1. The Epstein-Barr Virus Regulome in Lymphoblastoid Cells
Sizun Jiang, Hufeng Zhou, Jun Liang, Catherine Gerdt, Chong Wang, Liangru Ke, Stefanie C.S. Schmidt, Yohei Narita, Yijie Ma, Shuangqi Wang, Tyler Colson, Benjamin Gewurz, Guoliang Li, Elliott Kieff, Bo Zhao 
Cell Host & Microbe 
Volume 22, Issue 4, Pages e4 (October 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 22, 561-573. e4DOI: (10. 1016/j. chom. 2017
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3. Figure 1
RNAPII ChIA-PET Linked EBV Enhancers to Their Direct Target Genes
(A) ChIA-PET links between EBV enhancers and known EBNA2 target genes MTA1, SLC7A5, and IRAK1, and effects of enhancer CRISPR knockout on their expression. ChIP-seq tracks for the indicated TFs were listed on top. In ChIA-PET tracks, each magenta line represents a pair of genomic interactions mediated by RNAPII. ChIA-PET-linked genes were in red. gRNA-targeted regions are indicated by arrows. LCLs stably expressing Cas9 were transduced with gRNA or control lentiviruses and selected with puromycin. Total RNAs were prepared from these cells and qRT-PCR was used to determine the expression level normalized against β-actin as loading control. The levels of control gRNA-treated cells were set to 1. Error bars represented SE. ∗p < 0.05.
(B) Intersection of genes essential for LCL growth and survival and EBV enhancer-targeted genes.
(C) ChIA-PET links between EBV enhancers and genes essential for LCL survival, CDK6, CCND2, ZNF143, CTBP, and RBPJ, and a well-characterized EBNA2 target CR2. All data are represented as mean ± SE. See also Figure S1 and Tables S1 and S2.
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4. Figure 2 RNAPII ChIA-PET Linked ESEs to Their Direct Targets
(A) Snapshot of a 5 mb region on chromosome 18. ChIP-seq tracks for EBV TFs are shown on the top. The H3K27ac track is shown in the middle followed by RNAPII and CTCF ChIA-PET. Genes in the loci are listed, with MALT1, PMAIP1(NOXA), and BCL2 highlighted. Red arrows indicated ESEs at the loci.
(B) Snapshots of genome browser with ChIP-seq tracks and ChIA-PET links and cartoons representing the complexities of EBV-mediated enhancer-promoter interactions. Top, ChIP-seq tracks. Middle, red boxes indicate ESEs. RNAPII and CTCF ChIA-PET links are below ESEs followed by gene positions. Left panel, one ESE regulating one gene; middle panel, one ESE regulating two genes; right panel, three ESEs regulating one gene. In the cartoons, red arrows indicate ESEs and blue arrows indicate ESE-associated genes. Magenta lines represent ChIA-PET links.
(C) ESE and many other EBV enhancers were linked to CFLAR. Orange arrows indicate the EBV enhancers, the red arrow indicates ESE, and the green arrow indicates CFLAR gene body. See also Figure S2 and Table S3.
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5. Figure 3 MYC ESEs Were Essential for MYC Expression and Cell Growth
(A) RNAPII ChIA-PET linkages at the MYC locus. ChIP-seq tracks are shown on the top. MYCSE1 (−525 kb from TSS) and MYCSE2 (−428 kb from TSS) are indicated by red boxes below the tracks. RNAPII ChIA-PET links are shown by magenta lines. Orange boxes indicate the regions targeted by CRISPR.
(B) Validation of MYCSE1 deletion. Cas9 stable LCLs or BJAB were transduced with paired gRNA targeting the edges of ESE. After puromycin selection, genomic DNA was prepared from LCLs and BJAB cells transduced with dual gRNA. The targeted region was PCR amplified. The presence or absence of the MYC SE1 is shown.
(C) MYCSE1 deletion reduced MYC mRNA level. Two independent pairs of gRNAs both decreased MYC mRNA by qRT-PCR normalized to β-actin. Control gRNA-treated cells were set to 1. ∗p < 0.05.
(D) Growth of MYCSE1 deleted LCLs and LCLs with deletion in a control region. ∗∗p < 0.01.
(E) MYCSE2 deletion also reduced MYC mRNA level. Two independent pairs of gRNAs both decreased MYC mRNA by qRT-PCR. ∗p < All data are represented as mean ± SE. See also Figure S3.
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6. Figure 4
EBNA2, EBNA3A, and EBNA3C Were Essential for MYC ESEs Looping to MYC TSS
(A) qPCR primers spanning the MYC locus including the MYCSE1 and MYCSE2 enhancers for 3C are indicated on the top. The anchor primer was near MYC TSS. LCLs conditional for EBNA2, EBNA3A, and EBNA3C were grown under conditions permissive or non-permissive for their expression. Cells were fixed with formaldehyde and lyzed. Chromatin was digested with EcoRI, diluted, and ligated. After reverse crosslinking, DNA was purified. qPCR was used to determine the interaction frequency. The amount of DNA used in qPCR was normalized with GAPDH, and the qPCR primer efficiencies were normalized using ligated DNA from BAC clones covering the entire genomic region. EBNA2, EBNA3A, and EBNA3C inactivation significantly decreased MYC ESEs looping to MYC TSS. ∗p < Data are represented as mean ± SE.
(B) RBLs were infected with wild-type B958 or EBNA2 deletion mutant P3HR1 EBV for the time indicated. 3C-qPCR indicated that wild-type EBV-induced MYC ESEs looping to MYC TSS as early as 2 days post infection and continued to increase at day 7. However, EBV mutant deleted for EBNA2 and the EBNALP Y1Y2 exons failed to induce the looping. Data are represented as mean ± SE. ∗p < 0.05.
(C) ATAC-seq signals at the MYC ESEs. RBLs were infected with wild-type or P3HR1 mutant EBV for the indicated time. Nuclei were first prepared and mixed with transposase reaction. The DNA was then purified and PCR amplified. Deep sequencing was used to determine the accessibility of the MYC ESE regions. Published GM12878 LCLs ATAC-seq were also included.
(D) ATAC-seq signals at ESEs and the neighboring ±2 kb windows during the RBL infection time course experiment. LCL signals are indicated. See also Figure S4.
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7. Figure 5 EBNA3C Reduced Looping at the CDKN2A/B Loci
(A) RNAPII ChIA-PET linked the p14ARF, p15INK4B promoter, and p16INK4A gene body in LCLs. CDKN2A/B loci were also linked to MTAP promoter region.
(B) EBNA3C inactivation increased looping at the CDKN2A/B loci. Primers for 3C are listed on the top. Anchor primer is also indicated. EBNA3C conditional LCLs were grown under permissive or non-permissive conditions. 3C assays were done and the interaction frequencies between the anchor and other primers were determined by qPCR. The efficiencies of PCR primer pairs were normalized with BAC DNA. GAPDH was used to normalize the amount of DNA used in qPCR. All data are represented as mean ± SE. ∗p < See also Figure S5.
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8. Figure 6 EBNA3A Induced Looping at the CDKN2A/B Loci
(A) RNAPII ChIA-PET linked EBNA3A peaks >80k upstream of CDKN2A/B loci to the MTAP promoter and gene body. MTAP promoter and gene body were then linked to the CDKN2A/B loci. Major EBNA3C or EBNA3A ChIP-seq peaks are indicated.
(B) Primers used for 3C at the ∼150 kb region of the CDKN2A/B loci. Anchor primer near p16INK4A is indicated. 3C assay was done in GM12878 LCL (left) or EBNA3A conditional LCLs (right) grown under permissive or non-permissive conditions. qPCR was used to determine the interaction frequencies. EBNA3A inactivation significantly reduced EBNA3A site looping to the CDKN2A/B loci. ∗p < All data are represented as mean ± SE. See also Figure S6.
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9. Figure 7
EZH2 Inhibitor Decreased CDKN2A/B Looping and Stopped LCL Growth
(A) GM12878 LCLs were grown in 1% FBS and treated with EZH2 inhibitor 20 μM EPZ6438 for 4 days. 3C-qPCR was used to determine the interaction frequencies at the CDKN2A/B loci. ∗p < 0.05.
(B) GM12878 LCLs grown in 1% FBS were treated with 20 μM EPZ6438 or 10 μM UNC1999 for 4 days. Cell growth was determined using CellTiter-Glo. ∗∗p < All data are represented as mean ± SE. See also Figure S7.
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