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Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for KSHV LANA

ABSTRACT: LANA is essential for tethering the KSHV genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR), and to a few minor binding sites within the latency control region encoding that LANA transcript. We identified 256 LANA binding peaks with p < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Two candidate DNA sequence motifs were identified, and confirmed to bind purified LANA protein, although with weaker affinity compared to viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and TNF regulatory network. Further analysis revealed partial overlap of LANA binding sites with STAT1 binding sites in several interferon (IFN)-g regulated genes. We show that ectopic expression of LANA can down-modulate IFN-g mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9) required for class I antigen presentation. Our data provides a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements.

ORGANISM(S): Homo sapiens

PROVIDER: GSE34890 | GEO | 2012/01/06

SECONDARY ACCESSION(S): PRJNA150203

REPOSITORIES: GEO

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by 30-40 copies of 0.8 kb terminal repeat (TR) sequence. KSHV genomes persist in latently infected cells as episomes via tethering to the host cell chromosomes, and KSHV latency associated nuclear antigen (LANA) plays a crucial role in latent episomal DNA replication and segregation during host cell mitosis by binding to TR. While TR's function in plasmid maintenance is well-established, TR’s transcription regulatory roles as gene enhancer has not been fully explored. Gene enhancer harbors transcription enzymes via arrays of transcription factors bindings and often forms phase separate nuclear body in part through recruitment of BRD4 and MED1 that contain intrinsically disordered domain. Here we show KSHV TR possesses transcription regulatory function with LANA. A series of Cleavage Under Targets & Release Using Nuclease (CUT&RUN) demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modification were also conserved in unique region of the KSHV genome among three PEL cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TR's. In the Ori-Lyt region, the LANA protein complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and the nucleosome is franked by two K-Rta recruitment sites. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function when the reporter was ligated with TR. The deletion of LANA acidic repeat sequence, a highly-disordered protein domain, further increased gene repression functions. Combined, the TR region is (i) an epigenetically active DNA element that is stitched within 12.5 kb (20-40 copies), (ii) has an array of transcription factor (LANA) binding sites, (iii) recruited by transcription related enzymes including BRD4 (bromodomain containing 4), (iv) decorated by histone H3K27Ac marks, and (v) possesses orientation-independent transcription activation function. KSHV TR is therefore an enhancer domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps KSHV enhancer is predominantly regulated by the LANA nuclear body on the TR. We suggest that KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex.

Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is a g-herpesvirus which persists as circular extrachromasomal DNA in the nucleus during latent infection. During latency a limited number of viral proteins are expressed, including LANA (latency-associated nuclear antigen). LANA is a multi-functional protein known to interact with transcriptional regulators and chromatin remodelers, and to regulate the LANA and RTA promoters. We hypothesized that LANA may contribute to the establishment of latency through controlling chromatinization and histone modification of KSHV episomes. We performed ChIP-seq analysis and correlated H3K4me3, H3K27me3, polII, and LANA occupancy of the KSHV genome at nucleotide resolution. Epigenetic marks were analyzed in BCBL-1 lymphoid cells and in telomerase-immortalized vein endothelial TIVE-LTC cells. We found that the transcription active mark H3K4me3, but not silencing mark H3K27me3, was enriched at KSHV regions where LANA is bound. Co-occupancy of LANA and H3K4 was also detected on 167 host genes, of which 89 are actively transcribed. By comparing LANA occupancy with the profiling of 43 transcription factors from ENCODE, a subset of transcription factors was enriched at regions where LANA is bound, including znf143, CTCF, and Stat1. These results indicate that LANA also plays a role in modulating expression of host genes. Co-immunoprecipitation of LANA with the H3K4 methyltransferase hSET1 suggests that LANA recruits hSET1 to specific loci on the viral genome, leading to H3K4 methylation and ensuring transcription of latency-associated genes. Host gene expression may be regulated by the same mechanism. LANA binding was correlated with the distribution in the latent KSHV genome of the transcription active histone modification H3K4me3, the silencing mark H3K27me3, and RNA polymerase II, using cells of lymphoid and endothelial origin. A similar analysis was done for the human genome. Biological replicates (BR) and technical replicates (TR) were included.

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Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for KSHV LANA

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