Latent infection of B lymphocytes by Epstein-Barr trojan (EBV) results in their immortalization into lymphoblastoid cell lines (LCLs); this latency system is controlled from the EBNA2 viral transcriptional activator which focuses on promoters via RBPJ a DNA binding protein in the Notch signaling pathway. binding at sites near EBNA3A- or EBNA3C-regulated genes is definitely specifically regulated from the respective EBNA3. To investigate EBNA3 binding specificity we recognized sequences and transcription factors enriched at EBNA3A- EBNA3B- and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is definitely enriched at EBNA3A- and EBNA3C-bound sites and exposed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-bad BJAB cells we demonstrate that IRF4 is essential for EBNA3C but not EBNA3A or EBNA3B binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for unique subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with additional cell transcription factors. IMPORTANCE Epstein-Barr computer virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines test. Using this approach we confirmed 10 of 12 EBNA3A- 9 of 10 EBNA3B- and 9 of 12 EBNA3C-bound sites recognized by ChIP-seq (Fig. 2). In addition we found evidence of EBNA3B binding at 3 sites (EIF2AK3 QSK and ALOXE3) by ChIP-qPCR that were not observed by ChIP-seq. Based on these results we estimated the overall level of sensitivity and specificity of our EBNA3 ChIP-seq experiments relative to ChIP-qPCR to be 92% and 83% respectively. FIG 2 ChIP-qPCR validation of EBNA3 binding sites in LCLs recognized by ChIP-seq. The Scoparone pub plots display enrichment of genomic DNA from ChIP of EBNA3A EBNA3B or EBNA3C relative to input. Each EBNA3 was specifically ChIPed using HA antibody with either the EBNA3A-F-HA … EBNA3-bound sites are overrepresented at promoter and enhancer elements. We constructed average histone profile Scoparone plots for ±2-kb areas centered on Rabbit polyclonal to ITPK1. EBNA3 maximum summits for the activation marks H3K9Ac H3K27Ac H3K4me1 H3K4me2 and H3K4me3; the repressive marks H3K27me3 and H3K9me3; and the transcribed-region-associated mark H3K36me3 using ENCODE histone ChIP-seq data units (48). For each EBNA3 protein the signals from acetylation marks (H3K9Ac and H3K27Ac) were strong as were mono- di- and trimethylation of H3K4 (Fig. 3A). For those EBNA3s levels of repressive marks characteristic of facultative (H3K27me3) and constitutive (H3K9me3) heterochromatin were very low despite the prior observation that H3K27me3 levels are improved at EBNA3A- and EBNA3C-repressed genes such as CDKN2A and BCL2L11 (BIM). We also annotated EBNA3-bound peaks according to their locations within the epigenetic scenery (49). The results for EBNA3B were standard: 8% of EBNA3B sites reside within active promoters defined by high H3K4me3 and H3K9Ac 12 within poor and poised promoters characterized by high H3K4me3 and low H3K27Ac or high H3K27me3 33 within strong enhancers with high H3K4me1 and high H3K27Ac and 25% within poor enhancers with intermediate H3K4me1 and little H3K27Ac and 22% were found in heterochromatin regions characterized by the absence of these histone marks (Fig. 3B). Therefore the EBNA3 proteins despite their functions in the repression of multiple cell genes (19 -25) bind mainly at genomic sites bearing marks of transcriptionally active chromatin. FIG 3 Characterization of EBNA3-bound sites. (A) Average histone profile Scoparone plots for EBNA3A- EBNA3B- and EBNA3C-bound sites. The average densities of ChIP-seq reads for the indicated histone modifications are plotted for ±2-kb windows round the Scoparone summits … Overlap among EBNA3A EBNA3B EBNA3C EBNA2 and RBPJ sites in LCLs. Because the EBNA3 proteins cooperatively regulate many cell genes and because EBNA2 EBNA3A and EBNA3C must interact with the RBPJ transcription element to transform B lymphocytes we wanted to examine Scoparone the degree of overlap among EBNA3A- EBNA3B- EBNA3C- and EBNA2-bound sites. Significant overlap between EBNA3A and EBNA3C has been mentioned previously (35). Our experiments estimate that this overlap is about 26% of EBNA3A peaks that are Scoparone EBNA3C cobound and reveal the overlap extends to EBNA3B-bound sites. For EBNA3B 21 are shared with EBNA3A 22 with EBNA3C and 37% with EBNA2. McClellan et al. found that 80% of the genes closest to an EBNA3-bound site were also the closest genes to an EBNA2-bound site (33). Using their approach we found only 36% of EBNA3-bound genes to be EBNA2 cobound though this proportion increases to 50% for EBNA3B-bound genes (observe Table S4 in the supplemental material). However in LCLs there is considerable overlap among EBNA2-.