While there are currently drugs available to treat patients with human immunodeficiency virus (HIV), there is no known cure for this infection. As the HIV virus integrates into the host’s DNA, reservoirs of latent HIV are established. Because antiretroviral drugs only attack replicating HIV, the presence of these latent reservoirs prevents the eradication of the virus from the body. Once the use of anti-retroviral drugs is discontinued, the active virus reemerges in patients. The purpose of this project is to identify transcriptional and epigenetic pathways to reverse HIV latency, knowledge that could pave the way for new drug design to kill all existing virus in the body. To accomplish this goal, we are using a microplate-based matrix chromatin immunoprecipitation (ChIP) assay and quantitative polymerase chain reaction (qPCR) to measure more than one hundred different transcription and epigenetic events along the latent HIV genome upon its transcriptional activation. We applied cluster analysis to our data and have found that Protein Kinase A (PKA), a class of enzymes which phosphorylate proteins, and its target, the transcriptional factor CREB, are recruited to HIV genome. This finding, which has not previously been reported, suggests that PKA plays a role in HIV genome transcription, and if so, could be a possible path for developing pharmacological reversal of HIV latency.

Epigenetic analysis is a rapidly growing field of interest within the research community, due to its potential to advance our knowledge of the origins of a variety of diseases. It involves analyzing modifications of DNA as well as modifications of proteins associated with DNA, which affect overall chromatin structure and lead to potentially detrimental effects on gene expression. Aberrant gene expression has been shown to be involved in the development of diseases such as cancer, diabetes, and kidney disease. The most commonly used method for epigenetic analysis is chromatin immunoprecipitation (ChIP). However, current ChIP assays involve time-consuming and labor-intensive pre-analytical sample preparation and have low throughput, resulting in concerns about accuracy and sample loss. Thus, the current assays are limited in their clinical utility. The purpose of this project is to design an optimized protocol for DNA-protein crosslinking and chromatin shearing using novel high-throughput ultrasound technology for increased efficiency and reduced sample loss in ChIP. Various conditions were tested with different stages of ChIP, including DNA-protein cross-linking, and chromatin shearing. Effects of these conditions on the ChIP assay were analyzed utilizing a microplate-based matrix ChIP assay and quantitative polymerase chain reaction (qPCR). Efficacy of chromatin shearing was analyzed using gel electrophoresis. My findings will contribute to the development of a fully automated platform for chromatin shearing and ChIP, and lead to advances in epigenetic biomarker research of diseases that burden our society. Its application to human tissues will allow for quick and accurate evaluation of chromatin alterations and gene-bound enzymes, factors, and receptors in a wide variety of human biospecimens. It will also allow for discovery of innovative diagnostic and therapeutic biomarkers based on epigenetic pathways, and determination of genomic pathways that are associated with drug resistance development.