High Performance Liquid Chromatography

High-performance Liquid Chromatography (HPLC) is used as a method to separate DNA or protein molecules more commonly, by weight as well as by conformation. This is done through differences of the molecules in their distribution between a stationary phase and a mobile phase, which is carried out by the High-performance Liquid Chromatography (HPLC) Machine. Pressure changes in the system, allow for a greater resolution capacity. This method allows for the accurate quantification of the total amount of 5-methylcytosines in the entire genome.

High Performance Capillary Electrophoresis

This method allows for the accurate quantification of the total amount of 5-methylcytosines in the entire genome. High-performance Capillary Electrophoresis (HPCE) allows for separation of molecules, similar to the HPLC, but uses narrow-bore fused-silica capillary membranes to separate complex mixtures. Molecules are separated through the molecules size, structure, as well as charge and hydrophobic potential by electric fields.

Bisulphite Treatment Pyrosequencing

Bisulphite Treatment in Combination with Pyrosequencing

This method is great for detecting minimal amounts of aberrant DNA methylation. Conventional SNP typing is used through the C to T typing, as light is emitted through an enzymatic reaction which is used each time a nucleotide is incorporated into the DNA chain.


Ab Antibody


Bp Base pair
BSA Bovine serum albumin
CpG Cytosine-phosphate-Guanine
o C Degree Celsius
CDNA Complimentary deoxyribonucleic acid

Deoxyribonucleic acid


DNA methyltransferase

DNTPs Deoxynucleoside triphosphates
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
H3 Histone 3

Histone acetytransferase

HDAC Histone deacetylase
IPTG Isopropyl b -D-thiogalactoside
Ig Immunoglobulin
Kb Kilobase
Kda KiloDalton
KLK Kallikrein
L Liter
LB Luria-Bertani
LNCaP Lymph node-metastasize prostate cancer
Lys Lysine
M Molar
MRNA Messenger ribonucleic acid

Methyl-cytosine-binding proteins

MSP Methylation specific polymerase chain reaction
MW Molecular weight
NCBI National Center for Biotechnology Information
NES1 Normal epithelial cell-specific-1
OD Optical Density
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
RNA Ribonucleic acid
RT-PCR Reverse transcriptase polymerase chain reaction
s.c. Subcutaneous
SDS Sodium dodecyl sulphate
Ser Serine
TBS Tris-Buffered Saline
UV Ultraviolet

Epigenetic References – PUBMED Articles

Ahuja N, Mohan AL, Li Q, Stolker JM, Herman JG, Hamilton SR, Baylin SB, and Issa JP. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res. 57 , 3370-3374 (1997)

Antequera F, and Bird A. Number of CpG islands and genes in human and mouse. Proc. Natl. Acad. Sci. USA 90 , 11995-11999 (1993)

Asakai R, Davie EW, and Chung DW. Organization of the gene for human Factor XI. Biochemistry 26 , 7221-7228 (1987)

Avner P, and Heard E. X-chromosome inactivation: counting, choice and initiation. Nature Genet. Rev. 2 , 59-67 (2001)

Aznavoorian S, Murphy AN, Stetler-Stevenso WG, Liotta LA. Molecular aspects of tumor cell invasion and metastasis. Cancer 71 , 1368-1383 (1993)

Beard C, Li E, and Jaenisch R. Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev. 9 , 2325-2334 (1995)

Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 16 , 6-21 (2002)

Bird AP. CpG-rich islands and the function of DNA methylation. Nature 32 , 209-212 (1986)

Bird AP. The relationship of DNA methylation to cancer. Cancer Surv. 28 , 87-101 (1996)

Bird AP, and Wolffe AP. Methylation-induced repression – belts, braces, and chromatin. Cell 99 , 451-454 (1999)

Cameron EE, Bachman KE, Myohanen S, Herman JG, and Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21 , 103-107 (1999)

Chuang, L. Human DNA-(cytosine-5) methyltransferase-PCNA complex is target for p21 Waf1 . Science 277 , 1996-2000 (1997)

Cihak A. Biological effects of 5-azacytidine in eukaryotes. Oncology 30 , 405-422 (1974)

Courtier B, Heard E, and Avner P. Xce haplotypes show modified methylation ina region of the active X chromosome lying 3′ to Xist. Proc. Natl. Acad. Sci USA 92 , 3521-3535 (1995)

Dano K, Andreasen PA, Grohnadel-Hansen J, Kristensen PI, Nielsen LS, and Skriver, L. Plasminogen activators, tissue degradation and cancer. Adv. Cancer Res. 44 , 139-266 (1985)

Ehrlich M, Gama-Sosa MA, Huang LH, Medigett RM, Kuo KC, McCune RA, and Gehrke C. Amount and distribution of 5-methylcytosine in human DNA from different types of cells. Nucleic Acids Res. 10 , 2709-2721 (1982)

Esteller M, Hamilton SR, Burger PC, Baylin SB, and Herman JG. Inactivation of the DNA repair gene O 6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res. 793-797 (1999)

Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, and Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89 , 1827-1831 (1992)

Gardiner-Garden, M., and Frommer, M. CpG islands in vertebrate genomes. J. Mol. Biol. 196 , 261-282 (1987)

Goelz SE, Vogelstein B, Hamilton SR, and Feinberg AP. Hypomethylation of DNA from benign and malignant human colon neoplasms. Science 228 , 187-190, 1985

Heard E, Clerc P, and Avner P. X-chromosome inactivation in mammals. Annu. Rev. Genet. 31 , 571-610 (1997)

Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, Sidransky D, and Baylin SB. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55 , 4525-4530 (1995)

Herman JG, Graff JR, Myohanen S, Nelkin BD, and Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci.USA 93 , 9821-9826 (1996)

Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, Markowitz S, Wilson JK, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, and Baylin SB. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl. Acad. Sci. USA 95 , 6870-6875 (1998)

Horl WH. Proteinases: potential role in health and disease, in: Sandler M., and Smith HJ (Eds.), Design of enzyme inhibitors as drugs, Oxford University Press, Oxford, 1989, pp. 573-581

Hung MS, Karthikeyan N, Huang B, Koo HC, Kiger J, and Shen CJ. Drosophila proteins related to vertebrate DNA (5-cytosine) methyltransferases. Proc. Natl. Acad. Sci. USA 96 , 11940-11945 (1999)

Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE , and Baylin SB. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat. Genet. 7 , 536-540 (1994)

Jaenisch R, Schnieke A, and Harbers K. Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues. Proc. Natl. Acad. Sci. USA 82 , 1451-1455 (1985)

Jones PA, and Laird PW. Cancer epigenetics comes of age. Nat. Genet. 21 ,163-167 (1999)

Jones PA, and Baylin SB. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3 , 415-428 (2002)

Jones PL , Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, and Wolffe AP. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genet. 19 , 187-191 (1998)

Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, and Kolodner R. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defetive human tumor cell lines. Cancer Res. 57 , 808-811 (1997)

Kass SU, Landsberger N, and Wolfe AP. DNA methylation directs a time-dependent repression of transcription initiation. Curr. Biol. 7 , 157-165 (1997)

Kraut H, Frey EK, and Werle E. Der Nachweis eines Kreislaufhormon in de Pankreasdruse. Hoppe-Seylers Z Physiol. Chem. 189 , 97-106 (1930)

Ioshikhes IP, and Zhang MQ. Large-scale human promoter mapping using CpG islands. Nat. Genet. 26 , 61-63 (2000)

Lee JT, Davidow LS, and Warshawsky D. TsiX , a gene antisense to Xist at the X-inactivation center. Nature Genet. 21 , 400-404 (1999)

Lee WH et al. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA 91 , 11733-11737 (1994)

Leonhardt H, Page A, Weier H, and Bestor TH. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71 , 865-873 (1992)

Li E, Bestor TH, and Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69 , 915-926 (1992)

Li E, Beard C, and Jaenisch R. Role for DNA methylation in genomic imprinting. Nature 366 , 362-365 (1993)

Liang G, Gonzales FA, Jones PA, Orntoft TF, and Thykjaer T. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res. 62 , 961-966 (2002)

Lin X et al. Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide. Cancer Res. 61 , 8611-8616 (2001)

Lubbert M. DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr. Top. Microbiol. Immunol. 249 , 135-164 (2000)

McCawley LJ, Matrisian LM. Tumor progression: defining the soil round the tumor seed. Curr. Biol. 11 , R25-R27 (2001)

McGrath J, and Solter D. Comparison of mouse embryogenesis requires both the maternal and paternal genomes. Cell 37 , 179-183 (1984)

Merlo A, Herman J, Mao L, Lee D, Gabrielson E, Burger P, Baylin S, and Sidransky D. 5′ CpG island methylation is associated with transcriptional silencing of the tumor suppresssor p16/CDKN2/MTS1 in human cancers. Nat. Med. 1 , 686-692 (1995)

Mermoud JE, Constanzi C, Pehrson JR, and Brockdorff N. Histone macroH2A relocates to the inactive X chromosome after initiation and propagation of X inactivation. J. Cell Biol. 147 , 1399-1408 (1999)

Mohandas T, Sparkes RS, and Shapiro LJ. Reactication of an inactive X chromosome: evidence for X inactivation by DNA methylation. Science 211 , 393-396 (1981)

Momparler L, and Bovenzi V. DNA methylation and cancer. J. Cell. Physiol. 183 , 145-154 (2000)

Momparler RL. Molecular, cellular and animal pharmacology of 5-aza-2′-deoxycytidine. Pharmacol. Ther. 30 , 287-299 (2002)

Murzina N, Verreault A, Laue E, and Stillman B. Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol. Cell 4 , 529-540 (1999)

Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J. Clin. Oncol. 18 , 1135-1149 (2000)

Neumann B, Kubicka P, and Barlow DP. Characteristics of imprinted genes. Nature Genet. 9 , 12-13 (1995)

Ng HH, Zhang Y, Hendrich B, Johnson CA , Turner BM, Erdjument-Bromage H, Tempst P, Reinberg D, and Bird A. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nature Genet. 23 , 58-61 (1999)

Okano M, Xie S, and Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature Genetic . 19 , 219-220 (1998)

Okano M, Bell D, Haber D, and Li W. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99 , 247-257 (1999)

Paulsen M, El-Maarri O, Engemann S, Strodicke M, Franck O, Davies K, Reinhardt R, Reik W, and Walter J. Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse. Hum. Mol. Genet. 9 , 1829-1841 (2000)

Reik W, Collick A, Norris ML, Barton SC, and Surani MA. Genomic imprinting determines methylation of parental alleles in transgenic mice. Nature 351 , 665-667 (1991)

Reik W, and Walter J. Genomic imprinting: parental influences on the genome. Nature 2 , 21-32 (2001)

Robertson KD, Uzyolgyi E, Liang G, Talmadge C, Sumegi J, Gonzales FA, and Jones PA. The human DNA methyltransferases (DNMTs) 1, 3a, 3b: Coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res. 27 , 2291-2298 (1999)

Roberston KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL , and Wolffe AP. DNMT1 forms a complex with Rb, E2F1, and HDAC1 and represses transcription from E2F-responsive promoters. Nature Genet. 25 , 338-342 (2000)

Robertson, K.D. and Wolffe, A.P. DNA methylation in health and disease. Nature Rev. Genet. 1 , 11-19 (2000a)

Roundtree MR, Bachman KE, and Baylin SB. DNMT1 binds HDAC2 and a new co-repressor at replication foci. Nature Genet. 25 , 269-277 (2000)

Sado T, Fenner MH, Tan SS, Tam P, Shioda T, and Li E. X Inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation. Dev. Biol. 225 , 294-303 (2000)

Shapiro R, Braverman B, Louis JB, and Serivs RE. Nucleic acid reactivity and conformation. II. Reaction of cytosine and uracil with sodium bisulfite. J. Biol. Chem. 248 , 4060-4064 (1973)

Sheikhnejad G, Brank A, Christman JK, Goddard A, Alvarez E, Ford H, Marquez VE, Marasco CJ, Sufrin JR, O’Gara M, and Cheng X. Mechanism of inhibition of DNA (cytosine C5)- methyltranscferases by oligodeoxyribonucleotides containing 5, 6-dihydro-5-azacytosine. J. Mol. Biol. 285 , 2021-2034 (1999)

Simpkins, SB. MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum. Mol. Genet. 8 , 661-666 (1999)

Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald WL, Lazebruik YA, Cordon-Cardo C, and Lowe SW. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409 , 207-211 (2001)

Stallmach A, Wittig BM, Kremp WK, Goebel KR, Santourlidis S, Zeitz M, Menges, Raedle J, Zeuzem S and Schulz WA. Downregulation of CD44v6 in colorectal carcinomas is accosiated with hypermethylation of the CD44 promoter region. Exp. Mol. Pathology 74 , 262-266 (2003)

Strathdee G, and Brown R. Aberrant DNA methylation in cancer: potential clinical interventions. Exp. Rev. Mol. Med. ( Cam . UK ) 4 , 1-17 (2002)

Struhl K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 12 , 599-606 (1998)

Surani MAH, Barton SC , and Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308 , 548-550 (1984)

Tweedie S. Vestiges of DNA methylation system in Drosophila melanogaster. Nature Genetics 23 , 389-390 (1999)

Vu TH, Chuyen NV , Li T, and Hoffman AR. Loss of imprinting of IGF2 sense and antisense transcripts in Wilms’ tumor. Cancer Res. 63 , 1900-1905 (2003)

Wolf SF, and Migeon BR. Studies of X chromosome DNA methylation in normal human cells. Nature 295 , 667-671 (1982)

Yan L, Yang X, and Davidson N E. Role of DNA methylation and histone acetylation in steroid receptor expression in breast cancer. J. of Mammary Gland Biol. and Neoplasia 6 , 183-192 (2001)

Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, and Hirohashi S. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human cardcinomas. Proc. Natl. Acad. Sci. USA 92 , 7416-7419 (1995)


Packaging DNA in Higher Structural Forms

Packing of DNA is required are DNA molecules in eukaryotes is very large. DNA therefore packs itself up into structures referred to as chromosomes. This allows DNA to be stored within the nucleus of cells. The process is simple, with DNA being the lowest for of storage, moving to nucleosomes, which is protein interacting with DNA to condense it, then to a solenoid, which is an organized form of nucleosomes. The final structure is the chromosome.

Repetitive Sequences

Repetitive Sequences and Epigenetics

In comparison to CpG islands, which are found in the regulatory regions of genes, and are prime importance in the transcription of the regulators of cell growth and death, repetitive sequences, also known as endoparasitic sequences, which are transposable elements, inserting themselves into any sequence they please throughout the genome, are thankfully in normal cells, highly methylated at CpG dinucleotides. This, basically prevents them from propagating and disrupting the genome. The role of DNA methylation is thought to act as protector of genome fagility and activation of endoparasitic sequences (1, 2).


(1) Walsh, C.P. et al. 1998. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nature Genet. 20, 116-117

(2) Gaudet, F et al. 2003. Induction of tunors in mice by genomic hypomethylation. Science 300, 489-492

Histone Code

Histones are key regulators of gene expression, through numerous well-known chemical modifications. These include acetylation, phosphorylation, methylation, and ubiquitylation. For example, methylating and/or acetylating specific lysine residues of the nucleosomal cores of histones, such as lysine8, alter the chromatin structure, through interacting with the methylation machinery, and gene expression (1).

DNA Methylation Machinery, Histone Deacetylases and Histone Methyltransferases

Histone deacetylases and histone methyltransferases are the two key regulators of histone modifications.


(1) Wang Y, et al. 2004. Beyond the double helix: writing and reading the histone code. Novartis Found Symp. 259, 3-17

Insulators Loop Domain Model

Insulators and the Loop Domain Model

In the loop domain model, inactive DNA is heterchromatin, with methylated DNA and hypoacetylated histones. Active DNA, is euchromatin, and has hyperacetylated histones and unmethylated DNA. Chromatin fibers are attached within the nucleus to nuclear matrix component structures through specific DNA binding proteins. This creates ‘loop domains’ within the nucleus, as it compartmentalizes sections of the DNA into exposed regions versus structurally bound DNA that is inaccessible.

It is believed and currently slowly being supported that an enhancer in one of the loop domains, is unable to interact with a promoter in another loop domain. An enhancer is a DNA sequence that is capable of igniting transcription when placed upstream or downstream of a gene. Approximation of the enhancer with the gene is essential. Enhancers are known to be capable of working at 100’s of base pairs away from a gene, and even if they are inverted in orientation.