Isolating Mononucleosome Core DNAs To Be Used in the ENCODE Project

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Elliot Winters and Dr. Steven M. Johnson, MM Biology

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The purpose of this project has its roots in the nationwide mission named “ENCODE” for the “ENCyclopedia Of DNA Elements” the purpose of which is to map the functional elements of the human genome. The human genome was successfully sequenced in 2001, (Consortium T.I.H.G.S., 2001) but the functional units acting on the DNA are largely unknown in biomedical research. For example: the DNA contained in almost every cell of the human body is identical, encoding the same genes in each cell. How the same set of genes can be regulated to guide the development of all the various cells and tissues that compose a complete organism is just starting to be understood. The ENCODE project aims to provide researchers with a comprehensive catalog of genomic functional units, or “tools” with which to better understand and study the human genetic code. (ENCODE website). The way the DNA of the human genome is packaged and condensed in the nuclei of the cells has a tremendous influence on which genes can and will be turned on or off in any particular cell. Hence, this packaging is a regulating factor in development. (Valouev et al., 2008). The nucleosome is the first order of packaging and its position on the genome regulates genic expression. The ENCODE community of researchers were interested in having a nucleosome position map to add to their analysis of the human genome. Arend Sidow (a contributor to the ENCODE project at Stanford University) came to Dr. Johnson last year with a request to isolate mononucleosome core DNA to construct a comprehensive nucleosome position map for two specific human cell lines. With a comprehensive nucleosome position map, researchers will have a better idea of the impact nucleosomes have on gene expression at the cellular level.

I used a protocol developed by Dr. Johnson to isolate mononucleosomes, purify their associated DNA cores, and prepare these mononucleosome core DNAs for shipment to Dr. Sidow’s lab at Stanford. Isolating the mononucleosomes required a fairly difficult initial step. The cells were frozen in small pellets and in order to begin the protocol, it was necessary to crush the pellets with mortar and pestle with the aid of liquid nitrogen. I had to practice several times with frozen pellets containing only dye before I could actually use the sample DNA pellets. It took patience and experience to crush them into a fine powder to expose the DNA in the cell nuclei without allowing total thawing of the sample. Liquid nitrogen was vital to keep the sample in the solid state but using it too often could result in splashing and loss of material.

The protocol also called for several extractions using chloroform and phenol. I also had to practice with these steps because after centrifuging the sample it was often difficult to pipette the aqueous layer away from the organic layer of material. The DNA was suspended in the aqueous layer and often times it was difficult to distinguish the meniscus between the two. I had to develop dexterity and precision for this step.

Another major difficulty lay in the primary digestion of the DNA. I first used micrococcal nuclease to chew up the DNA between nucleosomes but not the DNA attached to the histones themselves. This step wasn’t mechanically difficult but it required very careful timing and meticulous attention to detail. Success requires that the DNA be exposed to the proper ratio of endonuclease, at the optimum functional temperature and only for the allotted incubation period. All of these factors must be adhered to. This step was the most likely cause of error in the protocol and the reason it required multiple attempts.

We used gel electrophoresis and spectrophotometry to make sure our DNA samples were of high density DNA with minimal contamination. The entire protocol takes about two weeks to complete and unfortunately I had to repeat it four times to get it right. Luckily there was ample material sent to us and Dr. Johnson trusted me enough to complete the project on my own. Once I successfully completed the first cell line on the fourth try, the protocol on the second cell line was completed easily on the first try.

The project was fully completed and it resulted in a high quality, high-resolution nucleosome position map composed of hundreds of millions of nucleosome core positions for each of the two cell lines. The nucleosome core DNAs I prepared provided important information on nucleosome positioning and enabled the formation of a genome-wide nucleosome position map for the ENCODE project. This aspect of the ENCODE research is ongoing and results dependent on my contribution have yet to be published. However, when a contributor uses the data I helped to submit, my name and Dr. Johnson’s will be listed and published as contributing researchers.

The project gave me a great deal of experience in laboratory science. In order to carry out the protocol I had to learn an enormous amount of material related to microbiology, laboratory instruments and modern lab techniques. This project was an invaluable introduction to help me gain Dr. Johnson’s trust and glean experience in a field I’m highly interested in. This project also helped me develop skills that I use everyday in the lab and especially on my new project. I’ve become an expert in mononucleosome isolation in the lab and I’ve helped several students carry out similar protocols in their own research. I’m also very grateful that this project gave me a chance to work closely with Dr. Johnson. He was very patient with my many mistakes, he was encouraging when I ran into problems and overall he is a joy to work with. I feel that I’ve gained a valuable friendship in this project with Dr. Johnson and I’ll always be appreciative of that.

Sources

  • Consortium T.I.H.G.S. 2001. Initial sequencing and analysis of the human genome. Nature 409:860–921.
  • ENCODE website: http://www.genome.gov/10005107.
  • Valouev, A., Ichikawa, J., Tonthat, T., Stuart, J., Ranade, S., Peckham, H., Zeng, K,, Malek, J.A., Costa, G., McKernan, K., Sidow, A., Fire, A., and Johnson, S.M. A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Research 18: 1051-1063.