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Epigenetics and the Immune System

What are Epigenetics?

As we all know, your physical traits and characteristics are largely determined by your DNA, which is referred to as your genetic material or code and is inherited from your parents. In general, your DNA cannot change over the course of your life. There are exceptions to this - some cells will undergo small spontaneous mutations that may result in the death of that cell or even the development of a tumor, and cerrtain cell types in the immune system intentionally delete segments of their DNA, but on average your DNA remains fixed at an organism level throughout your life time. While this provides a robust way to preserve genetic information so that it can provide a constant source for new cells and a way to effectively transmit the right information to your eventual offspring, this system lacks a good way to adapt to any change in the immediate environment. Genetic mutations require generations upon generations in order to allow one to effectively adapt to a new environment, and this mutatory process occurs at a species rather than an organismal level akin to, and in fact directly linked to, evolution. To circumvent this problem, cells have a way to modify the functionality of different parts of your DNA without actually changing the nucleotide content of these genes. This process of modifying DNA without altering genes themselves is referred to as epigenetic modification.

Epigenetic modifications provide a means by which specific signals encountered during the lifetime of an organism can mediate long-lived changes in gene expression in target tissues/cell types without the need for DNA mutation. In certain contexts these modifications can be relatively transient in nature, however some may last for the duration of an organsim's life and can be passed from parents to their offspring, thus allowing adaptation to the environment to be inherited far more rapidly than in the context of mutation alone. Epigenetic modifications consist of the addition or alteration of various chemical groups to the external portion of the DNA in affected cells, and depending on the site of modification in the DNA and the chemical makeup of said modification, the cell will treat that DNA in a different manner. For example, one common epigenetic modification of the DNA is known as methylation - the addition of a methyl group composed of carbon and hydrogen to a given nucleotide of the genome. If this methylation occurs at a promoter region that is normally important for expression of a specific gene, it can result in an alteration of the pattern of gene expression by either increasing or decreasing the rate of expression depending on the specifics of that given methylation event. Methyl groups and other epigenetic modifications are added to the DNA by a large array of enzymes, and their functionality is essential to ensure proper epigenetic memory formation.

There are several mechanisms by which gene expression can be altered epigenetically, many or all of which may potentially be involved in a given heritable epigenetic change. Initially, a transient environmental signal can interact with a sensor on a given cell type, as in the case of LPS inducing TLR4 macrophages. This signal is then transduced within the sensing cells, inducing certain molecules which cause these cells to undergo epigenetic modifications including promoter-specific methylation, histone modification, and nucleosome remodeling. These modifications can be maintained beyond the duration of the signal, as in the case of LPS altering the accessibility of promoters in macrophages such that upon restimulation inflammatory cytokines such as IL-6 are not significantly induced whereas other genes are induced with more rapid kinetics than in naïve cells [1]. This enables the cells to better respond to a microbial threat while reducing the chance of an excessive inflammatory response.

Epigenetic Inheritance

While epigenetic modifications can have an important fact on the immediate expression of genes during your lifetime, recent research has also shown that they can have a profound effect on the lives of your children and that this is because certain epigenetic modifications are transmissable through poorly defined mechanisms. One of the most common examples of epigenetic inheritance across generations come from studies of parents that went through periods of extreme hunger during their lives long before they had children. As it turns out, children that were born to parents whose own parents (ie. the child's grandparents) went through periods of starvation were more likely to be obese as they grow up. This “grandfather effect” is one of many examples of how the epigentic modification of genes can have a profound effect on the health of individuals.

While specific epigenetic modifications and their effects are increasingly well-studied and understood, there is still little information on how transgenerational epigenetic inheritance functions. A recent study demonstrated that such inheritance is independent of DNA sequence and specific chromatin modifications, but is dependent on the modifications being present within specific loci [2]. The authors further demonstrate that the longevity of an epigenetic modification is greatly enhanced if it is induced in a fetal mouse, suggesting that both timing and location are critical factors in transmissibility [2]. These results suggest the possibility that the specific chromatin structure of certain loci may shield them from further epigenetic modification such that these loci are capable of being reproducibly transmitted to offspring via female germline cells. It is also possible that the specific chromatin structures recruit specific methyltransferases and/or increase their retention time within these areas of the genome such that a given methylation event will likely be efficiently replicated and thus passed on upon DNA replication.

Testing the relationship between chromatin structure and epigenetic inheritance would be informative, though nontrivial. To attempt to identify the specific proteins which may be associated with epigenetic heritability, one could perform chromatin immunoprecipitation microarrays of the Col1a1 locus in fetal/adult cells which were either dividing (as assessed by CFSE or another marker) or not dividing. Proteins associated with this loci under conditions promoting stable epigenetic inheritance can then be compared to those from non-heritable regions in an effort to identify novel candidate BAF subunits or other proteins likely to be involved in this particular modification. Identification such proteins would allow for attempts are crystallization and strucutural analysis in order to identify unique chromatin binding motifs. Additionally, induction of such proteins could be experimentally modulated in an attempt to link their activity to epigenetic inheritance. While this approach will only have the potential to succeed if protein intereactions play a role in these processes, it is a valuable first step which can be modified in order to search for small RNAs or other factors playing a role in these processes.

Epigenetics in Bees: An Example

While epigenetic inheritance is all well and good, there are some other very remarkable examples of how epigenetic modifications can significantly alter the course of ones life. Specifically, all bees fall into different classes that are morphologically and phenotypically distinct such as worker/drone bees, soldier bees, and queen bees depending on the specific bee species being examined. These classes of bee are not determined genetically, meaning that any newborn bee larvae would have the potential to become a new queen bee. How then do bees produce multiple distinct classes of insect from a single pool of genetic material? The answer lies once again in epigentic modifications.

The epigenetic differences between the genetically identical worker and queen bees is likely to be a difference in cytosine methylation within the genome. This may manifest as a change in frequency of methylation between the two groups at a single set of methylation residues (such as CG residues), or it may be due to an increase in the frequency of non-CG methylation as seen in human embryonic stem cells [3]. Differential methylation frequency would allow different subsets of DNA-binding proteins to bind to genes and recognize methylated residues, thereby altering gene expression patterns in a broad manner producing the myriad changes regulating the queen bee phenotype. Alternatively, differential methylation of introns/exons could lead to alternative splicing, producing modified proteins necessary for queen bee development.

Royal jelly could induce changes in methylation by altering the activity level of the normal DNA methylation machinery by allosteric inhibition/activation, or by directly modifying the stability of the these proteins or their mRNA. Alternatively, royal jelly could function as a signal which leads to up- or down-regulation of these methylation proteins or novel methyltransferases which are not active in worker bees and which methylate alternative residues such as CHH or CHG (where H is A, C, or T).

The key experiment for determining whether or not differential methylation is likely to play a role in queen/worker bee differentiation would be to perform sodium bisulphite sequencing in order to produce single base pair maps of cytosine methylation in both queen and worker bees. Briefly, DNA extracts from these two castes are treated with sodium bisulphite which converts cytosine to uracil while leaving methylcytosine untouched. These DNA extracts can then be sequenced via high-throughput sequencing such that each cytosine in the consequent sequence will represent a site of cytosine methylation. As a control, each sample should include a small amount of unmethylated DNA derived from λ phage, such that bisulphite conversion saturation can be confirmed as having been reached when all of the λ phage cytosines have been converted to uracil.

An assortment of statistical analyses can then be employed to identify differences in methylation between these two castes. Important areas of focus should include CG versus non-CG methylation frequencies, differences in basal methylation rates, differences in sense versus antisense methylation, and differential exon methylation. Particular attention should be paid to the methylation of genes known to be involved in development and reproduction, as these genes are very likely to exhibit significant alterations in methylation in queen bees as compared to worker bees.

There are a number of approaches which could theoretically be taken to demonstrate that specific differences in DNA methylation lead to the unique queen and worker bee phenotypes. One approach would be to modulate the activity of DNA methyltransferases in bee larva exposed to royal jelly. If the effects of a reduction in methylation were of interest, siRNA knockdowns or homozygous/heterozygous knockout bees could be generated for each methyltransferase and combinations thereof. If increased frequency of methylation were of interest, transgenic bees overexpressing specific methyltransferases could be generated to assess the phenotype of these bees as they mature. Temperature-sensitive methyltransferase mutants obtained through mutation screens could be of additional value, allowing for a rough determination of what times during development specific methyltransferase activity is needed to produce a queen bee. These experiments would necessitate the development of genetic tools in bees that currently exist in other model organisms. Such tools include the ability to transfect with sequence specific siRNA, generate knockout/overexpressing bees, and the ability to perform large-scale mutagenesis screens to identify particular methyltransferase mutants.

While this approach would allow for the determination of the role of broad scale changes in methylation, it might be more informative to determine the effects of specific methylation events on certain genes as they relate to the queen bee development. An ideal experiment would be to introduce specific methylated genes of interest into larva not exposed to royal jelly in order to identify which, if any, gene-specific methylation event(s) can recapitulate the queen bee phenotype. Such methylated genes would need to be created and methylated synthetically and introduced into the bee embryos such that they undergo homologous recombination and offspring carrying the synthetic methylated allele can be selected for. Such an approach, while potentially very powerful, is not currently technologically feasible. Even with the ability to introduce genes into bee embryos, the technology necessary to synthesize stably methylated genes would still require further development. This approach would also suffer from the caveat that queen bee development is likely regulated by a large number of differentially methylated genes which would need to be introduced in combination with one another. Were the technology to carry out this experiment available, it should be combined with a genome-wide bisulphite screen of differentially methylated genes in order to identify candidates for specific methylation analysis.

References

  • 1. Spannhoff, A., et al., Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees. EMBO reports, 2011. 12(3): p. 238-243.
  • 2. Foret, S., et al., DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees. Proceedings of the National Academy of Sciences, 2012. 109(13): p. 4968-4973.
  • 3. Lyko, F., et al., The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS biology, 2010. 8(11): p. e1000506.
  • 4. Burdge, G.C. and K.A. Lillycrop, Nutrition, epigenetics, and developmental plasticity: implications for understanding human disease. Annual review of nutrition, 2010. 30: p. 315-339.
  • 5. Veening, J.-W., W.K. Smits, and O.P. Kuipers, Bistability, epigenetics, and bet-hedging in bacteria. Annu. Rev. Microbiol., 2008. 62: p. 193-210.
  • 6. Rodenhiser, D. and M. Mann, Epigenetics and human disease: translating basic biology into clinical applications. Canadian Medical Association Journal, 2006. 174(3): p. 341-348.
  • 7. Laird, P.W. and R. Jaenisch, The role of DNA methylation in cancer genetics and epigenetics. Annual review of genetics, 1996. 30(1): p. 441-464.
  • 8. Ohlsson, R., R. Renkawitz, and V. Lobanenkov, CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. TRENDS in Genetics, 2001. 17(9): p. 520-527.
  • 9. Gilbert, S.F. and D. Epel, Ecological developmental biology: integrating epigenetics, medicine, and evolution. 2009: Sinauer Associates Sunderland.
  • 10. Wolff, G.L., et al., Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. The FASEB Journal, 1998. 12(11): p. 949-957.
  • 11. Egger, G., et al., Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004. 429(6990): p. 457-463.
  • 12. Bird, A., Perceptions of epigenetics. Nature, 2007. 447(7143): p. 396-398.
  • 13. Jones, P.A. and D. Takai, The role of DNA methylation in mammalian epigenetics. Science, 2001. 293(5532): p. 1068-1070.
  • 14. Jones, P.A. and P.W. Laird, Cancer-epigenetics comes of age. Nature genetics, 1999. 21(2): p. 163-167.
  • 15. Foster SL, Hargreaves DC, Medzhitov RM. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature. 2007; 447: 972-8.
  • 16. Wan M, et al. Inducible mouse models illuminate parameters influencing epigenetic inheritance. Development. 2013; 140, 843-852.
  • 17. Lister R, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009; 462: 315-322.


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