ZIC2 Is Essential for Maintenance of Latency and Is a Target of an Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation.

Yuanzhi Lyu,Clifford G Tepper,Mel Campbell,Ryan R Davis,Yoshihiro Izumiya,Kazushi Nakano,Jae U Jung

doi:10.1128/jvi.00980-17

Abstract

ABSTRACTBivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modifications toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.IMPORTANCE Posttranslational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, a transcriptional factor essential for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts with and maintains polycomb repressor complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2-mediated local histone modification.

Highlights

Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region
We found that ZIC2 binds to the CAGC(A/G)GG sequence motif in BCBL-1 cells (E value, Ͻ10Ϫ10), which is highly similar to the findings for the motif identified in the mouse cerebellum (Fig. 5F)
As the polycomb repressive complex 2 (PRC2) complex is responsible for the deposition of the repressive H3K27me3 mark, we further examined the localization of enhancer of zeste homolog 2 (EZH2) and suppressor of zeste 12 homolog (SUZ12) on the KSHV replication and transcription activator (K-Rta) promoter by a Chromatin immunoprecipitation (ChIP) assay

Summary

Introduction

Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. In addition to its function as a DNA-binding transcription factor, K-Rta is known to target cellular and viral proteins for protein degradation. Mutation of the K-Rta SIM or the really interesting new gene (RING) finger-like domain significantly impairs its transactivation ability, linking the transactivation ability with the protein degradation function [20] These studies suggest that derepression through K-Rta-mediated protein degradation contributes to transactivation potency. This idea was first suggested by Yang et al [15] in a study which demonstrated that K-Rta targets K-RBP, a zinc finger (ZnF) protein, for degradation and proposed that promotion of repressor degradation by viral transactivators may be a mechanism for lytic gene activation in the herpesvirus family [15]. We identified ZIC2 (zinc finger protein of the cerebellum 2) as a key regulator of KSHV latency

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Conclusion