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. 2014 Jan 7;111(1):421-6.
doi: 10.1073/pnas.1321704111. Epub 2013 Dec 16.

Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A

Sizun Jiang  1 , Bradford Willox, Hufeng Zhou, Amy M Holthaus, Anqi Wang, Tommy T Shi, Seiji Maruo, Peter V Kharchenko, Eric C Johannsen, Elliott Kieff, Bo Zhao
Affiliations

Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A

Sizun Jiang et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.

Keywords: EBV; lymphoma; resting B lymphocyte; tumor suppressor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genome-wide distribution of EBNA3C sites and EBNA3C site overlap with EBNA2/RBPJ, BATF/IRF4, SPI1/IRF4, and RUNX3. (A) Genome-wide distribution of EBNA3C DNA binding by chromatin states (%). EBNA3C distribution calculated based on the chromatin state of GM12878 cells; 12,992 EBNA3C sites within annotated genomic regions were used to identify the genome-wide distribution of EBNA3C sites. (B) Venn diagram showing EBNA3C site overlap with RUNX3, SPI1/IRF4, EBNA2/RBPJ, or BATF/IRF4 (±250 bp of EBNA3C sites).
Fig. 2.
Fig. 2.
EBNA3C-associated cell TF clusters, chromatin state, and associated motifs at different clusters. Partitioning Around Medoids (PAM) clustering was used to cluster all nonheterochromatin EBNA3C sites together with EBNA2 and cell TFs into five unique clusters. ChIP-seq signals centered at the EBNA3C sites with neighboring ±2 kb were shown on a red (highest binding) to white (no binding) scale. The chromatin state distribution of EBNA3C sites are represented in the column titled “State.” Enriched motifs at each cluster are shown next to the clusters.
Fig. 3.
Fig. 3.
Anchor plots of EBNA3C (E3C) or RUNX3 centered at E3C or RUNX3 peaks. E3C (Left) or RUNX3 (Right) was intersected pairwise (±250 bp) with HOMER and the anchor plots of E3C without RUNX3, E3C with RUNX3, or RUNX3 without E3C ChIP-seq signals were plotted around a ±2-kb region.
Fig. 4.
Fig. 4.
Anchor plots at EBNA3C sites, showing the effects of AICEs (BATF and IRF4, Left) and EICEs (SPI1 and IRF4, Right) on EBNA3C ChIP-seq signals on DNA. ChIP-seq signals were plotted around a ±1-kb region.
Fig. 5.
Fig. 5.
GSEA analysis of EBNA3C/Sin3A binding and gene expression. The enrichment profile is shown on top, 229 genes with EBNA3C/Sin3A sites at ±2 kb of TSS were indicated with blue vertical lines in the center, and 9,193 genes profiled were ranked from EBNA3C induced (left) to EBNA3C repressed (right).
Fig. 6.
Fig. 6.
EBNA3C site at the p14ARF promoter and association with Sin3A. (A) EBNA3C binding at the p14ARF promoter. Normalized EBNA3C, Sin3A, and other TF tag density signals at the CDKN2A/B locus are shown. Red arrows indicate the direction of transcription. The peak heights are indicated at the left of and for each track. (B) EBNA3CHT LCLs were grown under permissive or nonpermissive conditions for 7 d. Sin3A enrichment at the p14ARF promoter over the control antibody was determined by ChIP-qPCR. (C) Anti-Flag agarose beads were used to immune precipitate EBNA3CFH and associated proteins from EBNA3CFH LCL with GM12878 LCLs as control. EBNA3C-associated Sin3A was detected by Western blotting.
Fig. 7.
Fig. 7.
EBNA3C sites upstream of MYC. EBNA3C, EBNA2, RBPJ, IRF4, BATF, SPI1, EBF, Pol II, and histone signals at MYC and up to ∼600 kb upstream are shown.
Fig. 8.
Fig. 8.
EBNA3C binds to promoters via RUNX3, IRF4, and E2F to recruit Sin3A repressor complexes (Sin3A, HDACs 1 and 2, and RBPJ). This complex represses the nearby p14ARF TSS. EBNA3C binds to H3K4me1-enriched enhancers via BATF/IRF4 and/or SPI1, RUNX3 to recruit p300 and drive distal target gene expression.
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