Epstein-Barr virus (EBV) establishes latent infection in human B cells and is associated with a wide range of cancers. The EBV nuclear antigen 3 (EBNA3) family proteins are critical for B cell transformation and function as transcriptional regulators. It is well established that EBNA3A and EBNA3C cooperate in the regulation of cellular genes. Here, we demonstrate that the gene STK39 is repressed only by EBNA3A. This is the first example of a gene regulated only by EBNA3A in EBV-transformed lymphoblastoid cell lines (LCLs) without the help of EBNA3C. This was demonstrated using a variety of LCLs carrying either knockout, revertant, or conditional EBNA3 recombinants. Investigating the kinetics of EBNA3A-mediated changes in STK39 expression showed that STK39 becomes derepressed quickly after EBNA3A inactivation. This derepression is reversible as EBNA3A reactivation represses STK39 in the same cells expressing a conditional EBNA3A. STK39 is silenced shortly after primary B cell infection by EBV, and no STK39-encoded protein (SPAK) is detected 3 weeks postinfection. Chromatin immunoprecipitation (ChIP) analysis indicates that EBNA3A directly binds to a regulatory region downstream of the STK39 transcription start site. For the first time, we demonstrated that the polycomb repressive complex 2 with the deposition of the repressive mark H3K27me3 is not only important for the maintenance of an EBNA3A target gene (STK39) but is also essential for the initial establishment of its silencing. Finally, we showed that DNA methyltransferases are involved in the EBNA3A-mediated repression of STK39.

IMPORTANCE EBV is well known for its ability to transform B lymphocytes to continuously proliferating lymphoblastoid cell lines. This is achieved in part by the reprogramming of cellular gene transcription by EBV transcription factors, including the EBNA3 proteins that play a crucial role in this process. In the present study, we found that EBNA3A epigenetically silences STK39. This is the first gene where EBNA3A has been found to exert its repressive role by itself, without needing its coregulators EBNA3B and EBNA3C. Furthermore, we demonstrated that the polycomb repressor complex is essential for EBNA3A-mediated repression of STK39. Findings in this study provide new insights into the regulation of cellular genes by the transcription factor EBNA3A.

爱泼斯坦-巴尔病毒（EBV）在人B细胞中建立潜在感染，并与多种癌症相关。EBV核抗原3（EBNA3）家族蛋白对于B细胞转化至关重要，并起着转录调节剂的作用。众所周知，EBNA3A和EBNA3C在细胞基因的调控中相互配合。在这里，我们证明了基因STK39仅被EBNA3A抑制。这是在没有EBNA3C帮助的情况下，仅由EBNA3A调控的基因在EBV转化的淋巴母细胞样细胞系（LCL）中的第一个例子。使用携带敲除，回复或条件性EBNA3重组体的各种LCL证明了这一点。研究EBNA3A介导的STK39表达变化的动力学过程表明，STK39EBNA3A失活后迅速变得抑制。当EBNA3A激活抑制表达条件性EBNA3A的相同细胞中的STK39时，这种抑制作用是可逆的。EBV感染原代B细胞后不久，STK39被沉默，感染后3周未检测到STK39编码蛋白（SPAK）。染色质免疫沉淀（ChIP）分析表明EBNA3A直接与STK39转录起始位点下游的调节区结合。首次，我们证明了带有阻遏标记H3K27me3的多梳阻抑复合物2不仅对维持EBNA3A靶基因（STK39），但对于其消音器的初始建立也必不可少。最后，我们表明DNA甲基转移酶参与EBNA3A介导的STK39抑制。

重要事项EBV以其将B淋巴细胞转化为连续增殖的淋巴母细胞样细胞系的能力而闻名。这部分是通过EBV转录因子（包括在该过程中起关键作用的EBNA3蛋白）对细胞基因转录进行重新编程来实现的。在本研究中，我们发现EBNA3A在表观遗传上使STK39沉默。这是第一个被发现具有EBNA3A抑制功能的基因，而无需其核心调控因子EBNA3B和EBNA3C。此外，我们证明了多梳阻遏物复合物对于EBNA3A介导的STK39抑制是必不可少的。这项研究的发现为转录因子EBNA3A对细胞基因的调控提供了新的见解。