EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells.
EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells.
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DOI：
10.1371/journal.ppat.1005383
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发表时间：
2016-01
期刊：
影响因子：
6.7
通讯作者：
Allday MJ
中科院分区：
文献类型：
--
作者：
Kalchschmidt JS;Gillman AC;Paschos K;Bazot Q;Kempkes B;Allday MJ
EB病毒核抗原3C（EBNA 3C）是一种有效的基因表达抑制因子，但对抑制过程中发生的事件顺序知之甚少。为了进一步探索EBNA 3C在基因抑制中的作用，特别是与组蛋白修饰和细胞因子相关的作用，研究了先前报道的被EBNA 3C抑制最强烈的三个宿主基因。COBLL 1是一种功能未知的基因，仅由EBNA 3C调节，两种共调节的去整合素/金属蛋白酶ADAM 28和ADAMDEC 1先前已被描述为EBNA 3A和EBNA 3C的靶标。在此首次显示EBNA 3C是原代B细胞感染后早期所有三种基因的主要调节因子。使用各种EBV重组体，仅当EBNA 3C表达时才看到超过数量级的抑制。出乎意料的是，直到感染后30天才实现完全抑制。这在已建立的携带EBNA 3C功能条件性EBV重组体的LCL中准确再现，证明了条件性系统在感染后早期复制事件的实用性。使用该系统，详细的染色质免疫沉淀分析显示，最初的阻遏与激活相关的组蛋白修饰（H3 K9 ac，H3 K27 ac和H3 K4 me 3）的丢失有关，并且与polycomb蛋白的募集和阻遏性H3 K27 me 3修饰的沉积无关，这仅在阻遏后期观察到。最值得注意的是，与目前RBPJ抑制模型相反，只有当EBNA 3C起作用时，这种DNA结合因子才在EBNA 3C结合位点积累。瞬时报告基因分析表明，这些基因的阻遏依赖于EBNA 3C和RBPJ之间的相互作用。这一点用编码不能结合RBPJ的EBNA 3C突变体的新型EBV重组体证实，其显示该病毒不能在新感染的原代B细胞中抑制COBLL 1或ADAM 28/ADAM DEC 1。Epstein-Barr核蛋白EBNA 3C是由EBV生长转化的B细胞中宿主基因表达的充分表征的阻遏物。也已经确定EBNA 3C可以与细胞因子RBPJ相互作用，RBPJ是从蠕虫到人类保守的Notch信号通路中的DNA结合因子。然而，在这项研究之前，很少有人知道这两种蛋白质之间的相互作用在宿主基因的抑制过程中的作用。因此，我们选择了三个基因的表达是非常强大的抑制EBNA 3C-探索分子相互作用。然而，这些基因尚未显示需要RBPJ进行EBNA 3C介导的抑制。我们已经描述了抑制过程中的事件序列，并挑战了一个广泛持有的假设，即如果蛋白质与RBPJ相互作用，它将被招募到DNA中，因为RBPJ具有结合特定序列的内在能力。我们表明，与RBPJ的相互作用是必不可少的所有三个基因的抑制期间，由EBV感染的B细胞，但RBPJ本身只招募的基因时，EBNA 3C是功能。这些数据表明，当EBNA 3C阻止细胞基因的表达时，多种蛋白质的相互作用出乎意料地复杂。
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 repression–particularly in relation to histone modifications and cell factors involved–the 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. The Epstein-Barr nuclear protein EBNA3C is a well-characterised repressor of host gene expression in B cells growth-transformed by EBV. It is also well established that EBNA3C can interact with the cellular factor RBPJ, a DNA-binding factor in the Notch signalling pathway conserved from worms to humans. However, prior to this study, little was known about the role of the interaction between these two proteins during the repression of host genes. We therefore chose three genes–the expression of which is very robustly repressed by EBNA3C –to explore the molecular interactions involved. Hitherto these genes had not been shown to require RBPJ for EBNA3C-mediated repression. We have described the sequence of events during repression and challenge a widely held assumption that if a protein interacts with RBPJ it would be recruited to DNA because of the intrinsic capacity of RBPJ to bind specific sequences. We show that interaction with RBPJ is essential for the repression of all three genes during the infection of B cells by EBV, but that RBPJ itself is only recruited to the genes when EBNA3C is functional. These data suggest an unexpectedly complex interaction of multiple proteins when EBNA3C prevents the expression of cellular genes.