Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

Abstract

EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up-or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10 −4 . lymphoma | Notch

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

“…Conversion of Affymetrix probe set identities into Ensembl gene codes identified 47 genes that were downregulated (see Table S1 in the supplemental material) and 122 genes that were upregulated by EBNA 3C by 2-fold or more (see Table S2 in the supplemental material). EBNA 3C was previously reported to upregulate expression of the B-cell activation marker complement receptor 2 (CR2, CD21) in BJAB E3C-4 cells and in an EBNA 3C transcomplemented LCL (50,56). Our analysis confirmed that this gene was upregulated 16-fold at the mRNA level, providing a good validation control for our study (see Table S2 in the supplemental material).…”

“…They comprise the genes encoding the heterodimeric B-lymphocyte ␣4␤1 integrin receptor (ITGB1 and ITGA4) and the novel ␣4␤1 integrin ligand, ADAM28 (7,31), along with a related gene, ADAMDEC1, located in the same gene cluster, that is downregulated during tumorigenesis (6,25). In support of our results, while this work was in progress, EBNA 3C was reported to repress ITGA4 expression in transcomplemented LCLs, although in another BL cell background EBNA 3C is required to maintain ITGA4 expression (52,56). Loss of EBNA 3B expression in LCLs results in downregulation of ITGB1, implicating EBNA 3B as a positive regulator of ITGB1 (52) ( Table 3).…”

“…Forty percent of the EBNA 3C-regulated genes we identified in our microarray analysis were associated with EBNA 3 binding peaks (P ϭ 7.8 ϫ 10 Ϫ8 ; hypergeometric), implicating targeted binding in their regulation. Seven hundred eighty-six (34%) out of a total number of 2,338 unique genes regulated by EBNA 3 proteins in our study or previous studies (8,14,52,56) were associated with a significant EBNA 3 binding peak.…”

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

“…3B). Thus, the EBNA3 proteins, despite their roles in the repression of multiple cell genes (19)(20)(21)(22)(23)(24)(25), bind predominantly at genomic sites bearing marks of transcriptionally active chromatin.…”

“…Although EBNA3s bind an RBPJ domain that is distinct from the EBNA2/ICN binding site, they nevertheless limit EBNA2 activation by competing for RBPJ binding (15)(16)(17)(18). In LCLs and Burkitt lymphoma tumor cells, the EBNA3 proteins have been shown to regulate distinct but extensively overlapping sets of cell genes (19)(20)(21)(22)(23)(24)(25)(26). EBNA3 proteins have been implicated in the pathogenesis of Burkitt lymphoma and in attenuating an antiproliferative DNA damage response during EBV transformation of primary B lymphocytes (19,(27)(28)(29).…”