Trevor Lythgoe Biology Lab Wed 1900 Summary of DNA Methylation Profiling of the Human Major Histocompatibility Complex: A Pilot Study for the Human Epigenome ProjectThe following is a summary of the Pilot Study for the Human Epigenome as wasconducted by Vardhman K. Rakyan, Thomas Hildmann, Karen L. Novik, Jorn Lewin,Jorg Tost, Antony V. Cox, T. Dan Andrews, Kevin L. Howe, Thomas Otto, AlexanderOlek, Judith Fischer, Ivo G. Gut, Kurt Berlin, and Stephan Beck.IntroductionThe Human Epigenome Project is intended to shed light on the complex process of geneexpression by mechanisms other than changes in the underlying DNA sequence. In short,the Project hopes to understand what process allows for a single fertilized egg cell tobecome neurons, muscle cells, blood cells etc. For instance, while the daughter cellacquires new functions from the parent cell, it retains the DNA code necessary tocontinue in its role of reproduction and growth. As a cell replicates it goes through aprocess called chromatin remodeling, which is accomplished through two mainmechanisms, one of which addressed in this study is the addition of methyl groups to theCpG sites, which convert cytosine to 5-methylcytosine. 5-methylscyotsine acts much thesame as cytosine in pairing with guanine, however, highly methylated areas tend to beless transcriptionally active. The question of what importance this step is to thedevelopment of the human cell expression was addressed in the study. The pilot studywas specifically aimed at the Human Major Histocompatibility Complex (MHC), acluster of genes located on chromosome 6 necessary to antigen production and critical tothe success of transplantation.Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of thehuman major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12):e405.
MethodsRakyan et al analyzed DNA methylation in seven human tissues (healthy and theirdiseased variants) from 32 separate individuals– adipose, brain, breast, liver, lung, muscleand prostate– using bisulphate sequencing and created a software program ESME todetermine the DNA methylation levels from the sequence trace files. This they comparedwith a known system called matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) and found that their results had a concordance rate of 88%.The concordance rate of 88% suggests the validity of the new method compared with theolder, but slower, known methods.ResultsA major goal of the pilot study was to create a database that would be publicly accessibleand adequately display the data learned through the study. The 253 unique ampliconssuccessfully analyzed were mapped to the human genome assembly using BLAST (BasicLocal Alignment Search Tool– an algorithm used for comparing primary biologicalsequence information such as the amino-acid sequences of different proteins or thenucleotides of DNA sequences.) (http://blast.ncbi.nlm.nih.gov/Blast.cgi, 18 March 2011)The results of the study were made available on the internet athttp://www.epigenome.org.Rakyan et al discovered that the methylation profile of the MHC region appears to bestrongly bimodal, 90% of the amplicons being hyper- (more than 70% medianmethylation of amplicon) or hypomethylated (less than 30% of median methylation ofCitation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of thehuman major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12):e405.
amplicon.) They confirmed the idea that these extremes of methylation profiles are themost stable states within the genome, finding also that the majority of the CpG islandswere hypomethylated throughout development. Hypermethalated regions result intranscriptional silencing effectively turning off the gene through an environment ofheterochromatin.While a majority of the genes that were associated with the tissue-specific methylationprofiles in this study showed no corresponding tissue-specific expression profiles in aprevious analysis, a significant portion (10%) of all amplicons displayed differentialmethylation between the tissue types. Of these 31% were located in the upstream regions,a proportion that is in the same range as the total number of upstream amplicons relativeto intragenic amplicons analyzed in this study. One reason for the lack of significant datasupporting the identification of tissue specificity of methylation profiles is that thedetection of some genes that are known to be associated with various mRNA isoforms isquite difficult with conventional microarray analysis. Another theory is that there areonly a small proportion of genes affected by methylation result in tissue specificity.Increasing evidence suggests that an individual’s epigenetic profile can influencephenotype and susceptibility to various diseases such as cancer. This is supported by theamount of intragenic amplicons found despite the fact that nearly all loci studiedevidenced some degree of heterogeneity.The primary purpose of epigenetic modifications is to control gene expression. AnCitation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of thehuman major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12):e405.
abnormal epigenetic modification, as in many cancers, occurs within in the CpG islandsthat overlap promoters, which may result in aberrant transcription of the cognate gene, atumor suppressor. If it were possible to control epigenetic modification the possibility ofeliminating genetic mutations like cancer, or even in the more moderate form ofcontrolling genetic disease in plants, would be an incredible boon to society as a whole.ConclusionThe biggest challenge of the Human Epigenome Project is acquiring all the elementsfrom several large-scale studies and obtaining a single holistic view of the humangenome. The HEP database will provide a central location to collect the results fromvarious studies. The results from the pilot study show a significant proportion of theanalyzed loci within the MHC show tissue-specific methylation profiles, which may helpus to understand certain cell phenotypes. Understanding certain cell phenotypes may leadto the ability to control epigenetic modification and the cure for cancer.Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of thehuman major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12):e405.