Cellular Methylation Machinery and DNA Methylatransferases

DNA methylation patterns are known to be established by a complex interplay of at least three independent DNA methyltransferases: DNMT1, DNMT3A and DNMT3B. Homozygous loss of any of the three currently known mammalian DNMTs, which includes DNMT1, 3a, and 3b, has been described to be lethal in mice (Li et al., 1992).

DNMT1 is the most abundant methyltransferase in somatic cells (Robertson et al., 1999), localizes to replication foci (Leonhardt et al., 1992), has a 10-40-fold preference for hemimethylated DNA(Pradhan et al., 1999), and interacts with the proliferating cell nuclear antigen (PCNA) (Chuang et al., 1997). It is thought to be the enzyme responsible for copying methylation patterns after DNA replication, and therefore is often referred to as the ‘maintenance’ methyltransferase (Robertson and Wolffe, 2000A). DNMT1 is essential for proper embryonic development, imprinting and X-inactivation (Li et al., 1992; Li et al., 1993; Beard et al.,1995).

Both of the DNMT3 methyltransferases are required for the wave of de novo methylation that occurs in the genome following embryonic implantation, and for the de novo methylation of newly integrated retroviral sequences in mouse ES cells (Okano, et al., 1999). It is believed that both these enzymes have an equal preference for hemi- and unmethylated DNA substrates, and that is why they are referred to as ‘de novo methyltransferases’ (Okano et al., 1998).

Recent studies have depicted that all three enzymes possess both de novo and maintenance functions and that, at least in somatic cells, specific methyltransferases will be responsible for the methylation of certain genomic regions by their interactions with other nuclear proteins (Robertson and Wolffe, 2000A). Purification of a DNMT1 complex that contains the retinoblastoma (Rb) gene product, E2F1 and histone deacetylase 1 (HDAC1) (Robertson, et al., 2000); and yeast two-hybrid experiments that show that DNMT1 can form a complex with HDAC2 and the co-repressor proteins DMAP1 and tumour susceptibility gene 101 (TSG101) (Roundtree, et al., 2000).

In experimental systems, it has been demonstrated that methylation at promoters does not lead to silenced transcription until chromatin proteins are recruited to the region, which mediate the gene silencing (Kass et al., 1997). It is therefore believed, that methylation initiates the process that results in a loss of transcription, by recruiting chromatin remodeling proteins. The majority of our genome is normally packaged in a transcriptionally repressive chromatin state, of the type found in pericentromeric heterochromatin regions. This type of chromatin is heavily methylated, and is packaged into compacted nucleosomes that contain deacetylated histones, particularly deacetylated histone H3. These histones are extensively deacetylated through the action of histone deacetylases (HDACs). This deacetylated state maintains the nucleosomes in a tightly compacted, regularly spaced, and transcriptionally silent state (Murzina et al., 1999; Struhl, 1998). The histone mark of a methylated Lys9 residue on the tail of histone 3 (H3) is believed to target DNA methylation to the region, and along with deacetylated histones, demonstrates that this is transcriptionally repressive chromatin. H3 Lys9 methylation is maintained by a histone methyltranscferase (HMT) that is recruited by binding of the chromodomain protein HP1 to the methylated H3 Lys9 (Jones and Baylin, 2002).

DNA methylation is involved in the forming of the transcriptionally silent state of pericentromeric heterochromatin. Methyl-cytosine-binding proteins (MBPs) associate with methylated cytosines and also with numerous chromatin-remodelling complexes (Jones and Baylin, 2002). These MBPs reside in complexes that contain HDACs; for example, the methyl-binding proteins methyl-CpG-binding protein 2 (MECP2) and methyl-CpG-binding-domain proteins MBD1 and MBD2 have been found to associate with transcriptional co-repressors, such as SIN3, which are known to bind HDACs directly (Bird and Wolffe, 1999; Jones et al., 1998; Ng et al., 1999).

Only a small fraction of the genome is transcriptionally competent. Transcriptionally active chromatin, euchromatin, consists of unmethylated CpG sites, that are protected from DNMTs and repressive complexes containing HDACs by transcription activator complexes. The nucleosomes around the promoter are more widely spaced than in heterochromatin and contain heavily acetylated histones. The histone mark, methylation of Lys4 residue in histone 3, is found associated with transcriptionally permissive chromatin. Lys4 is methylated by a different histone methyltransferase (HMT), than Lys9 (Jones and Baylin, 2002).

Immunohistochemistry Protocols

Immunohistochemistry Protocols

IMMUNOHISTOCHEMISTRY PROTOCOLS

Your source for protocols, techniques and information about immunohistochemistry, immunology, histology, pathology and chemistry.

History of Immunohistochemistry

Immunohistochemistry is a molecular technique that combines principles from both immunology and biochemistry techniques and principles to the study of histology and pathology by revealing molecules and patterns within cells and tissues.

The first to describe immunohistochemistry was Dr. Coons. The original immunohistochemistry method consisted of an antibody tagged with a fluorescent probe which was developed in rabbits, which was mixed with tissue sections and searched for, under a fluorescent microscope following a period of incubation. Since, numerous advancements and improvements have been done, to make the technique of immunohistochemistry fairly inexpensive and indispensable in both pathology departments and molecular laboratory benches worldwide.

Numerous different procedures are available, however the most commonly used are the peroxidase-antiperoxidase immune complex method and more so, the biotin-avidin immunoenzymatic technique.

DNA Methylation

DNA methylation is the addition of a methyl group to the carbon-5 position of cytosine residues. It is the only common covalent modification of human DNA and occurs almost exclusively at cytosines that are followed immediately by a guanine. DNA methylation results from the activity of a family of DNA methyltransferase (DNMT) enzymes that catalyze the addition of a methyl group to the cytosine residues at CpG dinucleotides (Bird, 1996). These so-called CpG dinucleotides, include approximately 3-5% of all the cytosine residues within the human genome (Ehrlich et al., 1982).

The bulk of the human genome displays a clear depletion of CpG dinucleotides. This is believed to be due to the high rate of deamination of 5-methylcytosine. Those CpG dinucleotides that are present are nearly always methylated. By contrast, seventy to eighty percent of these CpG dinucleotides are located in clusters termed CpG islands, which are up to a few kilobases in length and are nearly always free of methylation, unlike the bulk of DNA (Jones and Baylin, 2002). The exception to this pattern of methylation, is on the inactive X chromosome in females (Antequera and Bird, 1993). The genome consists of ~45,000 CpG islands and 50-60% of these are further clustered within control regions of a gene, mainly in the regulatory and promoter regions, but often in other parts of the gene, including exons (Bird, 1986). This pattern of DNA methylation is stably inherited from one cell generation to the next (Gardiner-Garden and Frommer, 1987).

DNA Methylation and Cancer

Alterations in DNA methylation are regarded as epigenetic and not genetic changes, because although epigenetic changes affect the structure of DNA, they do not materially affect the genetic code. In recent years, numerous studies have demonstrated that a close correlation exists between methylation and transcriptional inactivation, supporting the notion that not only genetic changes, but also epigenetic changes can contribute to the carcinogenic process (Strathdee et al., 2002; Yan et al., 2001). The pattern of methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. The methylation pattern in tumors consists of a global hypomethylation, in conjunction with localized hypermethylation at CpG islands (Goelz et al., 1985). This regional hypermethylation at CpG islands is associated with the transcriptional inactivation of cancer related genes (Momparler and Bovenzi, 2000).

Recent studies have demonstrated that hypermethylation of CpG islands may be implicated in tumorigenesis, acting as a mechanism to inactivate specific gene expression of a diverse array of genes (Baylin et al., 2001). Genes that have been reported to be regulated by CpG hypermethylation, include tumor suppressor genes, cell cycle related genes, DNA mismatch repair genes, hormone receptors and tissue or cell adhesion molecules (Yan et al., 2001). For example, tumor-specific deficiency of expression of the DNA repair genes MLH1 and MGMT (Herman, 1998; Simpkins, 1999) and the tumor suppressors, p16, CDKN2 and MTS1, has been directly correlated to hypermethylation (Jones, 1999; Merlo et al., 1995). Increased CpG island methylation can result in the inactivation of these genes resulting in increased levels of genetic damage, predisposing cells to later genetic instability which then contributes to tumor progression (Strathdee and Brown, 2002).

Hypermethylation is now the most well characterized epigenetic change to occur in tumors, and it is found in virtually every type of human neoplasm. Promoter hypermethylation is as common as the disruption of classic tumor-suppressor genes in human cancer by mutation and possibly more so (Baylin and Herman, 2000). Approximately 50% of the genes that cause familial forms of cancer when mutated in the germ line are also known to undergo methylation-associated silencing in various sporadic forms of cancer (Jones and Baylin, 2002).

In cancer, the dynamics of genetic and epigenetic gene silencing are very different. Somatic genetic mutation leads to a block in the production of functional protein from the mutant allele. If a selective advantage is conferred to the cell, the cells expand clonally to give rise to a tumor in which all cells lack the capacity to produce protein. In contrast, epigenetically mediated gene silencing occurs gradually. It begins with a subtle decrease in transcription, fostering a decrease in protection of the CpG island from the spread of flanking heterochromatin and methylation into the island. This loss results in gradual increases of individual CpG sites, which vary between copies of the same gene in different cells (Jones and Baylin, 2002).

Techniques to Study Epigenetics

TECHNIQUES TO STUDY EPIGENETICS

Links to Protocols, Methods and Techniques in order to study Epigenetic changes:

Epigenetic Cell Growth and Nucleic Acid Protocols:

Cell Lines and Cell Culture

Seeding, Passaging and Freezing Cells

Cell Stimulation Protocol

Preparation of Cytosolic Extracts

Total Cellular RNA Extraction

 

Polymerase Chain Reactions in Epigenetics:

Reverse Transcriptase Polymerase Chain Reaction RT-PCR

Methylation Specific PCR

Methods and Protocols to Analyze DNA Methylation and Epigenetic Changes:

Treatment of Cells with 5-Aza-2′-Deoxycytidine

Sodium Bisulfite Treatment of DNA

PCR Methods to study Epigenetics

Methylation Specific PCR

Methylation Sensitive Restriction Endonuclease Enzymes to study DNA Methylation and Epigenetic Changes

Methylation Sensitive Restriction Enzymes

Southern Blotting Method for DNA Methylation Detection

Combined Bisulfite Restriction Analysis COBRA

Bisulfite PCR Single Strand Conformation Polymorphism SSCP

PCR Fluorescence Melting Curve Analysis

Methylation Sensitive Single Nucleotide Primer Extension Ms-SNuPE

Identification of CpG Islands Exhibiting Altered Methylation Patterns (ICEAMP)

Hairpin-Bisulfite Polymerase Chain Reaction PCR

 

Histone Modification Protocols:

Chromatin Immunoprecipitation

 

Genome-Wide Approaches to studying Epigenetics:

Epigenomic Approaches

Treatment of Cells with 5-Aza-2′ – Deoxycytidine Protocol and Method

Treatment of Cells with 5-Aza-2′-Deoxycytidine

5-Aza-2′-Deoxycytidine: Demethylating Agents and Reactivation of Silenced Genes

Genes inappropriately silenced by structural chromatin changes that involve DNA methylation can be reactivated by demethylating agents, that can reverse these changes and, therefore, restore principal cellular pathways. This results in gene re-expression and reversion of some aspects of the transformed state. The demethylating agent 5-azacytidine and its deoxy derivative 5-aza-2’deoxycytidine were first synthesized in Czechoslavakia as potential chemotherapeutic agents for cancer (Cihak, 1974). These agents are incorporated into the nucleic acids of dividing cells, where they act as mechanism-based inhibitors of DNA methytransferases. They inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2′-deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme (Sheikhnejad et al., 1999).

These powerful inhibitors of DNA methylation, can restore gene function to treated cells in culture, which has indicated that they may have potential in treating patients with malignant disease (Lubbert, 2000; Jones and Baylin, 2002).

METHODS and MATERIALS: A Protocol for the Treatment of Cells with 5-Aza-2′-Deoxycytidine

Cells are seeded at a density of 5×10 5 /100-mm dishes, cultured for 48 hours, and treated with 0, 50, or 100 m M 5-aza-dC (Sigma Chemical Co., St. Louis, MD) (Li et al., 2001).

Forty-eight hours after treatment, cells are washed with PBS and fresh medium was added. Cells are further incubated for another 48 h before isolated total cellular RNA.

For protein studies, cells are seeded at a density of 5×10 5 /100-mm dishes, cultured for 24 h, and treated with 0, 1, 2, 3, 5, and 10 m M 5-aza-dC. After 5 days, cell supernatants are harvested and centrifuged at 1,800 rpm to pellet and remove any cell debris. Supernatants are subsequently transferred to a new tube and analyzed for protein concentrations or stored at -20 0 C .

Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-treated DNA

Sequencing of Sodium Bisulfite-treated DNA | DNA Methylation Analysis

 

SODIUM BISULFITE INDUCED OXIDATIVE DEAMINATION OF GENOMIC DNA

In single-stranded DNA, sodium bisulfite preferentially deaminates cytosine residues to uracil, compared with a very slow rate of deamination of 5-methylcytosine to thymine (Shapiro et al., 1973). Frommer et al. (1992) utilized this difference in bisulfite reactivity for genomic sequencing of 5-methylcytosine residues, by fully denaturing total genomic DNA and treating total genomic DNA with sodium bisulfite under conditions such that cytosine is converted stoichiometrically to uracil, but 5-methylcytosine remains nonreactive. The DNA is initially denatured by alkali treatment prior to treatment with bisulfite. The second part of the procedure involves PCR amplification of the region of interest in the bisulfite-reacted DNA to yield a fragment in which all uracil, formerly cytosine, and thymine residues have been amplified as thymine and only 5-methylcytosines have been amplified as cytosine. The bisulfite reaction yields DNA strand products, which are no longer complementary. PCR primers can therefore be designed, such that a specific pair can only bind to one of the bisulfite-reacted DNA strands. Primers for each strand will differ in every position where there is a C or G in the original sequence (Frommer et al., 1992).

If the PCR products from the bisulfite-treated DNA are cloned and individual clones are sequenced, the sequences will provide methylation maps of single DNA strands from individual DNA molecules in the original genomic DNA sample. The procedure yields a sequence and methylation pattern specific for each strand of the original genomic DNA. The position of each 5-methylcytosine will be given by a positive band on a sequencing gel (Frommer et al., 1992).

 

METHODS and MATERIALS: A Protocol for the Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-treated DNA

Genomic DNA is isolated using the Qiagen DNA KitTM and subjected to sodium bisulfite treatment to modify unmethylated cytosine to uracil, using the CpGenome TM DNA Modification Kit (Intergen Company, Oxford , UK ) and following the manufacturer’s instructions. The conditions for PCR were as follows: 1 cycle at 95 0 C for 15 min; 40 cycles of 94 0 C for 1 min, 60 0 C for 1 min and 72 0 C for 1 min; and 1 cycle of 72 0 C for 10 min. QIAGEN has recently developed an excellent kit for methylation analsys that should be sought if you are able to.

PCR products are then separated on a 1 % agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light. The PCR bands are subsequently cut from the gel with a sharp razor, pooled together and purified using the QIAGEN gel extraction kit (QIAGEN Inc., Valencia, CA) according to the vendor’s instructions. The purified DNA product is subsequently ligated to a TA cloning vector, TOPO pCR2.1 Sequencing Vector (Invitrogen, Grand Island , NY ). 3 m l of the dilution is then added to DH5 a -T1 competent cells and incubated in ice for 30 minutes. Cells are then heat-shocked for 45 seconds in a 42 o C heat block, and then placed on ice for a further 2 minutes. 250 m l SOC medium is added to each vial of cells. The vials are subsequently shaken at 225 RPM, at 37 o C for one hour, after which the content of each vial is spread on agar plates containing 100 m g/ml of ampicillin. Plates are then incubated overnight at 37 o C for the production of positive colonies.

Five-ten ampicillin resistant colonies grown on agar plates are selected and cultured overnight in Luria-Bertani (LB) medium composed of 1.0% NaCl, 1.0% tryptone and 0.5% yeast extract (DIFCO, MD, USA), pH 7.0 supplemented with 100 m g/ml of ampicillin (Sigma-Aldrich, St. Louis, MO). The ampicillin provides a selective pressure for the exclusive growth of positive colonies. Plasmid DNA is isolated using the QIAGEN Miniprep Kit (QIAGEN Inc., Valencia , CA ), following the manufacturer’s instructions. Screening for positive plasmids is done by a restriction digest. 1 m l of purified plasmid, with 1 m l of EcoRI (Fermentas Inc., MD, USA ), 1 m l of Buffer EcoRI and 17 m l of dH20, are incubated for 2 hours at 37 o C. The reaction mixture is subsequently seperated on a 2.5% agarose gel, stained with ethidium bromide and visualized under UV light. Positive clones are then sequenced using M13 primers (Invitrogen), and analyzed for percentage methylation of specific CpG dinucleotides. Data analsis consists of plotting CpG island dinucleotide positions and percentage of cytosines methylated.

Methylation-Specific PCR

Methylation-Specific PCR Protocol and Method

MSP can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of cloning or methylation-sensitive restriction enzymes. The assay consists of initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA.

MSP requires very small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed in DNA extracted paraffin-embedded samples (Herman et al., 1996).

METHODS and MATERIALS: A Protocol for Methylation-Specific PCR, MSP

One micro g of sodium bisulfite-treated genomic DNA is used for PCR amplification using MSP primers (Li et al., 2001) (MSP-Methylated and MSP-Unmethylated). The methylation-specific primers included in which nucleotides corresponding to potentially methylated cytosines where retained.

The primer combination to amplify unmethylated DNA included in which the nucleotides corresponding to cytosine nucleotides were changed to thymine (sense primer) or adenine (antisense primer) (Herman et al., 1996). The PCR conditions are as follows: 1 cycle of 95oC for 15 minutes; 40 cycles of 95 o C for 30 seconds, 50 o C for 30 seconds, and 72 o C for 45 seconds; and 1 cycle of 72 o C for 5 minutes.

The methylation-specific and nonmethylated DNA-specific primers yield different PCR products, respectively, when constructed. Depending on the primers created, differences in PCR temperatures and cycle optimizations are necessary.