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Epigenetics is a field of Molecular Biology that deals with regulation of genes expression and inheritance. This field basically focuses on studying the changes in DNA without involving changes in nucleotide bases (Tuchyurikov N.A. 2005). According to Young In Kiu and Esteller, the Epigenetic is "the inheritance of information based on expression level of genes." It is completely opposite to Genetics which involves the transmission of information based on sequences of DNA. Wu and Morris  defines Epigenetics as "the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence."(Altun, G 2010). This Molecular Biology field involves DNA, histone and DNA binding protein modification studies that brings changes in structure of chromatin without involving any changes in nucleotide bases in DNA (Egger, G. et al., 2004). Recent studies shows that alteration of gene expression takes place during differentiation and these changes get transferred to next generation by the process of mitosis. Same thing happens during ontogeny, the changes in expression of genes can be passed to next generation via meiosis. So in both the cases the changes in gene expression doesn't involve the sequences of DNA which indicates that the epigenetic changes doesn't involve DNA sequences (Tuchyurikov N.A. 2005).
The epigenetic factors play a crucial role in development pathways from stem cell to differentiated somatic cells. In this project, DNA methylation is being investigated at different stages of in vitro development of HSC (hematopoietic stem cell) from ES (embryonic stem cells).
Stem cells and their differentiation:
Cells of multicellular organisms which possessess the properties of self renewal and unlimited potency are called stem cells. Totipotency is generally shown by the early embryonic stem cell, but the adult stem cells are multipotent. (Avasthi, S. 2008). So stem cells possess the property of self renewable and pluripotency and are also capable to differentiate into different type of specialised cells (Ripon and Bishop, 2004 and Keller, 2005). The totipotent stem cells play an important role in human development as they provide raw material for organ and tissue in case of embryo (Ripon and Bishop, 2004). Because of potency and self renewability, these cells can be used in field of regenerative medicines (Keller, 2005). Identification of mechanism behind differentiation of embryonic cells helps us to generate cells, tissue and organ in lab which further help to cure various fatal diseases. Organ culture derived from ES can be used for pharmacological testing and reduces the demands of animal used in testing process (Ripon and Bishop, 2004).
According to Morgan et al., all somatic cells develop from embryonic stem cells and so the development pathways for each somatic cell are different. They acquire the specificity by genetic reprogramming during the process of differentiation. There are some factors which are responsible for maintaining switching on and off status of genes, these factors are called epigenetic factors which are different from DNA sequences (Morgan et al., 2005). Differential plasticity and multipotency of the stem cells can be exploited for therapeutic purposes (Avasthi, S. 2008).
Establishment of ES cell lines lead to the development of various experimental approaches in Developmental Biology (Keller 2005). ES cells are the cells that possess property of Totipotency (Evan et al, 1981) and so these cells are capable to differentiate and can give rise to cells of different lineages in vitro. The differentiation of ES cells in vitro helps to provide the answer related to lineage commitment (Keller 2005). To maintain ES cells in vitro, these cells are provided with embryonic fibroblast or LIF (leukemia inhibitory factor) when feeder layer is absent. There are various methods for achieving differentiation of ES cells. One of the commonly used methods involves the removal of ES cell from contact with LIF or feeder layer and further involve the culturing of cells on liquid media or media containing methyl cellulose. This condition prevents the adherence of cells to the surface and further helps in development of colony of differentiated cell known as Embryoid bodies. For studying hematopoietic development, slightly modified method is used which involves the differentiation of ES cells cultured on stromal cells. The aim of this modified method is to provide supportive environment for hematopoietic cells (Keller, 2005).
ES cells can be used to produce HSC which is a precursor of all blood cells. This involves the differentiation in 3 steps (Keller 2005, Dobbin et al 2008). First of all, ES cells get differentiated into primary embryoid bodies and the differentiation of primary embryoid bodies lead to the development of secondary blast like colony which gets differentiated to produce HSC. This differentiation process is basically controlled by the growth factors and optimal growth conditions required for differentiation process.
Gene silencing is an epigenetic process that regulates the expression of gene (Redberry, G. W. 2006). This term is generally used to indicate the genes which are switched "off". So it is a process of switching "off" of a gene other than genetic mutation. Regulation of genes is carried out at transcriptional and post transcriptional level which means, that silencing takes place only before and after transcription but always before translation (Morgan et.al., 2005; Redberry, G. W.2006).
Cytosine DNA methylation, histone tail modifications and genomic imprinting are the three main types of epigenetic information (Feinberg and Tycko 2004). Histone modifications are responsible for transcriptional gene silencing which creates a heterochromatin environment around gene, thus blocking transcription. Post transcription silencing is the result of mRNA destruction during silencing process. This breakdown of mRNA prevents translation and thus prevents formation of gene specific products i.e. proteins. Most important example of post transcriptional silencing is RNAi (Redberry, G. W., 2006). Silencing process takes place in both normal and abnormal cells (cancerous cells). Silencing mechanism can also protect genome from lethal or fate altering effect of transposable elements (Weber et al. 2007). If these transposable elements exist in active state these can alter gene functioning and can also produce lethal effects in cells (Tycko 2000).
Transmission of expression of genes takes place by epigenetic mechanism known as DNA methylation. DNA methylation involves addition of methyl group at 5 carbon position of pyrimidine base i.e. cytosine and found commonly in those cytosine bases which are followed by guanine base called as CpG dinucleotides. These dinucleotides are found abundantly at promoter regions of gene called CpG Island (Tchurikov 2004). Promoter is the region where RNA transcription factors bind to initiate transcription, but addition of methyl group during methylation process prevents the attachment of these factors to genes, that further blocks transcription process (Bird, 2002).
Fig A shows 3 methylatransferases1, 3a, 3b adds the methyl group at fifth carbon of cytosine by using SAM- CH3. B). Shows C-T transition initiated by sulphonation of cytosine (Zhu, J et al., 2006)
DNA methylation is of two types: 1) de novo DNA methylation: This methylation involves the addition of methyl group on cytosine base which was not present previously. 2.) Maintenance methylation: This type of DNA methylation maintains the methylation which was previously present (Bird 2002). The level of methylation keeps on changing during the embryogenesis process in mammals. During embryogenesis there is a sharp decrease in level of DNA methylation that can be seen, but de novo methylation recovers the methylation level at later stages (Kafri, T. 1992). Existence of regions with high CG content is the important feature of DNA methylation. These regions get methylated during development stages so as to maintain the stability of silenced genes. Maintenance and de novo methylation is carried out by the set of three enzymes, DNMTs (DNA methyl transferases) which are DNMT1, DNMT3a and DNMT3b (Broske, A. M et al. 2009). DNMT3a and DNMT3b control the de novo methylation and DNMT1 controls maintenance methylation so DNMT1 is also known as maintenance methyltransferase. This enzyme converts the newly replicated hemi-methylated DNA to the methylated one (Goll & Bastor, 2005). There is another enzyme called DNMT3L which affects the methylation process, but play an important role in trafficking imprinting. DNMT3L is essential for de novo methylation and in the absence of DNMT3L, DNMT3 can`t establish imprints (Diane J. Lees-Murdock., 2008).
DNA methylation and Cancer:
DNA hypomethylation, hypermethylation of tumour-suppressor genes and irregularities in the methyltransferase activities, leads to cancer. High activity levels of DNMTs is a major cause of multiple cancer and their increased level of activity is inhibited by using inhibitors such as 5 azadeoxycystidine and 5 azacystidine (Singal, R & Ginder, G.D. 1999). So we can say that Hypomethylation and hypermethylation contributes to cancer as these processes inactivate the tumour suppressor genes, activates oncogenes and alter the stability of chromosomes (Morgan et al., 2005, Tycko 2000). Hypomethylation activates the proto-oncogene, which exists in normal methylated and silenced form within genome. Proto-oncogenes activation is associated with cancer. H- ras oncogene is a good example which explains the effect of hypomethylation on cancer (Feinberg and Tycko 2004). This gene encodes a protein that plays an important role in transduction regulation to the cell nucleus, thus regulating the cell division. Genomic instability and loss of heterogeneity are the result of hypo-methylation (Chen, Patterson et al. 1998). Hypermethylation can be seen in promoters of several genes in case of cancer (Dunn 2003).
Methods to detect and quantify DNA methylation:
We can detect and quantify DNA methylation using different techniques which help us to visualise the methylated cytosine bases and also help us to quantify the methylation level in different genes. In my project, I am taking DNA from different developmental stages of embryonic stem cells to Hematopoietic stem cell. As I mentioned earlier that ES first gets converted to primary embryoid bodies and further gets differentiated into secondary blast like colony, the secondary blast like colony finally differentiates into HSC. For detecting and quantifying DNA methylation, it is necessary to isolate DNA from these different stages.
Before sequencing and DNA methylation quantification, the DNA needs to be isolated from cells at different stages. For extraction of DNA, cells are treated with lyses buffer which causes the breakdown of these cells and thus releases the DNA. Sodium dodecylsulphate is commonly used as the lyses buffer. SDS is a denaturing agent which interacts with lipid layer of membrane making it unstable. Lyses buffer also contains enzyme protenase K, which is resistant to the denaturing effect of SDS. This enzyme breakdowns the proteins and also digests nucleases (Liard, et al., 1991).
After completing the first step, next step is to extract DNA from lysed cells mixture so as to prevent damage caused by nucleases. This step also involves the removal of contaminating proteins. Phenyl: Chloroform: isoamyl alcohol is added to lysed mixture which is then vortexed and centrifuged. Centrifugation leads to the formation of organic and aqueous layer. DNA will be present in aqueous layer and rest of the cell components in organic layer that is below the aqueous layer. The supernatant with DNA is transferred to a clean tube. To obtain high purity, this step is repeated again and again (Liard, et al., 1991).
Next step involves precipitation of DNA which was purified by the procedure given above. This step involves the addition of precipitation mixture which contains ammonium acetate and ethanol. Precipitation takes place overnight at temperature -20 degrees C. After precipitation DNA can be seen at the bottom of the tube as a white fibrous material. Supernatant is decanted and DNA is isolated and resuspended in water (Liard, et al., 1991).
Next step is quantification of DNA using gel electrophoresis. This technique involves the separation of DNA molecule on the basis of charge and molecular weight. As DNA is negatively charged, when electric field is applied, it starts moving towards the opposite electrode and the movement depends on size, as smaller DNA molecule travel faster as compared to larger one. So for the quantification, DNA is run on 1 % gel, along with a ladder parallel to it. Bands are compared with the ladder to check the concentration of DNA (Waring, 1965). DNA gets visualized on gel when checked under U.V. because of the fluorescent dye ethydium bromide. This is an intercalating dye which forms a complex with DNA and RNA, thus providing visibility under U.V. (Olmsted et al., 1976).
Polymerase Chain Reaction (PCR):
PCR is a molecular Genetic method used commonly for amplification of sequences of nucleic acid. DNA strand serves as a template to synthesize the new strands of DNA. Amplification process is carried out using solution containing the mixture of reagents, which are essential for working of amplification process. Reaction mixtures used for PCR contains Taq polymerase, PCR buffer, MgCl2, dNTPs. PCR is carried out in three steps: denaturation, annealing and elongation. All these steps are carried out at different temperatures, so the thermocycler is used to provide all the conditions required for amplification. The cycle number can be adjusted to get the desired copies of DNA. Primers are very necessary for PCR and these are small sized sequences, about 20-30 bases long, and can easily be created in labs. Standard PCR uses two primers forward and reverse. Nested PCR enhances the sensitivity for the detection of methylated alleles (which is estimated as fifty times more than original methods (Divine, P et al., 2000). In nested PCR, the first stage products used are the templates during second stage.
Basic principle of PCR: The polymerase chain reaction is a method of cell free cloning. It is commonly used method for amplification of genes. PCR involves three steps. Denaturation- The first step of PCR, where DNA double helix breaks down at high temperature and forms single stranded DNA. High temperature enhances entropy and makes DNA unstable which further results in breakage of Hydrogen bonds between two strands. Denaturation generally takes place at 94-98 degrees C (Freeman et al., 1999). Next step after the strand separation is the primer annealing. During this step the small sized DNA sequences i.e. primer attaches to the strands by complementary base pairing and acts as a point where the DNA replication starts. Annealing is carried out at lower temperature i.e. 50-60 degrees C for a very short period of about 20-80 secs (Newton and Grahm 1994). Last step is the chain elongation step, where addition of nucleotides takes place because of the activity of Taq Polymerase. Besides dNTPs and Taq, level of Mg ions also affect the elongation process as Mg ion controls the activity of Taq polymerase. These ions are added as MgCl2 in mater mixture. Taq polymerase is the most commonly used enzyme for PCR. It is obtained from thermophilic bacteria called as Thermus aquaticus. The enzyme show high levels of activity between 75-80 degrees C, so elongation procedure takes place in thermocycler between or near to this temperature. These all three steps keep on repeating for 30-40 times. Agarose gel electrophoresis is carried out to check the amplification of gene.
Fig B: steps in PCR (Andy1999)
Methylation specific PCR: this is a widely used method to detect methylation and was developed by the Hopkin University. The method brings some modifications in normal PCR method to find out the CpG sites. DNA is first isolated and bisulphite converted then is used as template for PCR. Here in this case the primers are specific for methylated and unmethylated DNA; if primers match properly then amplification takes place (Herman Graff et al., 1996).
Fig C: methylation dependent PCR (Christina Dahl & Per Guldberg 2003).
Factors affecting PCR: there are various factors which affect the PCR effeciency and accuracy. One of them is contamination of sample which always leads to the failure of PCR (Roux 1995). The best way to avoid contamination is to prepare pre and post PCR samples in seperate areas in labs (Blanchard et al., 1993). Level of Mg ions also affect the effeciency of PCR. Low Mg levels are responsible for ineffective elongation process as they are needed for maintaing the activity of Taq polymerase (Gelfand 1989).
Template concentration: the amount of template present also affects the PCR end product. Small amount of template results in product which cannot be detected easily and the larger amount of templates inhibit the binding of primer to template. Generally 2 microliters of DNA is used as the optimal concentration of template for PCR (Blanchard et a., 1993).
Primer design: improper designed primers are not specific as they generate non specific products. So design of primer is an important thing related to PCR . We can manipulate the concentration of MgCl2 to optimise the procedure. Mg series is performed to check the concentration of MgCl2 required for efficient working of primers (Roux 1995).
Bisulphite conversion is a widely used technique for the analysis of methylation. It was first described by Frommer et al in 1992. This technique helps to distinguish methylated cytosine bases from the unmethylated ones. This is a highly effective and efficient method as 99% of cytosine bases get converted into uracil bases, which on amplification can be seen as T bases (Frommer et al., 1992; Gulshan Altun et al., 2010).
During bisulphite conversion, sodium bisulphite is added in DNA sample at an acidic pH. This treatment results in sulphonation of unmethylated cytosine bases followed by hydrolytic deamination to form Uracil sulphonate. Next step is the desulphonation step where uracil suphonate gets converted into uracil bases at alkaline pH. When the conversion is complete, cytosine bases left in treated sample are the methylated ones (Frommer et al., 1992). The sequencing methods are used to find out the cytosine bases, where methylation exists and other methods are used to quantify the methylation level in genes.
Combined Bisulphite restriction Analysis:
Because of Bisulphite treatment and PCR amplification, unmethylated cytosine basses get converted into thymine and methylated cytosine bases remains unchanged. The conversion of sequences results in retention of pre-existing restriction sites or creation of new restriction site which are methylation dependent (Xiong and Laird, 1997).
COBRA involves digestion of Bisulphite converted DNA using restriction enzymes such as BstUI. This enzyme possesses the property to cleave the regions with high level of C G bases because its cleavage site is CGCG. As we know, during bisulphite conversion unmethylated CGCG gets converted into TGTG and methylated CGCG remains as such, so the cleavage takes place at the point where methylation exists. The comparison between cleaved and uncleaved product determines the % of methylation present is genes. Products are run on 3% gel to check the cleavage.
Figure C: the figure on next page represents the full COBRA procedure. This involves sodium bisulphite PCR treatment, digestion with specific enzymes and Quantitation step. In first step unmethylated cytosine bases are converted to thymine and the methylated cytosine bases remains as such. BstUI enzyme is used for restriction because it has restriction site CGCG. Methylated sequences remain as such and get cleaved by enzymes. Cleaved and uncleaved product compared to check the % of methylation. The formula that is used for calculating the % of methylation is given in the figure on next page (Xiong and Laird, 1997).
C U T
5mC 5mC C
% methylation= (C/U+C) X 100
Fig D: shows COBRA analysis step wise (Fan Zhang et al., 2007).
There are various techniques for quantification of DNA methylation, but most of them are labour intensive and time consuming. It is not possible to analyse large number of samples by using those techniques. To overcome the limitations associated with these techniques, a new method is introduced known as Pyrosequencing, which is sequencing by synthesis. It is based on luminometric way of detection of phosphate which gets released during incorporation of nucleotide by the activities of four enzymes. It is real time method of sequencing and can be used for determining the frequency of SNPs in samples of DNA. It is said to be the best tool to study de novo methylation analysis along with quantification (Dupont J. M. et al., 2004).
Aim and Objective:
1. To study the timing and sequences of DNA methylation changes during HSC development. DNA and RNA from four stages of ES-HSC development in vitro will be extracted. Methylation at the promoter CpG Island will be examined using bisulphate modification followed by restriction analysis with methylation sensitive enzymes (COBRA) and sequencing of a limited set to confirm result from cobra.
2. Pyrosequencing for key gene regulator such as Gata1 and Cebpa gene.
3. Correlation of DNA methylation changes with gene expression: this involves the study of transcription of key genes Gata1, CD48, Cebpa, ID2, Scp3.