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. 2014 Jul 17;10(7):e1004240.
doi: 10.1371/journal.ppat.1004240. eCollection 2014 Jul.

LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex

Jianhong Hu  1 , Yajie Yang  1 , Peter C Turner  1 , Vaibhav Jain  1 , Lauren M McIntyre  2 , Rolf Renne  3
Affiliations

LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex

Jianhong Hu et al. PLoS Pathog. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genome-wide epigenome analysis of KSHV in BCBL-1 cells.
ChIP assays were performed in BCBL-1 cells with control IgG, H3K4me3, H3K27me3, and Pol II antibody. The resulting ChIP DNA was used for library construction followed by Illumina sequencing. Sequencing tags mapped to viral genome were visualized in UCSC Genome Browser. The number of sequence tags is indicated for each track (y-axis), which represents the number of times each region was recovered by the ChIP-seq. The highest coverage for H3K4me3 is 6,660 within the terminal repeat which was not shown in order to visualize the lower peaks.
Figure 2
Figure 2. H3K4me3 and H3K27me3 modifications and PolII occupancy at selected viral promoters in BCBL-1 cells.
The sequencing tags were mapped to the viral genome. The +2 to −2 Kb regions spanning the transcription start site were screened for the occupancy by H3K4me3, H3K27me3, and Pol II. The arrows indicate the transcription start site and orientation of transcription.
Figure 3
Figure 3. Validation of ChIP-seq data by quantitative PCR.
Quantitative PCR assays were performed with ChIP DNA to detect the enrichments of H3K4me3 and H3K27me3 (A), and Pol II (B) on promoters for LANA, vIRF1, vIL6, RTA, coding region of ORF19, and K7. The level of enrichment of chromatin immunoprecipitated by antibodies was normalized to input.
Figure 4
Figure 4. Occupancy by H3K4me3, H3K27me3, and Pol II on the viral episomes in TIVE-LTC cells.
ChIP-seq was performed in long-term infected TIVE cells with antibody against H3K4me3, H3K27me3, or Pol II. The corresponding library was sequenced without (A) or with (B) enrichment for viral sequences with SureSelect Target Enrichment System (details in Material and Methods). Tags were mapped to viral genome and visualized in UCSC Genome Browser. The number of sequence tags is indicated for each track (y-axis).
Figure 5
Figure 5. UCSC Genome Browser view of LANA binding on the KSHV genome.
ChIP-seq was performed in BCBL-1, TIVE-LTC with or without SureSelect Target Enrichment System with LANA-specific antibody. Sequencing tags were mapped to viral genome and visualized in UCSC Genome Browser.
Figure 6
Figure 6. LANA transactivates viral promoters in a dose-dependent manner.
The −2 Kb promoter regions of individual viral genes were PCR amplified and cloned upstream of Luciferase reporter gene. 293 cells were co-transfected with fixed amount of reporter plasmid and increasing amount of LANA expression vector as indicated. Results from three independent experiments are shown and error bars indicate standard deviations.
Figure 7
Figure 7. Positions of LANA peaks to the transcription start sites of known genes in BCBL-1 and TIVE-LTC cells.
LANA peaks within the +/−2 kb region of known genes are calculated and plotted for distance from center of the peaks to the transcription start sites in BCBL-1 cells (A) and TIVE-LTC cells (B).
Figure 8
Figure 8. LANA binds to two sites within the IQGAP3 promoter and upregulates IQGAP3 expression.
(A) Using transient transfection assays LANA upregulates IQGAP3 promoter in a dose-dependent manner. (B) The proximal and distal similar LANA binding sites form complexes with purified LANA-C protein. At the top the sequences are shown for the high-affinity LBS1 site, the low affinity LBS2 site, and the BSpro and BSdis regions of IQGAP3. Bases differing from LBS1 are shown in red. Below are the result of gel shift analysis. Lane 1, LBS1 probe; Lane 2, LBS1 probe incubated with V5-tagged LANA-C; Lane 3, LBS1 probe incubated with V5-tagged LANA-C and V5 monoclonal antibody; Lanes 4 to 6, and 7 to 9, same incubations with BSpro and BSdis from IQGAP3 promoter; Lanes 10 to 12, and 13 to 15, same incubations with either negative control fragment, or LBS2, the low affinity LANA binding site from TR. Lanes 16 to 18, longer exposure of lanes 13 to 15. Complexes with LANA-C are indicated by single arrowheads, and supershifted complexes with LANA-C and V5 mAb by double arrowheads. (C) Transcription level of IQGAP3 is induced in cells expressing LANA. LANA/Tet-on cells were treated with doxycycline and total RNA extracted was analyzed by real-time RT-PCR using IQGAP3-specific primers.
Figure 9
Figure 9. Identification and biochemical characterization of novel LANA binding motif.
Peak Motif analysis of LANA enriched sequences retrieved to highly homologous consensus sequences from BCBL-1 (A) and LTC-TIVE (B) cells. (C) LANA-C binds to a novel sequence motif. Lane 1, LBS1 probe incubated with V5-tagged LANA-C; Lane 2, LBS1 probe incubated with V5-tagged LANA-C and V5 monoclonal antibody; Lane 3, LBS1 probe. Lanes 4 to 6, and 7 to 9, same incubations with (TCCAT)3 probe. Lanes 7 to 9 longer exposure to visualize bands. Comparison to Fig. 8 shows similar binding to LBS2.
Figure 10
Figure 10. Distance of LANA peaks and four transcription factor binding site at LANA-bound promoter regions using ENCODE data.
Distance was plotted based on analysis of ENCODE transcription factor ChIP-seq data set for GM12878, a B cell lymphoma: (A) ZNF143, (B) CTCF, (C) Whip, and (D) STAT1. Between 32% (Whip) and 45% (CTCF) of LANA binding peaks are located within 200 bp of their respective transcription factor binding sites.
Figure 11
Figure 11. LANA and H3K4me3 but not H3K27 overlap at many regions of the KSHV and human genome in BCBL-1 cells.
(A) ChIP-seq was performed in BCBL-1, with LANA-, H3K4me3-, H3K27me3-specific antibodies. Sequencing tags were mapped to viral genome and visualized in UCSC Genome Browser. Cluster analysis (seqMINER) using sequence regions (plus/minus 5 kbp around TSSs) that were enriched by LANA ChIP-seq upstream of 1295 annotated transcripts in BCBL-1 cells (Table 2 and Table S1). (B) Heterogeneous LANA binding patterns relative to TSSs; (C) H3K4me3 distribution at LANA-enriched sites and (D) H3K27me3 distribution at LANA-enriched sites.
Figure 12
Figure 12. LANA forms complex with H3K4me3 methyltransferase hSET1 in vivo.
Nuclear extract was harvested from BCBL-1 cells (A) or BC-3 cells (B) and LTC-TIVE (C). The nuclear extract was immunoprecipitated with control IgG or monoclonal rat antibody against LANA. The immunoprecipitated complex was separated in 8% SDS-PAGE gel and immunoblotted with RbBP5, ASH2L, and hSET1, or MLL-1 antibody.
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