EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells

abstract: ...It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repressionparticularly in relation to histone modifications and cell factors involvedthe three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1 , a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.

... act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repressionparticularly in relation to histone modifications and cell factors involvedthe three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1 , a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and

... RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells. Author Summary The Epstein

... ADAMs, a microarray study of EBNA3C-conditional LCL identified COBLL1 as the gene most robustly repressed by EBNA3C ( S1 Table , [ 52 ]). The function of the COBLL1 gene product is unknown and it has not previously been characterised as an EBNA3C repressed gene. Based on the previous studies and the microarrays, we selected ADAM28 , ADAMDEC1 and COBLL1 in order to explore in more detail the temporal sequence of events and factors that are involved in EBNA3C-mediated gene regulation. Here, we show

... EBNA3C-mediated transcriptional repression has been described in such detail and it provides novel insights into temporal sequence of events occurring early after infection and the dynamic role of RBPJ in EBV-mediated gene repression. Results EBNA3A and EBNA3C co-repress ADAM28 and ADAMDEC1 , but only EBNA3C is required to repress COBLL1 in primary B cell infection Interrogation of Affymetrix Exon 1.0 ST microarray analysis from the EBNA3C conditional system (3CHT) indicated that COBLL1 , ADAM28 and ADAMDEC1 required expression of functional EBNA3C for very significant levels of repression in LCL ( S1 Table ; http://www.epstein-barrvirus.org.uk ). In order to establish that ADAM28 , ADAMDEC1 and COBLL1 are regulated by EBNA3C during viral infection of B cellsand to determine whether EBNA3A and/or EBNA3B are involved in the regulationprimary CD19 + cells were infected with previously characterised wild type (B95.8-BAC) EBV or recombinant EBNA3 KO or revertant (Rev) viruses ( Fig 1A1C ) [ 54 ]. Infections with wild type, Rev and EBNA3B KO viruses resulted in a reduction of both ADAM28 (23 log fold) and ADAMDEC1 (12 log fold) levels of mRNA, over a period of 30 days after infection. In the absence of EBNA3C (3CKO) and to a lesser extent EBNA3A (3AKO) there was a failure to repress ADAM28 ( Fig 1A ) and ADAMDEC1 ( Fig 1B )this is consistent with reports derived from stable cell lines [ 51 ]. All infections with EBNA3C competent viruses led to a remarkable 34 log fold reduction of COBLL1 mRNA over the same period of time, but this was not seen after infection with 3CKO virushere the levels detected in primary B cells were maintained ( Fig 1C ). These differences in gene expression between the various virus infections were not observed for the two control genes ALAS1 ( S1A Fig ) and GNB2L1 ( S1B Fig ), neither of which are known to be targets of EBNA3 proteins or EBV. 10.1371/journal.ppat.1005383.g001 Fig 1 Repression of ADAM28 , ADAMDEC1 and COBLL1 after infection of primary B cells with

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Gene: COBLL1 - cordon-bleu WH2 repeat protein-like 1 - (NCBI)

Protein : 2daj

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