Source: UNIV OF WISCONSIN submitted to
INTERPRETING CATTLE GENOMIC DATA: BIOLOGY, APPLICATIONS AND OUTREACH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0201661
Grant No.
(N/A)
Project No.
WIS04895
Proposal No.
(N/A)
Multistate No.
NC-1010
Program Code
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Project Director
Khatib, H.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
DAIRY SCIENCE
Non Technical Summary
While most of our genes are simultaneously expressed from both maternally and paternally expressed chromosomes, there is a subset of genes that are only expressed from one of the parental alleles. This is called genomic imprinting. The number of imprinted genes currently defined in mammalian species is about 75, most of which were studied and identified in human and mouse. There is only one reported imprinted gene (M6P/IGF2R) in cattle. It is of interest to investigate the imprinting status of additional bovine genes for a number of reasons. First, examining the extent of conservation among mammals would offer a powerful tool for identifying novel imprinted genes and their regulatory elements. Secondly, a number of studies have mapped imprinted quantitative trait loci (QTL) in livestock species. The main goal of this project is to investigate the imprinting status of cattle genes and identify potential imprinted genes affecting production traits in dairy cattle. For the fraction of identified genes through the positional candidate gene approach, the potential for useful application in genetic improvement programs is truly enormous. Identification of quantitative genes associated with improved milk yield and composition will provide a powerful tool for genetic improvement through marker assisted selection in the Holstein population and in other related populations.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30334991040100%
Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3499 - Dairy cattle, general/other;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
1. Determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle. 2. Interpret and apply genomics and proteomics information by developing statistical/bioinformatics methods and utilizing molecular tools in cattle. 3. Develop and deliver educational materials about bovine genomics research to consumers and stakeholders.
Project Methods
Objective 1: 1. Identification of 15-20 provisional imprinted genes in bovine genome. 2. Developing SNPs in bovine genes: a. primer design- primers will be designed to amplify cDNAs to investigate the expression of genes. b. RNA extraction from various bovine tissues (obtained from local slaughterhouse) by standard procedures used routinely in our lab. RNA will be extracted from 10-12 tissues from each individual (5-10 individuals). RT-PCR will be performed on each RNA and RT-PCR products will be sequenced. c. SNP sequence analysis of imprinting status of genes- sequences will be analyzed for homozygosity (monoallelic expression) and heterozygosity (biallelic expression) of genes. Objective 2: 1. Construction of DNA pools from individuals in the high and low tails of protein %, fat %, protein yield, fat yield, and milk yield traits. 2. Determination of SNP allele frequencies in DNA pools at 40 candidate genes already identified in our lab. 3. Analysis of the results- estimation of SNP allele effects on trait values. 4. Genes showing significant results will be tested for imprinting.

Progress 10/01/04 to 09/30/07

Outputs
OUTPUTS: Imprinted genes are monoallelically expressed in a parent-of-origin-specific manner under epigenetic regulation. Identification of species-specific imprinted genes is necessary to understand the evolution of genomic imprinting and to elucidate mechanisms leading to allele-specific expression. The main goal of this project is to investigate the imprinting status of cattle genes and identify potential imprinted genes affecting production traits in dairy cattle. PARTICIPANTS: Ismail Zaitoun, a graduate student TARGET AUDIENCES: Dairy cattle breeding associations, dairy producers and farmers in Wisconsin and in the US

Impacts
Comparative expression analysis and imprinting status are powerful tools for investigating biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. In livestock species, the imprinting status of gene has important role in fertility, growth and development. Imprinted genes are monoallelically expressed in a parent-of-origin-specific manner under epigenetic regulation. Although it is generally believed that genomic imprinting is conserved among mammalian species, there is accumulating evidence that suggests such an assumption is false. Identification of species-specific imprinted genes is necessary to understand the evolution of genomic imprinting and to elucidate mechanisms leading to allele-specific expression. In a recent study, we analyzed the imprinting status of the CD81, TSSC4, and OBPH1 genes clustered on bovine chromosome 29; the PEG10 and ASB4 genes clustered on bovine chromosome 4; and the HTR2A microdomain gene on bovine chromosome 12 using a sequencing-based approach. CD81 and OBPH1 showed biallelic expression in all cattle tissues examined, while TSSC4 showed monoallelic expression in placental tissues, like its mouse orthologue. Comparative expression analysis showed that the imprinting pattern of the CD81, TSSC4, and OBPH1 cluster was not conserved among mouse, human, and cattle. None of these genes was imprinted in all three species. The PEG10 gene was imprinted in all three species, while ASB4, reported to be imprinted in mouse, was not imprinted in cattle. The HTR2A gene was not imprinted in cattle, and human imprinting data has shown conflicting results. It is more likely that imprinting in the genes examined in this study is species-specific. In addition, we studied the expression and tissue distribution of transcripts of these genes in 174 fetal and adult cattle tissues.

Publications

  • Zaitoun I and Khatib H. 2007. Comparative genomic imprinting and expression analysis of six cattle genes. J. Anim Sci.86:25-32.
  • Khatib H. 2007. Invited. Is it genomic imprinting or preferential expression. BioEssays 29:1-7.
  • Khatib H Zaitoun I and Kim E. 2007. Invited. Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle. Mamm Genome 18:538 547.


Progress 01/01/06 to 12/31/06

Outputs
Imprinted genes are monoallelically expressed in a parent-of-origin-specific manner under epigenetic regulation. Identification of species-specific imprinted genes is necessary to understand the evolution of genomic imprinting and to elucidate mechanisms leading to allele-specific expression. The main goal of this project is to investigate the imprinting status of cattle genes and identify potential imprinted genes affecting production traits in dairy cattle. To study the imprinting status of cattle genes and asses their conservation among mammalian species, the expression patterns of COPG2, DCN, and SDHD genes were examined in a total of 128 fetal and adult tissues. Two single nucleotide polymorphisms (SNPs) were identified in COPG2, three SNPs in DCN, and one SNP in SDHD. In all 128 tissues, COPG2, DCN, and SDHD transcripts showed biallelic expression. This is the first report of genes that are not imprinted in cattle while the corresponding genes in human or mouse are imprinted. In a different study, we assessed the imprinting status and pattern of expression of SLC38A4, NNAT, NAP1L5, and H19 in a total of 75 tissues obtained from 12 fetuses and dams. In contrast to the mouse gene which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle. Also, we found that it is expressed in all adult tissues examined in contrast to expression pattern in adult mouse. The two transcripts of NNAT showed paternal expression like their counterparts in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of fetal and adult tissues which suggests additional functions to those known in rain development. NAP1L5 is an intron-less gene located in the intron of HERC3 gene. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene is paternally expressed in all examined tissues, in contrast to mouse where it is tissue-specific imprinted. H19 was maternally expressed like its counterparts in human, sheep, and mouse. Recently, we analyzed the imprinting status of the CD81, TSSC4, and OBPH1 genes clustered on bovine chromosome 29; the PEG10 and ASB4 genes clustered on bovine chromosome 4; and the HTR2A microdomain gene on bovine chromosome 12 using a sequencing-based approach. CD81 and OBPH1 showed biallelic expression in all cattle tissues examined, while TSSC4 showed monoallelic expression in placental tissues, like its mouse orthologue. Comparative expression analysis showed that the imprinting pattern of the CD81, TSSC4, and OBPH1 cluster was not conserved among mouse, human, and cattle species. None of these genes was imprinted in all three species. PEG10 was imprinted in all three species, while ASB4, reported to be imprinted in mouse, was not imprinted in cattle. HTR2A was not imprinted in cattle, and human imprinting data has shown conflicting results. It is more likely that imprinting in the genes examined in this study is species-specific. In addition, we studied the expression and tissue distribution of transcripts of these genes in 174 fetal and adult cattle tissues.

Impacts
Comparative expression analysis and imprinting status are powerful tools for investigating biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. In livestock species, the imprinting status of gene has important role in fertility, growth and development.

Publications

  • Zaitoun I. and Khatib H. 2006. Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle. BMC Genetics 7:49
  • Khatib H and Schutzkus V. 2006. The expression profile of the H19 gene in cattle. Mamm Genome 17, 991-6.


Progress 01/01/05 to 12/31/05

Outputs
The number of imprinted genes currently defined in mammalian species is about 75, most of which were studied and identified in human and mouse. The main goal of this project is to investigate the imprinting status of cattle genes and identify potential imprinted genes affecting production traits in dairy cattle. To study the imprinting status of cattle genes and asses their conservation among mammalian species, the expression patterns of COPG2, DCN, and SDHD genes were examined in a total of 128 fetal and adult tissues. Two single nucleotide polymorphisms (SNPs) were identified in COPG2, three SNPs in DCN, and one SNP in SDHD. These polymorphisms were employed to distinguish between monoallelic and biallelic expression using a primer extension method and a sequencing-based approach. In all 128 tissues, COPG2, DCN, and SDHD transcripts showed biallelic expression. This is the first report of genes that are not imprinted in cattle while the corresponding genes in human or mouse are imprinted. We assessed the imprinting status and pattern of expression of SLC38A4, NNAT, NAP1L5, and H19 in a total of 75 tissues obtained from 12 fetuses and dams. A polymorphism-base approach was used to assess the imprinting status of the bovine genes. In contrast to the mouse gene which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle. Also, we found that it is expressed in all adult tissues examined in contrast to expression pattern in adult mouse. Two single nucleotide polymorphisms (SNPs) were identified in NNAT and used to distinguish between monoallelic and biallelic expression in fetal and adult tissues. The two transcripts of NNAT showed paternal expression like their counterparts in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of fetal and adult tissues which suggests additional functions to those known in rain development. NAP1L5 is an intron-less gene located in the intron of HERC3 gene. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene is paternally expressed in all examined tissues, in contrast to mouse where it is tissue-specific imprinted. H19 was maternally expressed like its counterparts in human, sheep, and mouse.

Impacts
Comparative expression analysis and imprinting status are powerful tools for investigating biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. In livestock speceis, the imprining status of gene has important role in fertility, growth and development.

Publications

  • Khatib H. (2005). The COPG2, DCN, and SDHD genes are biallelically expressed in cattle. Mamm Genome 16, 545-552.
  • Khatib H. (2005). Characterization and analysis of the imprinting status of the ovine SDHD and COPG2 genes. Anim Genet. 36, 186-8.


Progress 01/01/04 to 12/31/04

Outputs
The objectives of our research are to study the imprinting status of cattle genome. Identification of imprinted genes will allow better understanding of gene expression regulation. We have identified one imprinted gene NESP55 as maternally expressed. In addition, we've investigated the protease inhibitor gene and found that gene displays monoallelic expression in some tissue, whereas displaying biallelic expression in other tissues. Both genes were published in two different publications in Mammalian Genome Journal.

Impacts
It is of interest to investigate the imprinting status of additional bovine genes for a number of reasons. First, examining the extent of conservation among mammals would offer a powerful tool for identifying novel imprinted genes and their regulatory elements. Second, studies have mapped imprinted QTL in other livestock species. Thus, it would be of interest to examine imprinted genes as sources of quantitative genetic variation. In mammals in particular, imprinted genes have an important function in the regulation of fetal growth, development, function of the placenta, and postnatal behavior. Consequently, variation in imprinting status of imprinted genes may be a source of genetic variation in difficult birthing and perinatal mortality.

Publications

  • Khatib H. (2004) Imprinting of Nesp55 gene in cattle. Mamm Genome. 15(8):663-7.
  • Khatib H. (2005) Monoallelic expression of the protease inhibitor gene in humans, sheep, and cattle. Mamm Genome. 16(1):50-8.