DNA methylation_2023.pptx
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This document discusses DNA methylation. It begins by explaining that DNA methylation is an epigenetic mechanism where a methyl group is added to cytosine residues in DNA, without changing the DNA sequence. This modification regulates gene expression and is involved in cell differentiation. The document then focuses on DNA methylation in more detail, describing where in the genome it occurs, the enzymes involved like DNMTs, and techniques to detect methylation like bisulfite sequencing. It explains that DNA methylation patterns are important for normal cell functions and development.
DNA methylation_2023.pptx
- 2. Epigenetics Study of heritable changes in gene expression that occur without a change in a DNA sequence. Stable alteration in gene expression pattern. Dynamic process that plays a key role in normal cell growth and differentiation. To date, the best understood epigenetic mechanisms are: 1. DNA methylation 2. Histone modifications
- 3. DNA methylation DNA methylation is one of the most commonly occurring epigenetic events taking place in the mammalian genome. This change, though heritable, is reversible, making it a therapeutic target. Methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues.
- 4. DNA methylation DNA structure is maintained from generation to generation. This structure is modified by base methylation in nearly all cells and organisms.
- 5. DNA methylation The DNA of most organisms is modified by a post-replicative process which results in three types of methylated bases in DNA: C5-methylcytosine(5-mc) N4-methylcytosine N6-methyladenine. This Modification is called DNA methylation. DNA methylation is a covalent modification of DNA that does not change the DNA sequence, but has an influence on gene activity. Wide spread in prokaryotes
- 8. DNA Methylation In eukaryotes DNA methylation occurs in the cells of fungi, plants, non-vertebrates and vertebrates. In vertebrates, 3-6% of DNA cytosine is methylated. No methylation in many insects and single-celled eukaryotes. In plants, 30% of DNA cytosine is methylated.
- 9. DNA methylation •Addition of methyl group to C-5 position of cytosine residues. • Most cytosine methylation occurs in the sequence context 5'CG3' •Occurs almost exclusively at cytosines that are followed immediately by a Guanine- CpG Dinucleotide.
- 10. Mechanism Methyl groups are transferred from S-adenosyl methionine in a reaction catalysed by a DNA methyl transferases(DNMT) or methylases. SAM is then converted to SAH (S-adenosyl homocysteine).
- 11. Enzymes involved in DNA methylation Enzymes involved- DNA METHYLTRANSFERASES(DNMTs) DNMTs catalyze this reaction at different times during the cell cycle. In Mammals, 1. DNMT1- Maintainance methylase 2. DNMT 2 3. DNMT3a and DNMT3b-‘de novo’methylases 4. DNMT3L
- 12. PRE-IMPLANTATION Genome undergoes Demethylation AFTER IMPLANTATION OF EMBRYO AND DURING CARCINOGENESIS New Methylation patterns are set by de-novo methylation. DURING REPLICATION Methylation patterns must be maintained. Therefore, DNMT1, methylates the hemimethylated DNA after strand synthesis.
- 13. Mammalian Genome The human genome is not methylated uniformly and contains regions of unmethylated segments interspersed by methylated regions. In contrast to the rest of the genome, smaller regions of DNA, called CpG islands, ranging from 0.5 to 5 kb and occurring on average every 100 kb, have distinctive properties. These regions are unmethylated normally. Approximately half of all the genes in humans have CpG islands, and these are present on both housekeeping genes and genes with tissue-specific patterns of expression.
- 14. CpG Dinucleotides Occur at low abundance throughout the human genome. Tend to concentrate in regions known as CpG islands (found in 50% of promoter regions of genes). Typically methylated in non-promoter regions and unmethylated in promoter regions. Methylation within the promoter region correlates with transcriptional silencing. Methylation of CpG islands is believed to dysregulate gene transcription through the inhibition of transcription factor binding either directly or via altered histone acetylation.
- 15. Bisulfite Sequencing Bisulfite sequencing is used to detect methylation in DNA. Bisulfite deaminates cytosine, making uracil. Methylated cytosine is not changed by bisulfite treatment. The bisulfite-treated template is then sequenced. 15
- 16. Bisulfite Sequencing The sequence of treated and untreated templates is compared. GTC Methylated sequence: GTC Me GGC Me GATCTATC Me GTGCA … Treated sequence: Me GGC Me GATUTATC Me GTGUA … DNA Sequence: (Untreated) reference: ...GTCGGCGATCTATCGTGCA… Treated sequence: ...GTCGGCGATUTATCGTGUA… This sequence indicates that these Cs are methylated. 16
- 17. The Steps to Determining the Methylation Status of Cytosine in a Known DNA Sequence by The Bisulfite Conversion Method 17 Singal, R. & Ginder, G.D. DNA Methylation. Blood Journal 93, 4059-4070 (1999).
- 18. Techniques for Enrichment of Methylated or Target Regions Prior to BS Sequencing 18 Lister, R. & Ecker, J.R. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res 19, 959-66 (2009). Genomic DNA Deep Sequencing Harrison, A. & Parle-McDermott, A. DNA methylation: a timeline of methods and applications. Front Genet 2, 74 (2011).