Marcus Vranes and Dr. Steven Johnson, Molecular Biology
Introduction
Recent studies have attempted to discover the correlation that exists between DNA methylation and nucleosome positioning, but none have explored the direct effect of DNA methylation on nucleosome formation and positioning. This research is directly testing the effects of DNA methylation on nucleosome positioning, and whether the histone octamer has preferred sequences to which it binds, which will add to our understanding of gene expression and regulation. A better understanding of these concepts will help to aid efforts in gene therapy to improve the quality of life for many people who suffer from various genetic conditions.
Unfortunately, the project is not complete, but is ongoing. I will explain the reasoning for this in the following sections:
i. Methodology
ii. Discussion
iii. Conclusion
Methodology
As reported in the ORCA proposal, we (Ashley Wright, graduate student; Jordan Ritchie, undergraduate student; and I) began the research by using the restriction enzymes HincII and RsaI to digest the C. elegan DNA into fragments. After the C. elegan DNA was fragmented, we then treated it with Methyltransferase (MSssI) in order to methylate the naked DNA fragments. We then used a bisulfite treatment to convert the non-methylated cytosines to uracils. In order to confirm the methylation, we were going to use PCR amplification. However, we encountered problems with obtaining the correct primers for the PCR amplification, and after several attempts, we decided to change methods.
Instead of using restriction enzymes HincII and RsaI to fragment the C. elegan DNA, we obtained the Covaris Sonicator m22 to create fragments of exactly 800 base pairs. We then treated the fragments with Methyltransferase (same as before) to methylate them. However, instead of using bisulfite treatment and PCR amplification to confirm methylation, we used restriction enzymes HhaI and BstUI.
After confirming the methylation of the C. elegan DNA, we proceeded with the reconstitution protocol. In vitro nuclesome reconstitution means that the now methylated DNA is combined with human recombinant histone octomer in a salt solution. As the salt is decreased, the natural attraction between the octomer and DNA causes the DNA to wrap around the octomer. This is the step where the project is at present.
Discussion
Because we changed our digestion method (from restriction enzymes to sonication) and our methylation confirmation method (from bisulfite and PCR to restriction enzymes), the project has been delayed several months. However, these changes are more efficient for our project and have been useful for our purposes. The Covaris Sonicator m22 allows us to quickly obtain the desired fragments. The restriction enzymes HhaI and BstUI do not digest methylated DNA, so by using it, we are able to quickly confirm methylation by the lack of digestion.
In February 2013, I was able to travel to the Utah Conference of Undergraduate Research (UCUR) in Logan, UT, and present a poster on this project. I spent several hours answering questions and presenting the information to both faculty and students from all over the state of Utah.
Conclusion
In conclusion, there is still work to be done. Upon successful completion of the reconstitution, the nucleosomes created in vitro will be sequenced to observe the effects of DNA methylation on nucleosome positioning. Jordan Ritchie, undergraduate student, will be continuing the project in my absence.
