Epigenetics. © INRA, INRA

Livestock epigenetics: laying the foundation for future benefits

Determining bovine epigenotypes

Scientists at the INRA research center in Jouy-en-Josas have developed a tool that allows them to characterize the methylomes of cattle, a crucial step if we want to have a better grasp of the epigenetic mechanisms that control phenotype expression in this group of domestic animals.

By Pascale Mollier, translated by Jessica Pearce
Updated on 07/10/2014
Published on 05/22/2014

Just as we can sequence genomes, that is to say, describe the nucleotide sequences of which they are composed, we can determine the methylation patterns of DNA sequences. All of the methylation patterns found throughout the genome are referred to, en masse, as the “methylome.” However, it is important to note that, of the four nucleotides that make up DNA (adenine, cytosine, guanine, and thymine), only cytosines situated in front of guanines are targets for methylases.

The importance of characterizing epigenotypes

The addition and removal of epigenetic tags (methyl groups placed on DNA and histones) are the basic mechanisms by which gene expression is regulated. During development, these tags serve as the basis for cell differentiation because they determine which genes are expressed in the different tissue types. Over the course of an organism’s life, epigenetic tags, and thus gene expression, will shift depending on what is going on in the environment (e.g., dietary changes or stress), which allows individuals to respond dynamically to their surroundings. However, this genomic “plasticity” can be challenging for researchers because it is necessary to figure out at which moment and in which tissue epigenetic modifications are taking place. It is for this reason that it is necessary to develop high-throughput-sequencing tools. Such tools are not currently available to the cattle industry. Researchers at Jouy-en-Josas have refined certain approaches (a microarray and software to perform statistical and bioinformatics analyses) that can quickly and affordably reveal the methylation patterns associated with different factors (see the examples provided below). Using these tools, they hope to be able to help elucidate the epigenetic mechanisms underlying a variety of the issues facing livestock systems.

Comparison of bovine tissues reveals that methylation may play a role in the complement system

The researchers compared the methylome of liver cells with those of two other cell types: fibroblasts and spermatozoa. They found 5,634 differences between the methylation patterns of spermatozoa and liver cells and thus evidence for cell-type-specific epigenetic profiles. There were significant differences in these profiles when it came to genes that encode proteins belonging to the complement system, which is a component of the innate immune system. In liver cells, these genes are hypomethylated, which means that they can be more easily expressed, thus facilitating the production of complement system proteins. The pattern of methylation in the liver therefore underscores the important role played by this organ in the immune system.

Methylome differences between two cattle breeds

When the researchers compared the methylomes of liver cells taken from Holstein cows and Japanese Black cows, they found 3,642 differences between the two breeds. Unexpectedly, these differences were largely associated with genes involved in gestation and placental implantation. These different epigenetic profiles may stem from genetic differences between the two breeds, which have been subject to very different forms of artificial selection with different production goals in mind. They may also reflect differences in the metabolic processes that kick in when gestation begins. For instance, in Holsteins, metabolic demands are very high at the peak of milk production.

Age-related epigenetic profiles in liver cells are disrupted by cloning

These same tools have also allowed researchers to uncover age-related differences in epigenetic profiles: 257 genome regions have different methylation patterns in young (perinatal) versus adult cattle. These differences likely reflect the liver having become metabolically accustomed to the animal eating autonomously and consuming an herbivorous diet after birth. These profiles are disrupted in an interesting way when cattle are cloned—in cloned animals, a certain number of genomic regions do not reflect the expected epigenetic “age” of the animal. Furthermore, 87 epigenetic differences attributable to the cloning process itself were identified, which confirms that cloning via somatic-cell nuclear transfer can cause epigenetic disruptions at the genome level.  

Next-generation sequencing tools are currently being developed thanks to research platforms that bring together several research units to collaborate on various projects. The GenEpi project is examining epigenetic tags that can be used to diagnosis an animal’s physiological state as well as those that may be useful in predicting an animal’s response to an immune challenge using easily accessible cells, namely white blood cells (monocytes). The SeQuaMol project, which is funded by a national research grant (ANR Labcom) and which is being conducted in collaboration with UNCEIA, seeks to characterize the epigenetic patterns associated with fertility in bulls.

Contact(s)
Scientific contact(s):

  • Hélène Kiefer UMR1198 BDR Joint Research Unit for Developmental Biology and Reproduction
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Animal Physiology and Livestock Systems
Associated Centre(s):
Jouy-en-Josas

A MICROARRAY THAT CAN REVEAL THE METHYLATION PATTERNS OF REGULATORY REGIONS IN CATTLE

Design of a microarray for cattle that includes the putative regulatory regions of 21,416 genes.. © INRA
Design of a microarray for cattle that includes the putative regulatory regions of 21,416 genes. © INRA

The genomic regions targeted by the microarray (regulatory regions (1)) are broken up into a number of small fragments called probes that are attached to the biochip. At the same time, DNA from the cells of interest is also broken up into fragments and exposed to an antibody that specifically recognizes methylated DNA fragments, allowing them to be filtered out. These fragments are then labeled with green fluorescent molecules. The original DNA, which has not been filtered using antibodies, is labeled with red fluorescent molecules. After the labeled DNA has hybridized with DNA attached to the chip, researchers can visualize the pattern of fluorescence. “Greener” probes have greater quantities of methylated DNA attached to them. In this way, it is possible to identify which regions of the genome are methylated and thus compare, for instance, methylation patterns among cell types and individuals as well as those generated in response to different environmental factors and over time. This microarray created for cattle contains probes for the regulatory regions of over 20,000 genes.

(1) The target regions represented on the microarray are putative gene regulatory regions, to which epigenetic tags are frequently added. These regions are chosen in silico, by employing sequences located to either side of the transcription start site for each gene; as a result, they are likely to contain a gene’s promoter and its untranslated 5’ sequence, which are both involved in gene regulation. These chromosomal coordinates are then sent off to the company that manufactures the microarrays, which uses algorithms to choose the best probes to use (e.g., based on parameters such as genome specificity, the presence of DNA secondary structures, and sequence size).