Bio Lab Paper
- 1. Trevor Lythgoe Biology Lab Wed 1900 Summary of DNA Methylation Profiling of the Human Major Histocompatibility Complex: A Pilot Study for the Human Epigenome Project The following is a summary of the Pilot Study for the Human Epigenome as was conducted by Vardhman K. Rakyan, Thomas Hildmann, Karen L. Novik, Jorn Lewin, Jorg Tost, Antony V. Cox, T. Dan Andrews, Kevin L. Howe, Thomas Otto, Alexander Olek, Judith Fischer, Ivo G. Gut, Kurt Berlin, and Stephan Beck. Introduction The Human Epigenome Project is intended to shed light on the complex process of gene expression 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 to become neurons, muscle cells, blood cells etc. For instance, while the daughter cell acquires new functions from the parent cell, it retains the DNA code necessary to continue in its role of reproduction and growth. As a cell replicates it goes through a process called chromatin remodeling, which is accomplished through two main mechanisms, one of which addressed in this study is the addition of methyl groups to the CpG sites, which convert cytosine to 5-methylcytosine. 5-methylscyotsine acts much the same as cytosine in pairing with guanine, however, highly methylated areas tend to be less transcriptionally active. The question of what importance this step is to the development of the human cell expression was addressed in the study. The pilot study was specifically aimed at the Human Major Histocompatibility Complex (MHC), a cluster of genes located on chromosome 6 necessary to antigen production and critical to the success of transplantation. Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of the human major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12): e405.
- 2. Methods Rakyan et al analyzed DNA methylation in seven human tissues (healthy and their diseased variants) from 32 separate individuals– adipose, brain, breast, liver, lung, muscle and prostate– using bisulphate sequencing and created a software program ESME to determine the DNA methylation levels from the sequence trace files. This they compared with a known system called matrix-assisted laser desorption/ionization mass spectrometry (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 the older, but slower, known methods. Results A major goal of the pilot study was to create a database that would be publicly accessible and adequately display the data learned through the study. The 253 unique amplicons successfully analyzed were mapped to the human genome assembly using BLAST (Basic Local Alignment Search Tool– an algorithm used for comparing primary biological sequence information such as the amino-acid sequences of different proteins or the nucleotides 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 at http://www.epigenome.org. Rakyan et al discovered that the methylation profile of the MHC region appears to be strongly bimodal, 90% of the amplicons being hyper- (more than 70% median methylation of amplicon) or hypomethylated (less than 30% of median methylation of Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of the human major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12): e405.
- 3. amplicon.) They confirmed the idea that these extremes of methylation profiles are the most stable states within the genome, finding also that the majority of the CpG islands were hypomethylated throughout development. Hypermethalated regions result in transcriptional silencing effectively turning off the gene through an environment of heterochromatin. While a majority of the genes that were associated with the tissue-specific methylation profiles in this study showed no corresponding tissue-specific expression profiles in a previous analysis, a significant portion (10%) of all amplicons displayed differential methylation 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 relative to intragenic amplicons analyzed in this study. One reason for the lack of significant data supporting the identification of tissue specificity of methylation profiles is that the detection of some genes that are known to be associated with various mRNA isoforms is quite difficult with conventional microarray analysis. Another theory is that there are only a small proportion of genes affected by methylation result in tissue specificity. Increasing evidence suggests that an individual’s epigenetic profile can influence phenotype and susceptibility to various diseases such as cancer. This is supported by the amount of intragenic amplicons found despite the fact that nearly all loci studied evidenced some degree of heterogeneity. The primary purpose of epigenetic modifications is to control gene expression. An Citation: Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, et al. (2004) DNA methylation profiling of the human major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12): e405.
- 4. abnormal epigenetic modification, as in many cancers, occurs within in the CpG islands that overlap promoters, which may result in aberrant transcription of the cognate gene, a tumor suppressor. If it were possible to control epigenetic modification the possibility of eliminating genetic mutations like cancer, or even in the more moderate form of controlling genetic disease in plants, would be an incredible boon to society as a whole. Conclusion The biggest challenge of the Human Epigenome Project is acquiring all the elements from several large-scale studies and obtaining a single holistic view of the human genome. The HEP database will provide a central location to collect the results from various studies. The results from the pilot study show a significant proportion of the analyzed loci within the MHC show tissue-specific methylation profiles, which may help us to understand certain cell phenotypes. Understanding certain cell phenotypes may lead to 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 the human major histocompatibility complex: A pilot study for the human epigenome project. PLoS Biol 2(12): e405.