Dna methylation


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Dna methylation

  2. 2. Epigenetics <ul><li>Study of heritable changes in gene expression that occur without a change in a DNA sequence. </li></ul><ul><li>Stable alteration in gene expression pattern. </li></ul><ul><li>Dynamic process that plays a key role in normal cell growth and differentiation. </li></ul><ul><li>To date, the best understood epigenetic mechanisms are </li></ul><ul><li>1 . DNA methylation </li></ul><ul><li>2. Histone modifications </li></ul>
  3. 3. DNA methylation <ul><li>DNA methylation is one of the most commonly occurring epigenetic events taking place in the mammalian genome. </li></ul><ul><li>This change, though heritable , is reversible, making it a therapeutic target . </li></ul><ul><li>Methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. </li></ul>
  4. 4. DNA methylation <ul><li>DNA structure is maintained from generation to generation. </li></ul><ul><li>This structure is modified by base methylation in nearly all cells and organisms. </li></ul>
  5. 5. DNA methylation <ul><li>The DNA of most organisms is modified by a post-replicative process which results in three types of methylated bases in DNA: </li></ul><ul><li>C5-methylcytosine(5-mc) </li></ul><ul><li>N4-methylcytosine </li></ul><ul><li>N6-methyladenine. </li></ul><ul><li>This Modification is called DNA methylation. </li></ul><ul><li>DNA methylation is a covalent modification of DNA that does not change the DNA sequence, but has an influence on gene activity. </li></ul>Wide spread in prokaryotes
  6. 6. DNA Methylation <ul><li>It occurs in the cells of fungi, plants, non-vertebrates and vertebrates. </li></ul><ul><li>In vertebrates, 3-6% of DNA cytosine is methylated. </li></ul><ul><li>No methylation in many insects and single-celled eukaryotes. </li></ul><ul><li>In plants, 30% of DNA cytosine is methylated. </li></ul>
  7. 7. DNA methylation <ul><li>Addition of methyl group to C-5 position of cytosine residues. </li></ul><ul><li>Most cytosine methylation occurs in the sequence context 5'CG3 ' </li></ul><ul><li>Occurs almost exclusively at cytosines that are followed immediately by a Guanine- CpG Dinucleotide. </li></ul>
  8. 8. Mechanism <ul><li>Methyl groups are transferred from S-adenosyl methionine in a reaction catalysed by a DNA methyl transferases(DNMT) or methylases. </li></ul><ul><li>SAM is then converted to SAH (S-adenosyl homocysteine). </li></ul>
  9. 9. Enzymes involved in DNA methylation <ul><li>Enzymes involved- </li></ul><ul><li>DNA METHYLTRANSFERASES(DNMTs) </li></ul><ul><li>DNMTs catalyze this reaction at different times during the cell cycle. </li></ul><ul><li>In Mammals, </li></ul><ul><li>1 . DNMT1- Maintainance methylase </li></ul><ul><li>2. DNMT 2 </li></ul><ul><li>3. DNMT3a and DNMT3b- ‘ de novo ’methylases </li></ul><ul><li>4. DNMT3L </li></ul>
  10. 10. Enzymes <ul><li>DNMT1: </li></ul><ul><li>maintains the pattern of DNA methylation after DNA replication. </li></ul><ul><li>requires a hemi-methylated DNA substrate and will faithfully reproduce the pattern of DNA methylation on the newly synthesized strand . </li></ul><ul><li>DNA methylation- ‘an automatic semi conservative mechanism’ </li></ul><ul><li>DNMT3a and DNMT3b: </li></ul><ul><li>Will add methyl groups to CG dinucleotides which are previously unmethylated on both the strands. </li></ul><ul><li>Re-establish the methylation pattern. </li></ul>
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  12. 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. 13. Mammalian Genome <ul><li>The human genome is not methylated uniformly and contains regions of unmethylated segments interspersed by methylated regions. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul>
  14. 14. CpG Dinucleotides <ul><li>Occur at low abundance throughout the human genome. </li></ul><ul><li>Tend to concentrate in regions known as CpG islands (found in 50% of promoter regions of genes). </li></ul><ul><li>Typically methylated in non-promoter regions and unmethylated in promoter regions. </li></ul><ul><li>Methylation within the promoter region correlates with transcriptional silencing. </li></ul><ul><li>Methylation of CpG islands is believed to dysregulate gene transcription through the inhibition of transcription factor binding either directly or via altered histone acetylation . </li></ul>
  15. 15. CpG ISLANDS PROMOTER REGIONS NON-PROMOTER REGIONS Non-methylated Methylated Binding of TF Transcription Inhibition of TF binding Transcriptional silencing Methylated Non-methylated Silence parasitic genetic elements Genomic stability Binding of TF Transcription
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  17. 17. Role of DNA methylation <ul><li>Plays a role in long term silencing of gene. </li></ul><ul><li>Plays a role in silencing of repetitive elements ( eg: transposons). </li></ul><ul><li>Plays a role in X-chromosome inactivation. </li></ul><ul><li>In the establishment and maintenance of imprinted genes. </li></ul><ul><li>Suppresses the expression of viral genes and other deletorious elements that have been incorporated into the genome of the host over time. </li></ul><ul><li>In Carcinogenesis. </li></ul>
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  20. 20. METHYLATION IMBALANCE may contribute to TUMOR PROGRESSION GLOBAL HYPOMETHYLATION DNA HYPERMETHYLATION Observed in neoplastic cells May induce neoplastic transformation Genomic instability, Abnormal chromosomal structures and Activating oncogenes. Inactivation of tumor-suppressor genes: p16, BRCA1 Inactivation of DNA repair genes: MLH1, MGMT
  21. 21. A CpG island hypermethylation profile of human cancer
  22. 22. Global Hypomethylation <ul><li>There will be significant decrease in 5-meC. </li></ul><ul><li>Occurs in numerous solid tumors and in some haematological malignances. </li></ul><ul><li>eg: Chronic lymphocytic leukaemia (CLL), </li></ul><ul><li>Chronic myelogenous leukaemia (CML), </li></ul><ul><li>Acute Myelogenous leukaemia (AML). </li></ul><ul><li>Occurs in early stages of chest tumors, colorectal cancer and chronic lymphocytic leukaemia </li></ul>
  23. 23. Exceptions <ul><li>There are some examples where a CpG island in a promoter is unmethylated while the gene is still kept silent. </li></ul><ul><li>eg: The CpG island in human α-globin gene promoter is unmethylated in both erythroid and non-erythroid tissues (Bird et al., 1987). </li></ul><ul><li>Reason : role of histone modifications in gene silencing. </li></ul>
  24. 24. <ul><li>Carcinogens : Chronic exposure of human bronchial epithelial cells to tobacco-derived carcinogens drives hypermethylation of several tumor suppressor genes. </li></ul><ul><li>The reactive oxygen species (ROS) associated with chronic inflammation is another source of DNA damage. </li></ul><ul><li>Cigarette smoke : causes hypomethylation. </li></ul><ul><li>Aging. </li></ul>RISK FACTORS
  25. 25. Detection of DNA methylation <ul><li>Sodium bisulfite conversion (SBC) </li></ul><ul><li>SBC LC-MS-MS </li></ul><ul><li>cDNA microarray </li></ul><ul><li>Restriction landmark genomic sequencing </li></ul><ul><li>CpG island microarray </li></ul>
  26. 26. 1. Bisulfite conversion (SBC) <ul><li>Offers highest degree of resolution of the methylation status of a given sample, allowing to determine the positional CpG genotype for individual samples. </li></ul><ul><li>Involves the chemical modification of DNA by bisulfite treatment, where sodium bisulfite deaminates cytosine to uracil. </li></ul><ul><li>Methylated cytosine is resistant to this conversion. </li></ul>
  27. 27. Major Advance: Conversion of unmethylated cystosines to uracil using sodium bisulfite Sequencing: unemethylated cytosines read as thymidine in sense strand; adenine in the anti-sense strand. Other technologies evolved from here.
  28. 28. <ul><li>After bisulfite modification, there are a number of methods available to study CpG island methylation. </li></ul><ul><li>These include </li></ul><ul><li>sequencing, </li></ul><ul><li>methylation-specific polymerase chain reaction, </li></ul><ul><li>combined bisulfite restriction analyses, </li></ul><ul><li>methylation-sensitive single nucleotide primer extension, and </li></ul><ul><li>methylation-sensitive single-strand conformational polymorphism. </li></ul>1. SBC contd..
  29. 29. 1. SBC contd.. <ul><li>To be useful as a routine diagnostic tool, the actual methylation detection method has to be sensitive, quick, easy, and reproducible. </li></ul><ul><li>After bisulfite modification, PCR is performed using two sets of primers designed to amplify either methylated or unmethylated alleles . </li></ul><ul><li>Of the various techniques available , methylation-specific polymerase chain reaction (MSP) seems to be most useful at present. </li></ul>
  30. 30. Methylation specific PCR
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  32. 32. DNA methylation inhibitors (clinical approach) <ul><li>Agents targetted against DNMTs </li></ul><ul><li>5-azacytidine </li></ul><ul><li>2'deoxy-5-azacytidine (also known as Decitabine) </li></ul>
  33. 33. References <ul><li>Wentao Gao et al, Carcinogenesis vol.29 no.10 pp.1901–1910, 2008 </li></ul><ul><li>J.A. McKay*1, E.A. Williams† and J.C. Mathers *, Biochemical Society Transactions (2004) Volume 32, part 6 </li></ul><ul><li>Robin Holliday, Biochemistry (Moscow), Vol. 70, No. 5, 2005, pp. 500-504. </li></ul><ul><li>Ibáñez de Cáceres and P. Cairns, Clin Transl Oncol (2007) 9:429-437 </li></ul><ul><li>Melissa Conerly1 and William M. Grady2,3,*, Disease Models & Mechanisms 3, 290-297 (2010) </li></ul><ul><li>Steven s. smith*, Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4744-4748, May 1992,Biochemistry. </li></ul>
  34. 34. References <ul><li>Manel Esteller, Human Molecular Genetics, 2007, Vol. 16, Review Issue 1 R50–R59. </li></ul><ul><li>Yaping Li et al, Journal of Dermatological Science 54 (2009) 143–149. </li></ul>