Benjamin Jorgensen and Dr. Steven Johnson, Mirco and Molecular Biology

Main Text

Gene therapy is a technique that uses gene insertions or transgenes to alter and correct genetic mutations that cause hereditary diseases. Gene therapy holds hope in curing diseases such as Cystic Fibrosis and Duchenne Muscular Dystrophy or any disease caused by a recessive mutation in a single gene target. Transgenes may be incorporated into the nuclei of the patient’s cells, however, problems occur with the genes being turned off after time, negating the positive effects of the therapy. The goal of my research is to elucidate the contribution of underlying DNA sequences in the formation of nucleosomes (the basic unit of chromatin compaction) and to use specific sequences to prevent transgenes from becoming inactivated through heterochromatinization (dense bundling of the chromatin that results in inactivation of genes). Ultimately, this may have a useful application in gene therapy by aiding genes in retaining their euchromatic state (loose packaging of genes allowing proper gene expression) through manipulation of histone positioning, and thus maintaining the expression of these transgenes.

In almost every cell of the human body there is close to two meters of DNA. This DNA must all fit into the nucleus of the cell, while still allowing transcription factors (proteins which either promote or prevent RNA polymerases which translate DNA to RNA molecules) to access the DNA. Nucleosomes provide the first order of compaction. It has been shown in several studies that the underlying DNA sequence can influence the positioning of nucleosomes while the position of the nucleosomes can influence transcription by hiding or making available transcription factor binding sites (Albert et al. 2007). Thus altering the nucleosome positions can alter the expression of the underlying genes present.

The nematode roundworm, Caenorhabditis elegans is a model system in which to study the effect of chromatin architecture (specifically nucleosome positioning) on transgene expression. Early studies demonstrated that transgenes inserted into the worm are readily expressed in somatic tissues (non-germline tissues), but not germline tissues (the cells that become the sperm and the eggs) (Kelly et al. 1997). Germline transgene silencing is thought to be caused by bundling of the DNA into tightly packed nucleosomes (heterochromatinization) resulting in a state not competent to be activated or turned on even in the presence of appropriate transcription factors. Thus C. elegans is an ideal experimental system to test our hypothesis, allowing us to see if modification of the nucleosome positions (chromatin architecture) via manipulation of the underlying sequence, will allow maintenance or establishment of expression of transgenes in the germline.

My project is on going and will continue in Dr. Johnson’s lab after I graduate this April. Thus far we have successfully prepared the plasmid (the means by which transgenes are inserted into suject’s DNA). This proved to be difficult and took the majority of a semester. With the plasmid prepared, we have now begun injecting negative control plasmids into the worms to show the inactivation of transgenes with time. The learning curve for injection of C. elegans is quite steep and so, much time has been spent learning to successfully inject. We will continue to inject with negative controls in order to grow up colonies of worms to compare our experimental colonies to. If our injections are successful and the worms take up the transgenes without any problems, our negative controls will show the silencing of the transgenes with time while our experimental colonies with repositioned nucleosomes will show the maintenance of the transgenes overtime. We will begin injecting the experimental plasmid which we have prepared once we have shown the inactivation of transgenes with the negative control.

If our resulting data are consistent with our hypotheses, and we can demonstrate nucleosome positioning and gene regulation in both somatic and germline systems, we would anticipate publication of these findings in a mid- to high tier peer-reviewed journal such as Nucleic Acids Research or Genome Research. Additionally, I will present my work and data at appropriate venues locally (both on campus and around the state). We also anticipate additional studies and application of our findings.

Sources

• Albert, I., Mavrich, T.N., Tomsho, L.P., Qi, J., Zanton, S.J., Schuster, S.C., and Pugh, B.F. 2007. Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome. Nature 446: 572–576.
• Kelly, W.G., Xu, S., Montgomery, M.K., and Fire, A. Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146: 227-228.