Comparative transcript profiling of EBNA2 target gene expression in CBF1 proficient and deficient DG75 cells. Homo sapiens

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently growing lymphoblastoid cell lines (LCLs). The viral Epstein-Barr virus nuclear antigen 2 (EBNA2) plays key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Both, CBF1 independent EBNA2 target genes and chromatin binding sites are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 in CBF1 proficient and deficient B cells and requires EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2. In order to test, if EBNA2 can exert any functions in the absence of its DNA adaptor CBF1, a microarray based genome wide screen for EBNA2 target genes in DG75 B cells that are either proficient (wt) or deficient (ko) for CBF1 was performed. CBF1 deficient DG75 cells (SM224.9) cells had been generated by gene targeting using homologous recombination in the somatic B cell line DG75. Both cell lines, the CBF1 proficient DG75 parental cell line and the CBF1 deficient somatic knock-out cell line constitutively express an estrogen receptor (ER) hormone binding domain EBNA2 fusion protein (ER/EBNA2). ER/EBNA2 is retained in the cytoplasm of the cell but is rapidly activated and translocated to the nucleus in response to estrogen. For expression profiling, DG75, DG75 CBF1 ko (SM224.9), DG75 ER/EBNA2 CBF1 wt (SM295 D6) and DG75 ER/EBNA2 CBF1 ko (SM296 D3) cells were cultured in estrogen supplemented media for 24 h, total cellular RNAs were harvested and processed for the hybridization of gene arrays. The cellular system used for this study has been published: Maier S, Santak M, Mantik A, Grabusic K, Kremmer E, Hammerschmidt W, et al. A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent. Journal of Virology. 2005 Jul;79(14):8784-92. PubMed PMID: 15994772. Overall design: Gene expression profiling of EBNA2 induced target genes by Affymetrix microarrays

爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）感染可将静息态人B细胞转化为永生化增殖的淋巴母细胞系（lymphoblastoid cell lines, LCLs）。EB病毒核抗原2（EBNA2）在此过程中发挥关键作用，其优先结合B细胞增强子区域，并在LCLs中建立独特的病毒与宿主细胞基因表达程序。宿主细胞DNA结合因子CBF1/CSL可作为EBNA2的序列特异性染色质锚定蛋白。该蛋白高度保守且普遍表达，这引发了一个关键问题：是否存在其他宿主因子，可使EBNA2仅在B细胞中选择性结合染色质。本研究利用CBF1缺陷型B细胞，鉴定了受EBNA2调控的宿主差异表达基因（上调或下调），以及不依赖CBF1的EBNA2染色质结合位点。相较于依赖CBF1的EBNA2功能，不依赖CBF1的EBNA2靶基因与染色质结合位点均更为少见。不依赖CBF1的EBNA2结合位点中，EBF1结合基序显著富集。本研究证实，在CBF1正常与缺陷的B细胞中，EBNA2均可与EBF1结合，且EBF1是EBNA2结合不依赖CBF1位点的必需因子。本研究结果表明，EBF1作为EBNA2的辅助因子，可赋予EBNA2 B细胞特异性结合的特性。为验证EBNA2在缺失DNA衔接因子CBF1的情况下是否仍能发挥功能，本研究针对两种DG75 B细胞系开展了基于芯片的全基因组EBNA2靶基因筛选：一种为CBF1正常表达型（野生型, wt），另一种为CBF1敲除型（ko）。CBF1缺陷型DG75细胞（SM224.9）是通过在体细胞B细胞系DG75中利用同源重组进行基因靶向敲除构建得到的。CBF1正常表达的DG75亲本细胞系与CBF1缺陷型体细胞敲除细胞系，均组成型表达雌激素受体（estrogen receptor, ER）激素结合结构域与EBNA2的融合蛋白（ER/EBNA2）。ER/EBNA2通常滞留于细胞胞质中，但在雌激素刺激下可快速激活并转位进入细胞核。为进行基因表达谱分析，本研究将DG75、DG75 CBF1敲除型（SM224.9）、DG75 ER/EBNA2 CBF1野生型（SM295 D6）以及DG75 ER/EBNA2 CBF1敲除型（SM296 D3）四种细胞在添加雌激素的培养基中培养24小时，随后收集总细胞RNA并进行基因芯片杂交实验。本研究使用的细胞模型已发表：Maier S, Santak M, Mantik A, Grabusic K, Kremmer E, Hammerschmidt W, 等. 人B细胞系中CBF1的体细胞敲除实验证实，EBNA2诱导CD21与CCR7的表达完全依赖CBF1，而免疫球蛋白M的下调则部分不依赖CBF1。《病毒学杂志》（Journal of Virology）. 2005年7月;79(14):8784-92. PubMed PMID: 15994772. 实验整体设计：采用Affymetrix基因芯片开展EBNA2诱导靶基因的表达谱分析