Sjöblom-Hallén A, Yang W, Jansson A, Rymo L
Department of Clinical Chemistry and Transfusion Medicine, Göteborg University, Sahlgrenska University Hospital, SE 413 45 Gothenburg, Sweden.
J Virol. 1999 Apr;73(4):2983-93. doi: 10.1128/JVI.73.4.2983-2993.1999.
The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.
爱泼斯坦-巴尔病毒（EBV）基因组中的肿瘤相关潜伏膜蛋白1（LMP1）基因由EBV编码蛋白和作为一般信号转导途径一部分的细胞因子激活。如先前所示，LMP1启动子调控序列（LRS）的近端区域包含一个负性顺式元件，在EBNA2介导的B细胞中LMP1基因表达调控中起主要作用。在此，我们表明这种沉默活性与包含E盒同源基序的LRS序列中的转录增强子重叠。推定的阻遏物结合位点的突变在启动子近端背景和完整的LRS背景下均解除了抑制作用，表明阻遏物具有功能作用。凝胶阻滞试验表明，包括Max、Mad1、USF、E12和E47在内的碱性螺旋-环-螺旋转录因子家族成员以及共阻遏物mSin3A与含E盒的序列结合。增强子活性与USF的结合相关。此外，报告基因构建体中LMP1启动子的活性通过USF1和USF2a的过表达而上调，并且Max和Mad1的同时表达抑制了反式激活。这表明Max-Mad1介导的包括mSin3A和组蛋白脱乙酰酶在内的多蛋白复合物锚定到E盒位点构成了抑制的基础。从组蛋白H3和H4上去除乙酰基部分应导致转录因子无法接近的染色质结构。因此，用曲古抑菌素A抑制脱乙酰酶活性可诱导EBV转化细胞中内源性LMP1基因的表达。
