<b>Measuring Methylation</b> Two techniques have traditionally been employed to measure DNA methylation. <br><br><i>Restriction Based.</i> The first, called Combined Bisulfite Restriction Analysis (COBRA), involves chemically converting unmethylated cytosines into uracils (a process called bisulfite conversion), while leaving methylated cytosines intact. Performing PCR that amplifies the region of interest leaves methylated cytosines intact and unmethylated cytosines converted to thymines (Figure 1). Samples are then digested using an enzyme that will only cut the unconverted (originally methylated) cytosines. The enzyme can no longer recognize unmethylated sites, as they are “TG” instead of “CG”. Designing primers for this process is not always feasible, even with the help of advanced algorithms, and the process needs to be optimized each time a new site is to be analyzed. Furthermore, the workflow takes several days, is expensive, and is not high throughput enough to accomodate screening libraries of candidate DNA-binding-domain-methyltransferase fusion proteins. It has recently fallen out of favor because it is difficult to interpret and does not consider all CpG sites, but only ones which fall within a restriction enzyme’s recognition sequence. (Xiong 1997 and Li 2002). Our methylation assay, MaGellin, is also restriction-based but is much simpler than COBRA because it does not require bisulfite conversion of the DNA. This eliminates most of the problems that made COBRA unwieldy.<br><br>
<b>Epigenetics.</b> The code of life is more than a sequence of A’s, C’s, T’s, and G’s. Heart cells in the human body contain the same DNA as skin cells in the foot, yet these two cell types behave in radically different ways. Both contain the DNA for every one of over 20,000 human genes but express only the ones needed for their own form and function. These differences are due to epigenetic controls. Epigenetics refers to any regulation of gene expression and phenotype that is not based on the sequence of bases in DNA. In addition to governing cellular differentiation, epigenetic mechanisms facilitate the proper functioning of a cell. When these mechanisms go awry, neurodevelopmental disorders, immunodeficiency, and cancer can result. Epigenetic phenomena are amongst the primary ways gene expression is regulated; yet, our current understanding of them is limited, especially due to the challenge of studying them in noisy mammalian systems. By virtue of its emphasis on the isolation and testing of biological networks in engineered systems with reduced complexity, synthetic biology offers the promise of fostering understanding of phenomena difficult to study in their native environments. It is thus an ideal forum for epigenetic studies. At its core, synthetic biology also involves the engineering of non-native networks for useful purposes. Well-tuned gene expression is essential to the proper functioning of synthetic biological circuitry, yet epigenetics has not yet been fully explored as a tool for this application.
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Figure 1: COBRA. This restriction based assay detects methylation at CpG sites that fall within restriction enzyme recognition sequences. <br><br>
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<i>Sequencing Based. </i>The second, and more commonly employed technique, is bisulfite sequencing, which employs the same bisulfite conversion step previously described, and is immediately followed by sequencing. Unmethylated cytosines are read as thymines and methylated cytosines are read as cytosines. Comparing converted and unconverted sequences reveals the methylation pattern with high resolution. Despite its advantages, this method is time consuming and can become very expensive as more and more constructs are screened for activity and specificity (Darst 2010).
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<b>DNA Methylation.</b> DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).
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<b><center><h1>An Unmet Need</h1></center></b>
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<b>Epigenetic Engineering.</b> Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/8/8d/In_Vivo_Methylation.jpg" alt="In Vivo Methylation" width="700" height="395"><figcaption><i>In Vivo Methylation</i></figcaption></figure></div>
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<b>Epigenetic Disease.</b> Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/4/47/Hypomethylation.jpg" alt="Hypomethylation" width="700" height="395"><figcaption><i>Hypomethylation can lead to cancer.</i></figcaption></figure></div>
Epigenetics. The code of life is more than a sequence of A’s, C’s, T’s, and G’s. Heart cells in the human body contain the same DNA as skin cells in the foot, yet these two cell types behave in radically different ways. Both contain the DNA for every one of over 20,000 human genes but express only the ones needed for their own form and function. These differences are due to epigenetic controls. Epigenetics refers to any regulation of gene expression and phenotype that is not based on the sequence of bases in DNA. In addition to governing cellular differentiation, epigenetic mechanisms facilitate the proper functioning of a cell. When these mechanisms go awry, neurodevelopmental disorders, immunodeficiency, and cancer can result. Epigenetic phenomena are amongst the primary ways gene expression is regulated; yet, our current understanding of them is limited, especially due to the challenge of studying them in noisy mammalian systems. By virtue of its emphasis on the isolation and testing of biological networks in engineered systems with reduced complexity, synthetic biology offers the promise of fostering understanding of phenomena difficult to study in their native environments. It is thus an ideal forum for epigenetic studies. At its core, synthetic biology also involves the engineering of non-native networks for useful purposes. Well-tuned gene expression is essential to the proper functioning of synthetic biological circuitry, yet epigenetics has not yet been fully explored as a tool for this application.
DNA Methylation. DNA methylation is one of the most prominent and powerful mechanisms of epigenetic control. In mammals, enzymes called methyltransferases catalyze the addition of methyl groups to cytosines in CpG sites (a cytosine next to a guanine); these sites are abundant throughout the human genome (Venter 2001). Methylated cytosines in CpG sites can block transcription factors from binding to DNA and repress gene expression (Jones 1998 and Nan 1998). Although epigenetic factors do not affect genotype, they do affect phenotype. Specific methylation patterns are necessary for healthy development and disruption of methylation patterns has been shown to lead to many diseases (Cooper 1988, Rideout III 1990, Baylin 1998, Jones 1999, and Amir 1999).
An Unmet Need
Epigenetic Engineering. Synthetic biology largely involved engineering genetic networks in bacterial chasses at its inception, but there have been increasing efforts to engineer more complex mammalian systems. Yet, despite the dramatic effects that subtle epigenetic modifications can have on phenotype, there are no robust and well-characterized tools for engineering the epigenome. If synthetic biologists intend to successfully transition from bacterial to mammalian chasses, they must appreciate the epigenetic control of gene expression. An epigenetic toolbox for synthetic biology would enable the creation of engineered organisms that more closely rival their natural counterparts with regard to the subtlety and robustness of cellular control. We decided to make one of these tools, an enzyme that can direct DNA methylation to specific sequences.
In Vivo Methylation
Epigenetic Disease. Methylation abnormalities are linked to a wide range of diseases. Many types of cancer can be characterized by their DNA methylation profiles. In fact, DNA methylation has been called “the hallmark of cancer” (Syzf, 2004). Specifically, hypomethylation of oncogenes has been linked to tumorigenesis and loss of CpG methylation at specific sites has been implicated as a main cause of cancer. No drugs, approved or in testing, can restore methylation. We determined to engineer enzymes that are up to the task.
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