Host cell factor (HCF) was first discovered as a cellular cofactor required by the herpes simplex virus transactivator VP16 to activate the viral immediate-early promoters. HCF is a large multidomain protein and is expressed ubiquitously. Recent analysis of a cell line (tsBN67) with a temperature-sensitive point-mutation in HCF has revealed an essential role in promoting progression through the G1 phase of the cell cycle. These cells grow normally at the permissive temperature but undergo a G1/G0 arrest when incubated at the non-permissive temperature. The tsBN67 mutation in HCF does not alter protein stability but prevents association with VP16. This suggests the virus has targeted a critical surface on HCF required for cell cycle progression, providing a mechanism to link the control of viral replication with the proliferative capacity of the host cell. A strong candidate for the cellular equivalent of VP16, is a ubiquitous basic-leucine zipper protein known as LZIP. Like VP16, LZIP is an extremely potent transcriptional activator and recent work from this laboratory has shown that this activity is largely dependent on recruitment of HCF. This suggests that HCF functions as a coactivator or bridging factor connecting LZIP to other components of the general transcription apparatus. This proposal addresses the mechanism by which HCF augments activation domain function using a combination of in vitro and in vivo approaches. Experiments will be performed to identify additional coactivator proteins that function in concert with HCF and to determine whether HCF facilitates their recruitment to LZIP through direct protein-protein contacts. Domains in HCF involved in coactivator function will be mapped using a battery of HCF mutants. Finally, the LZIP-coactivator complex will be reconstructed using defined components and the role of enzymatic functions such as histone acetyltransferase activity will be examined to determine whether HCF may facilitate the recruitment or processivity of RNA polymerase . Overall, these studies will significantly enhance our understanding of the cellular function of HCF and provide deeper insight into the molecular mechanisms that underlie transcriptional activation and the regulation of the cell cycle.
The control of gene expression during development, differentiation and normal cell physiology is governed by a complex array of sequence-specific transcription factors. Exactly how these proteins stimulate the process by which specific target genes are copied into RNA (transcribed) remains unclear. An important layer of complexity not anticipated by earlier studies of bacteria, involves a heterogeneous class of accessory proteins that modulate the activity of individual transcription factors. The proposed research will focus on understanding the mechanism of action of host cell factor (HCF), a new and interesting example of this important class of regulatory protein. HCF is an essential player in the initiation of a cascade of gene expression leading to replication of herpes simplex viruses and also for cell cycle progression in mammalian cells. The long-term goal of these studies is to understand the molecular mechanisms by which HCF regulates gene expression and cellular proliferation.
