Project 2. Dissecting the chromatin boundaries from the HSVgenome Latently infected HSV contains nucleosomal DNA similar to the host chromatin. Recent studies hasdemonstrated that the histones in the LAT promoter and intron regions are hyper acetylated, whereas theICPO gene, which lies only 5 kb away, is hypo acetylated and silenced. The key question is how this activechromatin kept separated from the repressed chromatin in the nearby ICPO region. Our preliminary studyindicates that the 2.0 kb LAT contains an insulator activity, suggesting that the LAT intron may work as achromatin boundary to keep active and repressed chromatin separate during latency. The goals of thisproposal are to describe molecularly the mechanism by which repressed chromatin in the latent HSVgenome are separated from the active chromatin in the LAT region, and to analyze the biologicalconsequence after experimentally manipulation of the boundary region, for example deleting the potentialinsulator(s). The long-term goal is to explore biochemical or pharmacological means to manipulate theboundary or other chromatin functions near the LAT region to disrupt the activation of the lytic cycle.
In Specific Aim I we will Identify and functionally characterize the insulator from the LAT region of theHSV genome.
In Specific Aim II, we will identify the molecular mechanism of the insulator by testing whetherknown insulator proteins function through the LAT insulators.
In Specific Aim III, we will study the biologicalfunction of the insulators by deleting they from the virus and measure the effect such as the establishment-.and maintenance of latency, histone modification profile and expression of viral LAT gene and host genes.. Our proposed studies are well integrated with the rest of the program projects. With project one, we will >first collaborate on testing nucleosome free region for potential chromatin boundaries. Then we willcollaborate on detecting the effect on mutating the insulator on the latency of the virus. With project three, wewill collaborate on histone modification studies on the chromatin boundary in the wildtype latentvirus andhow histone modification changes after deleting the insulator. With project 4, we will collaborate.on usingPC12 latency model to test the effect of insulator on histone modifications. Through these studies, we expectto gain insight into the molecular mechanism controlling the latency cycle of infection, and hope to identifypotential molecular targets for therapy.
