Progress 02/01/06 to 06/30/09
Outputs
OUTPUTS: Genomic imprinting is expression from only one of the two parental copies of a gene and is involved in intra-uterine growth and possibly calf birth weight. Significant differences between the offspring of reciprocal crosses of Bos taurus x Bos indicus cattle contribute to losses estimated at over $500 million in the US annually via difficult calving. The molecular genetics of birth weight in cattle is still largely unknown. Our working hypothesis is that the expression of imprinted genes during placentation and embryogenesis may play important roles in determining birth weight in cattle. Since imprinted genes profoundly affect placental and fetal development, the results of our project may contribute to the following long-term applications of genetic markers to select calves at birth that will favorably exploit parent-of-origin effects, better understanding of the role of imprinted genes on the pathological condition of large offspring syndrome (LOS) associated with nuclear cloning, embryo culture and nuclear transfer in ruminants and comparative molecular genetics of imprinting. We have cloned 4 out of the 5 genes, identified several variants in 2 of the 4 genes and determined gene expression patterns for one of the genes. Additional bioinformatics work has analyzed structural genomic features of homologous imprinted regions in the bovine genome. Results were presented at Plant and Animal Genome meetings in 2007, 2009, 2010 and the Epigenetics Meeting in Melbourne, Australia in Dec 2009. PARTICIPANTS: Dr Gary Hansen, North Carolina State University, Plymouth, NC 27962. Dr Marcos De Donato, Laboratorio Genetica Molecular, IIBCA, Universidad de Oriente, Cumana. Venezuela. Dr Chris Bates, Assistant Professor, Dept of Biology, Augusta State University, Augusta, GA 30904. Several technicians hired at Spelman College with full or partial support from this project. Training opportunity was provided to several undergraduate students at Spelman College who were trained in animal molecular genetics. TARGET AUDIENCES: The target audience for this project is primarily fellow researchers in animal molecular genetics and genomics. Our results have been presented at professional meetings and a few papers have been submitted. We expect several more papers to be submitted from this project. PROJECT MODIFICATIONS: We could not conduct association analysis between identified genetic variants and birth weight due to several reasons: (1) the Principal Investigator moved from Spelman College to Cornell University in summer of 2009. The lab was packed and moved before the work could be completed. As a result, funds to carry out genotyping could not be utilized because Spelman College kept the left-over funds since Cornell University as a major research University is not eligible to receive USDA-NRI seed award funds; (2) our collaborator at University of Florida who was going to provide animal resources moved to North Carolina State University and did not have access to the cattle herd anymore. The remainder of the project is now being completed with non-USDA funds and will be completed in 2010.
Impacts
We successfully cloned bovine cDNAs of 4 out of 5 genes proposed in this project. We cloned 1,551 bp of MAGEL2, 770 bp of PHLDA2, 1,180 bp of CDKN1C and 949 bp of MASH2. We also determined that MAGEL2 is only expressed in cerebral cortex and not in bovine liver, pancreas, skeletal muscle, kidney, lung, ovary, spleen and stomach. These sequences are being prepared for submission to GenBank. We screened the 4 cloned genes for SNPs and found 8 SNPs in 2 genes and did not find any SNPs in the other two. Two coding SNPs and 4 intronic SNPs were found in ASCL2 and 2 coding SNPs in MAGEL2. As a first step to determine epigenetic status of these genes, we performed DNA methylation of the CpG islands in their promoter regions. Our analysis using MS-PCR or pyrosequencing showed that MAGEL2 and ASCL2 are methylated while PHLDA2 and CDKN1C appear to be non-methylated. Due to the lack of access to informative families we could not verify imprinting status of these genes. However, from the results of methylation analysis, MAGEL2 and ASCL2 may be imprinted while the PHLDA2 and CDKN1C may not be.
Publications
- Imumorin, I.G. and De Donato, M. Comparative in-silico positioning of human and mouse imprinted genes in the bovine genome. In-Silico Biology. Submitted. 2010.
- De Donato, M., Young, E. Z., Dubose, P., Jeff, J. Bates, C. and Imumorin, I. G. Comparative analysis of the imprinted region on human chromosome 11 demonstrates differences among mammalian genomes. Epigenetics. Submitted. 2010.
- Imumorin, I.G., Jain, A., Bates, C.S., De Donato, M., and Hansen, G.R. Molecular cloning, methylation analysis and tissue expression of bovine Magel2 gene. Gene. Submitted. 2010.
- Imumorin, I.G. Jain, A., De Donato, M., Bates, C., Tomlin, B., Young, E., Jeff, J., Eubanks, J., Dubose, P., Farooqi, I.M., Okpeku, M and Hansen, G.R. Epigenetics studies in cattle: DNA methylation, gene expression and imprinting analysis of some placental genes in cow. Epigenetics 2009 Australian Scientific Conference. Melbourne, Australia. December 2009.
- Imumorin, I.G. Jeff, J., Dubose, P., De Donato, M., Bates, C. and Hansen G.R. Comparative in-silico analysis and functional classification of putative and verified imprinted bovine genes. 17th International Conference on Plant and Animal Genomes (Plant and Animal Genomes XVI) San Diego, CA. January 2009.
- De Donato, M., Young, E. Z. and Imumorin, I. G. Comparative analysis of the imprinted region on human chromosome 11 demonstrates differences among mammalian genomes. 15th International Conference on Plant and Animal Genomes Plant and Animal Genomes XV), San Diego, CA. January 2007.
- Imumorin, I.G. Dubose, P., and De Donato, M. Comparative in-silico positioning of human and mouse imprinted genes in the bovine genome. 15th International Conference on Plant and Animal Genomes (Plant and Animal Genomes XV), San Diego, CA. January 2007.
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Progress 02/01/06 to 02/01/07
Outputs
Since this proposal was funded, the bovine genome sequence has been released and has provided a unique resource to address an ancillary objective of expanding the repertoire of putative imprinted genes in cattle. Using comparative bioinformatics methods, we have localized 59 human and mouse imprinted genes onto 18 bovine chromosomes with most genes aggregating into 7 blocks. The largest blocks were found on BTA4 and BTA29, which are homologous to human chromosomes 7 and 11, and mouse chromosomes 6 and 7, respectively. The bovine positions were concordant with defined regions of homology in the human and mouse genomes. About 20 genes were matched to bovine contigs that have yet to be assigned to specific chromosome locations. The localization of these genes in the bovine genome will assist further characterization of these genes to determine their imprinting status. To gain better comparative understanding of BTA29 which corresponds to the HSA11p15 region with the most
imprinted genes, we carried out a comparative analysis of the annotated genomic sequences from the human, chimpanzee, mouse, rat, dog and bovine genomes, as well as the genomes of chicken and zebrafish as outgroups. We found very high conservation of synteny, gene order and transcription orientation in all but one of the mammalian genomes, while the chicken and zebrafish genomes showed some rearrangements in this region. The average density of genes in the region was about 80% higher than in the rest of the chromosome for most mammals, except for rodents and chicken that were only about 10 and 20% higher, respectively. Also, the average density of CpG islands was about 2 times higher than in the rest of the chromosome for most mammals, except for rodents which remained about the same. Objective 1: We have used a PCR strategy to obtain sequences of bovine homologues of MAGEL2, RASGRF1, PHLDA2, CDKN1C and MASH2. Through collaboration with Gary Hansen of the University of Florida-Mariana
Station, we have obtained placental tissues for RNA isolation and analysis and currently generating cDNA transcripts for 3 prime and 5 prime amplifications to obtain full length cDNAs. Our next step is allele-specific methylation analysis to determine imprinting status, to be followed by SNP polymorphism screening, and genotyping of SNPs in about 300 Brangus cattle to determine effects of these genes on birth weight.
Impacts
Recent reports have strengthened the evidence that almost if not all imprinted genes are expressed in placenta and fetal tissues. This raises the intriguing possibility that differences in birth weights in mammalian livestock may be due in part to imprinted genes and interactions with environment. The use of in-vitro fertilization, nuclear cloning and transfer in ruminants has revealed a myriad of pathologies including large offspring syndrome. The emerging consensus points to alterations in epigenetic modifications manifested in aberrant reprogramming during pre-implantation development in cultured and cloned embryos. At the center of this consensus is the undeniable role of imprinted genes in the multiple regulatory pathways accompanying mammalian embryonic and fetal development, yet most of these have not been studied in cattle. Our in-silico work has expanded the list of putative imprinted genes in cattle to 59, and our experimental work on 5 of these genes will
hopefully demonstrate imprinting and show their effects on birth weight in cattle. Demonstrating the imprinting status of these and other putative genes will address some of the above problems and may in the long term lead to marker-assisted selection using validated markers derived from these genes.
Publications
- Imumorin, I.G. Dubose, P., and De Donato, M. Comparative in-silico positioning of human and mouse imprinted genes in the bovine genome. 15th International Conference on Plant and Animal Genomes (Plant and Animal Genomes XV), San Diego, CA. January 13 - 17, 2007.
- Dubose, P., Jeff, J., De Donato, M. and Imumorin, I.G. PCR cloning and sequencing of bovine ASCL2, MAGEL2, PHLDA2 and RAGSRF1 genes. Gordon Research Conference on Quantitative Genetics and Genomics, Ventura, CA. Feb 18 - 23. 2007.
- De Donato, M., Young, E. Z. and Imumorin, I. G. Comparative analysis of the imprinted region on human chromosome 11 demonstrates differences among mammalian genomes. 15th International Conference on Plant and Animal Genomes (Plant and Animal Genomes XV), San Diego, CA. January 13 - 17, 2007.
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