class: center, middle, inverse, title-slide .title[ # Methylation introduction ] .author[ ### Mikhail Dozmorov ] .institute[ ### Virginia Commonwealth University ] .date[ ### 2026-04-28 ] --- <!-- HTML style block --> <style> .large { font-size: 130%; } .small { font-size: 70%; } .tiny { font-size: 40%; } </style> <!-- https://gemini.google.com/u/2/app/27600da0f5d8f386 --> ## Epigenomics .pull-left[ - **Epigenomics** can be defined as the genome-wide investigation of stably heritable phenotypes resulting from changes in a chromosome without alterations in the DNA sequence. - Term coined by Conrad Hal Waddington in 1942. ] .pull-right[ <img src="img/waddington_epigenetics.jpg" alt="" width="80%" style="display: block; margin: auto;" /> ] .small[ Berger, S. L., Kouzarides, T., Shiekhattar, R., and Shilatifard, A. (2009). An operational definition of epigenetics. Genes Dev. 23, 781–783. doi: 10.1101/gad.1787609 ] --- ## DNA methylation - DNA methylation is a type of chemical modification of DNA which involves the addition of a methyl group to the number 5 carbon of the cytosine (5C), to convert cytosine to 5-methylcytosine (5mC). - The most well-characterized epigenetic mechanism. - In humans, DNA methylation occurs in cytosines that precede guanines (hence, CpG). - Invertebrates (Drosophila, yeast) do not exhibit cytosine methylation. <img src="img/methylation.png" alt="" width="50%" style="display: block; margin: auto;" /> --- ## DNA modifications - Other nucleotides (Adenine, Guanine, Thymine) can also be modified. - Mainly in bacteria, but genomes of eukaryotes may contain base modifications on bases other than cytosine, such as methylated adenine or guanine. <img src="img/methylation.png" alt="" width="50%" style="display: block; margin: auto;" /> .small[ Sood AJ, Viner C, Hoffman MM. 2016. "**DNAmod: the DNA modification database.**" bioRxiv 071712. https://www.pmgenomics.ca/hoffmanlab/proj/dnamod/ ] --- ## CpG Sites and CpG islands - CpG sites are not randomly distributed in the genome—the frequency of CpG sites in human genomes is 1% (~28 million CpGs), which is less than the expected (~4-6%). - Around 60-90% of CpGs are methylated in mammals. - DNA methylation frequently occurs in repeated sequences and may help to suppress transcription from these sequences and aid chromosomal stability. --- ## CpG Sites and CpG islands - There are regions of the DNA that have a higher concentration of CpG sites (> 60%), named CpG islands, which tend to be located in the promoter regions of many genes. - Between 200-1000 bp in length. - Usually not methylated. - Less than 10% of CpGs occur in CG-dense regions. --- ## non-CG methylation - Embryonic stem cells (ESCs) have ~25% non-CG methylation (mCHG and mCHH, where H=A, C, T). - non-CG methylation correlates with gene expression in ESCs. - non-CG methylation is on the anti-sense strand of gene bodies and correlates with increased _intronic_ transcription in ESCs. - non-CG methylation is depleted in enhancers in ESCs. .small[ Lister, Ryan, Mattia Pelizzola, Robert H. Dowen, R. David Hawkins, Gary Hon, Julian Tonti-Filippini, Joseph R. Nery, et al. “Human DNA Methylomes at Base Resolution Show Widespread Epigenomic Differences.” Nature 462, no. 7271 (November 19, 2009): 315–22. https://doi.org/10.1038/nature08514. ] --- ## Creation and maintenance of DNA methylation - In humans, DNA is methylated by three enzymes: DNA methyltransferase DNMT1, DNMT3a, and DNMT3b. - **DNMT1** is the maintenance methyltransferase responsible for copying DNA methylation patterns to the daughter strands during DNA replication. - **DNMT3a and 3b** are the _de novo_ methyltransferases that, in combination with DNMT3L, set up DNA methylation patterns early in development. --- ## Removal of DNA methylation - Loss of 5mC can be achieved **passively** by dilution during replication or exclusion of DNMT1 from the nucleus. - **Ten-eleven translocation (TET)** family of proteins can **actively** convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) in vertebrates—demethylation. - Iterative oxidations of 5hmC catalyzed by TET result in 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The 5caC mark is excised from DNA by G/T mismatch-specific thymine-DNA glycosylase (TDG), which returns the cytosine residue back to its unmodified state. .small[ Guo, Junjie U., Yijing Su, Chun Zhong, Guo-li Ming, and Hongjun Song. “Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain.” Cell 145, no. 3 (April 29, 2011): 423–34. https://doi.org/10.1016/j.cell.2011.03.022. ] --- ## Roles of DNA methylation .pull-left[ - Transcriptional gene silencing - Maintain genome stability - Embryonic development - Genomic imprinting - X chromosome inactivation (females) ] .pull-right[ <img src="img/methylation1.png" alt="" width="100%" style="display: block; margin: auto;" /> ] .small[ http://learn.genetics.utah.edu/content/epigenetics/imprinting/ ] --- ## Factors associated with changes in DNA methylation - Aging (developmental stage) - Diet - Inflammatory patterns - Environmental exposures - Smoking - Alcohol --- ## DNA methylation and cancer **Hypomethylation** – decrease methylation levels - A lower level of DNA methylation in tumors was one of the first epigenetic alterations to be found in human cancer (Feinberg AP, et al., 1983). - Demethylation of the promoter region of proto-oncogenes will activate normally repressed gene expression. - Global hypomethylation of DNA sequences that are normally heavily methylated may result in: - Chromosomal instability - Increased transcription from transposable elements - An elevated mutation rate due to mitotic recombination --- ## DNA hypermethylation **Hypermethylation** – increase methylation levels - Hypermethylation of CpG islands in the promoter regions of tumor-suppressor genes is a major event in the origin of many cancers. - Hypermethylation of promoters can inactivate tumor-suppressor genes, affecting genes involved in the cell cycle, DNA repair, and the metabolism of carcinogens. - The profiles of hypermethylation of CpG islands in tumor-suppressor genes are specific to the cancer type. --- <img src="img/laird.png" alt="" width="80%" style="display: block; margin: auto;" /> .small[ Laird PW "**Oncogenic mechanisms mediated by DNA methylation.**" Mol Med Today. 1997 http://www.cell.com/moltod/pdf/S1357-4310(97)01019-8.pdf ] --- ## Technologies for Measuring DNA Methylation - **Bisulfite Sequencing (BS-Seq):** The "Gold Standard." Bisulfite treatment converts unmethylated cytosines to uracil, while 5mC remains intact. - **MeDIP-seq:** Methylated DNA Immunoprecipitation followed by sequencing; uses antibodies to isolate methylated fragments. - **BeadChip Arrays (e.g., Illumina EPIC):** High-throughput hybridization-based method targeting specific known CpG sites across the genome. - **Nanopore Sequencing:** Direct detection of modified bases by measuring electrical current changes as DNA passes through a pore. --- ## Application of DNA methylation assays **Early diagnosis** - Detection of CpG-island hypermethylation in biological fluids and serum. **Prognosis** - Hypermethylation of specific genes. Whole DNA methylation profiles. **Prediction** - CpG island hypermethylation as a marker of response to chemotherapy. **Prevention** - Developing DNMT inhibitors as chemopreventive drugs to reactivate silenced genes.