Evaluation of the DNA microarray data by the nuclear run-on and Northern blot analyses

Since the molecular probes for the genes identified by the ExpressCode™ were available, a “self-made” array was generated. This “self-made” arrays contained PCR products representing 40 up-regulated genes in the proliferating cells tested on both ExpressCode™ and GeneChip®. In addition, PCR products for 14 genes up-regulated in lymphochip and Gene Chip® were generated by the amplification of gene fragments cloned into the plasmid vectors, and were spotted on the membrane. A secondary screen using a nuclear run-on technique was performed for the P493-6 cells growing in two different growth programmes. By this experiment, the target genes were sorted into EBV dependent and independent pathways.

Since all genes which were spotted onto these arrays were candidate or well known EBNA2 target genes derived from the previous screens, it would be expected that all of them generate a signal. This was not the case. Even signals of the well characterized target gene LMP1, known to be transcriptionally activated by EBNA2

were weak, indicating that the efficiency of the nuclear run-on technique can vary significantly depending on the individual gene analyzed.

Genes which seemed to belong to the EBNA2 pathway were further evaluated by the Northern blot analysis. In order to sort these genes into primary and secondary EBNA2 target genes, three cell lines were used: EREB2-5 in the presence and absence of de novo protein synthesis, P493-6 and BL41-K3. The results of the Northern blot analyses suggested that some of the genes are not primary EBNA2 target genes. One group, which encompasses the genes EGR-1, DUSP2, SLAM, lymphotactin, IP-10, MIP-1 alpha, PLAB, PI3K and ATF5, showed no change in gene expression in BL41-K3 thus, indicating the requirement of another cellular or viral protein for the induction of these genes. These genes were considered as secondary EBNA2 target genes and were not subjected to promoter analysis. Furthermore, two novel c-Myc target genes have been identified. CDC37 and NUDT4 were identified as genes induced downstream of c-Myc and thus were also excluded from any further promoter analysis. The rest of the genes formed three groups according to the results of the Northern blot analyses.

Group I contained the genes c-myc, CCR7 and RDC1 since these genes are induced in the absence of de novo protein synthesis. This indicates that these genes are primary EBNA2 target genes. The transcription factor and proto-oncogene c-myc and the chemokine receptor, a member of the G-protein-coupled receptor family, CCR7 had been shown to be primary target genes of EBNA2 (Kaiser et al., 1999, Burgstahler et al., 1995, respectively).

RDC1 is a novel primary EBNA2 target gene. It is an orphan G-protein coupled receptor. The data presented herein convincingly show a strong induction of RDC1 in EREB2-5 and BL41-K3. P493-6 cells failed to induce RDC1 according to the Northern blot analysis. Since the P493-6 cells are an EREB2-5 transfectants, there is no obvious explanation for this finding. The product of this gene is expressed in various cell types. RDC1 can act as a co-receptor for HIV (Shimizu et al., 2000), but its cellular function is not known.

Group II included two genes: bfl-1 and MIP-1 alpha. The bfl-1 protein is a member of the Bcl-2 gene family which comprises anti- and pro-apoptotic regulators. The protein encoded by this gene is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and it blocks caspase activation. This gene is a direct transcription target of NF-κB (Grumont et al., 1999, Edelstein et al., 2003) in response to inflammatory

extracellular signals, such as GM-CSF, CD40, PMA, TNF and IL-1, which suggests a cytoprotective function essential for lymphocyte activation as well as cell survival. It

was also shown by D’Souza et al., 2000 that the EBV protein LMP1 can transcriptionally up-regulate bfl-1. A second member of group II, the macrophage inflammatory protein 1 beta (MIP-1 beta) directly participates in the activation and directional migration of lymphocytes and monocytes to sites of inflammation. It is up- regulated in human T cells by the induction of the NF-κB pathway (Guo et al., 2002, Guo et al., 2003). Given that the induction of these genes is regulated by the NF-κB elements, which are among other factors activated by the EBV protein LMP1, it becomes clear why the induction of these genes is decreased in the absence of viral proteins in comparison to an EBV/EBNA2 situation.

Group III is formed by the genes CD21, CD83 and PLEK. CD21 is a human complement receptor type 2 (CR2). Additionally, it is the receptor for EBV and mediates EBV infection. The regulation of expression of this gene has been extensively studied (Makar et al., 1998, Ulgiati and Holers, 2001) and it will be discussed later in the context of promoter analysis. CD83 is a cell surface glycoprotein, expressed mainly on dendritic cells. It had been shown that the induction of this gene is strictly dependent on the NF-κB element (Berchtold et al., 2002). PLEK encodes a protein called pleckstrin, a major substrate of protein kinase C in blood platelets (Abrams et al., 1995) which is involved in the reorganisation of the actin skeleton (Ma and Abrams, 1999). It contains a pleckstrin homology domain (PH) which is commonly found in eukaryotic signalling molecules. The transcriptional regulation of this gene has not been described to date. All three genes are strongly induced by EBNA2. They require neither viral genes nor c-Myc to be induced. However, it is not possible to claim that they are directly regulated by EBNA2 as their mRNA is induced and/or stabilised by cycloheximide. Thus a comparison of the induction level between the activated and non-activated EBNA2 state in the presence of the protein synthesis inhibitor could not be indicative.

Although the results described above provide strong evidences that all genes belonging to group I, II or III are directly activated by EBNA2, the mechanism by which EBNA2 does so could only be indirectly addressed. Very recently, a direct biochemical approach towards the identification of target genes of a given transcription factor has been developed. This methodology identifies transcription

immunoprecipitation (ChIP). Briefly, cells are lysed, chromatin associated transcription factors and DNA are chemically crosslinked and protein/DNA complexes are immunoprecipitated. DNA fragments bound to the immunoprecipitate are quantified by PCR. This technique can now be used to identify genomic regions to which EBNA2 is bound and thus provide a direct biochemical approach by which EBNA2 target genes can be identified (Weinman et al., 2001).

3.3 A search for potential cis-acting elements relevant for EBNA2 function