The Role of H3.3 histone variant in the pathogenesis of oral Kaposi's Sarcoma

Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS),
and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's
disease (MCD). KS is the most common oral cancer in human immunodeficiency virus (HIV)-infected
patients. The oral cavity is the major site for virus transmission via saliva. Although the incidence of
AIDS-KS cases has been reduced in the US due to combination antiretroviral therapy (cART), recent
studies suggest recurrence of oral KS in long-term treated AIDS patients. While KS tumor cells are
mostly latently infected, and express a limited number of viral genes, gingival epithelial cells in the
oral cavity actively replicate KSHV which is important for a) seeding the latency reservoir in B cells
after primary infection and b) shedding in the oral cavity and transmission via saliva. Epigenetics and
chromatin structure play a central role in the regulation of both transcription and replication.
Deregulation, aberrant deposition, and mutations of histone variants such as H3.3, CENP-A, and
H2A.Z and their associated chaperones have been implicated in multiple human cancers
including head and neck. After de novo infection, KSHV genomes rapidly associate with
nucleosomes which acquire specific epigenetic modifications that partition the viral episome into
transcriptionally active and silenced domains. While multiple KSHV epigenetic marks have
been mapped in lymphoid, epithelial and endothelial cells, the processes and potential host-
viral interactions leading to the formation of latency permissive episomes are still poorly
understood. Given that the KSHV latency-associated nuclear antigen (LANA) interacts with
both chromatin remodelers and H3.3 histone chaperones Daxx, HIRA, and DEK, we
hypothesize that H3.3 deposition plays an important role in the establishment and maintenance
of KSHV latency. Our preliminary data demonstrate that H3.3 deposition on KSHV episomes can
be detected early after de novo infection on episomes of long-term infected cells. Moreover, we
demonstrated that genetically disrupting the H3.3 chaperone pathways HIRA and Daxx by
CRISPR/Cas9 leads to marked changes in KSHV latency control, associated with alterations in
the epigenetic status. We further hypothesize that latency-associated gene products including
LANA modulate and or de-regulate histone chaperone pathways during de novo infection and
latency. To directly address this hypothesis we propose to mechanistically study histone H3.3
deposition and the formation of histone post translational modifications (PTMs), with the goal of
understanding their relationship with respect to viral gene expression and latent/lytic
replication. A main aspect of these studies is to investigate how viral gene products interact with
and modulate histone variant chaperone pathways. In addition, we use human oral primary epithelial
cells which are more lytic than transformed epithelial cell lines to characterize epigenetic
changes responsible for their lytic phenotype. Importantly, through a collaboration with Dr.
Donald Cohen, College of Dentistry, we propose to establish in vivo epigenomes of oral KS in both
formalin-fixed and snap frozen tumor samples. The overall goal is to investigate how histone
variant deposition determines latent and lytic infection in a cell type-specific manner. The results
may point to KSHV-specific novel therapeutic targets for oral KS.