ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3-a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFβ), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFβ with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFβ in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored.

译文

:ChIP-seq在淋巴母细胞细胞系(LCL)上进行,表达来自EBV重组子的表位标记的EBNA3A,EBNA3B或EBNA3C,揭示了EBNA3与染色质结合的重要原理。当与整体染色质循环数据结合时,发现结合EBNA3的基因座具有奇异的特征,每个基因座都直接与EBNA3抑制或EBNA3激活的基因相关,而与这两者均不相关。 EBNA3A和EBNA3C与受抑制和激活的基因显着相关。 EBNA3B基因座的直接直接关联只能与EBNA3B抑制的基因一起显示。在LCL GM12878中将EBNA3结合位点与已知转录因子结合位点进行比较,发现EBNA3s与RUNX3-a蛋白在B细胞转化过程中由EBV大量共定位。与RUNX3异源二聚体的核心结合因子(CBFβ)的β亚基可以强力共免疫沉淀EBNA3B和EBNA3C,但仅弱免疫共沉淀。慢病毒递送的shRNA耗尽RUNX3或CBFβ会损害多个受调控基因位点上被表位标记的EBNA3B和EBNA3C的结合,这表明在靶基因调控过程中,EBNA3募集过程中需要CBF异二聚体。 ShRNA介导的EBNA3C条件性LCL中CBFβ的耗竭证实了CBF在EBNA3C诱导和抑制的基因调控中的作用。这些结果揭示了迄今为止尚未发现的RUNX3 / CBF在B细胞转化和EBV潜伏期中的重要作用。

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