were estimated with general linear models. xiv All studied imprinted genes were subject to developmental control of gene expression with the transcript abundance being downregulated in postnatal tissues. We identified IGF2 promoter-specific transcripts and the bovine orthologue of human P0 promoter, and found a developmental shift in tissue specificity of P0 from fetal skeletal muscle to postnatal liver. Fetal body weight and absolute weights of fetal tissues, except brain, were subject to significant parent-of-origin effects, and were higher in fetal groups with B. taurus maternal genetics compared to B. indicus maternal genetics. Fetal placental weight, lung weight and relative muscle mass showed significant heterosis. Heterosis in fetal placental weight was associated with a polar overdominance imprinting pattern of IGF2R and AIRN in cotyledon with highest expression in B. indicus (sire) × B. taurus (dam) group, whereas the transcript abundance in the other reciprocal was close to purebred groups. In hybrid genetic groups, expression of IGF2R and AIRN in cotyledon was positively correlated with weight of fetal placenta and negatively correlated with placental efficiency, defined as fetal to placental weight ratio. H19 expression in skeletal muscle was significantly affected by the interaction between parental genomes. With respect to significant negative association between H19 expression and muscle mass, the negative heterotic effect on H19 expression may explain the positive heterosis in relative muscle mass. Fetal genetics consistently influenced H19 expression, which in B. indicus was between 1.5 to 2.2-fold higher than in B. taurus in all examined tissues, except brain. A negative relationship was observed between transcript abundance of H19 and weights of fetal tissues, except brain. These results suggest that H19 could be a molecular driver for differential subspecies-specific fetal phenotypes. By in silico search, we found B. indicus-specific nucleotide polymorphisms at CpG sites of the consensus sequence for the first CTCF binding site within the imprinting control region (ICR) located upstream of H19 promoter. This led us to speculate that higher expression of H19 in B. indicus tissues could be attributable to partial or complete relaxation of imprinting resulting from epigenetic changes in the ICR. xv In conclusion, our results provide insight into the interplay between genetics and epigenetics and its consequences for genetic programming of phenotypic variation and heterosis. Significant interaction between parental genomes on expression of H19, a miRNA precursor and master regulator of an imprinted gene network, and negative relationship between H19 expression and fetal muscle mass suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.