ABSTRACT
Kaposi's sarcoma (KS) is a highly-vascularized tumor characterized by inflammation and extensive neo-angiogenesis. The KS tumor microenvironment is rich in inflammatory and pro-angiogenic cytokines. Here, we report that the expression of Epidermal growth factor-like domain 7 (EGFL7) is upregulated in Kaposi's sarcoma-associated herpes virus (KSHV) infected cells. EGFL7 is a secreted pro-angiogenic cytokine that has been implicated in angiogenesis and the proliferation of endothelial cells during many pathological conditions. Our data show that KS tumors as well as primary effusion lymphoma cells have increased levels of EGFL7 compared to the uninfected cells. We determined that the expression of a KSHV latent protein, LANA (latency-associated nuclear antigen), is the main viral factor responsible for this upregulation. The modulation of EGFL7 expression by LANA involves sequestration of death domain-associated protein 6 (Daxx) from the EGFL7 promoter. Daxx acts as a suppressor of promoter activity by binding to the avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1), which is the core transcription factor required for the expression of EGFL7. We additionally show that the upregulation of EGFL7 by LANA contributes to the promotion of angiogenesis since siRNA-mediated knockdown of EGFL7 reduced in vitro tubulogenesis in LANA-expressing HUVEC cells. EGFL7 promotes angiogenesis through autocrine as well as paracrine mechanisms as the supernatant from LANA expressing cells depleted of EGFL7 showed reduced tubulogenesis. This study for the first time demonstrates EGFL7 to be an important angiogenic molecule secreted during KSHV infection that could be exploited for blocking KSHV associated malignancies in conjugation with other anti-angiogenic therapies.
ABSTRACT
Post-translational modifications to histone tails contribute to the three-dimensional structure of chromatin and play an important role in detegrmining the relative expression of nearby genes. One such modification is symmetric di-methylation of arginine residues, which may exhibit different effects on gene expression including blocking the binding of transcriptional activators, or recruiting repressive effector molecules. Recent ChIP-Seq studies have demonstrated the importance of cross-talk between different histone modifications in gene regulation. Thus, to acquire a comprehensive understanding of the combined efforts of these epigenetic marks, ChIP-Seq must be utilized for identifying specific enrichment on the chromatin. Tumorigenic herpesvirus KSHV, employs epigenetic mechanisms for gene regulation, and by evaluating relative abundance of multiple histone modifications in a thorough, unbiased way, using ChIP-Seq, we can get a superior insight concerning the complex mechanisms of viral replication and pathogenesis.
ABSTRACT
Kaposi's sarcoma associated herpesvirus (KSHV) persists in a highly-ordered chromatin structure inside latently infected cells with the majority of the viral genome having repressive marks. However, upon reactivation the viral chromatin landscape changes into 'open' chromatin through the involvement of lysine demethylases and methyltransferases. Besides methylation of lysine residues of histone H3, arginine methylation of histone H4 plays an important role in controlling the compactness of the chromatin. Symmetric methylation of histone H4 at arginine 3 (H4R3me2s) negatively affects the methylation of histone H3 at lysine 4 (H3K4me3), an active epigenetic mark deposited on the viral chromatin during reactivation. We identified a novel binding partner to KSHV viral DNA processivity factor, ORF59-a protein arginine methyl transferase 5 (PRMT5). PRMT5 is an arginine methyltransferase that dimethylates arginine 3 (R3) of histone H4 in a symmetric manner, one hallmark of condensed chromatin. Our ChIP-seq data of symmetrically methylated H4 arginine 3 showed a significant decrease in H4R3me2s on the viral genome of reactivated cells as compared to the latent cells. Reduction in arginine methylation correlated with the binding of ORF59 on the viral chromatin and disruption of PRMT5 from its adapter protein, COPR5 (cooperator of PRMT5). Binding of PRMT5 through COPR5 is important for symmetric methylation of H4R3 and the expression of ORF59 competitively reduces the association of PRMT5 with COPR5, leading to a reduction in PRMT5 mediated arginine methylation. This ultimately resulted in a reduced level of symmetrically methylated H4R3 and increased levels of H3K4me3 marks, contributing to the formation of an open chromatin for transcription and DNA replication. Depletion of PRMT5 levels led to a decrease in symmetric methylation and increase in viral gene transcription confirming the role of PRMT5 in viral reactivation. In conclusion, ORF59 modulates histone-modifying enzymes to alter the chromatin structure during lytic reactivation.
