Bloch D B, Chiche J D, Orth D, de la Monte S M, Rosenzweig A, Bloch K D
Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts.
Mol Cell Biol. 1999 Jun;19(6):4423-30. doi: 10.1128/MCB.19.6.4423.
The nuclear body is a cellular structure that appears to be involved in the pathogenesis of acute promyelocytic leukemia and viral infection. In addition, the nuclear body is a target of autoantibodies in patients with the autoimmune disease primary biliary cirrhosis. Although the precise function of the nuclear body in normal cellular biology is unknown, this structure may have a role in the regulation of gene transcription. In a previous investigation, we identified a leukocyte-specific, gamma interferon (IFN-gamma)-inducible autoantigen designated Sp140. The objectives of the present study were to investigate the cellular location of Sp140 with respect to the nuclear-body components PML and Sp100 and to examine the potential role of Sp140 in the regulation of gene transcription. We used adenovirus-mediated gene transfer to express Sp140 in human cells and observed that the protein colocalized with PML and Sp100 in resting cells and associated with structures containing PML during mitosis. In cells infected with the adenovirus expressing Sp140 and incubated with IFN-gamma, the number of PML-Sp100 nuclear bodies per cell increased but immunoreactive Sp140 was not evenly distributed among the nuclear bodies. Sp140 associated with a subset of IFN-gamma-induced PML-Sp100 nuclear bodies. To examine the potential effect of Sp140 on gene transcription, a plasmid encoding Sp140 fused to the DNA-binding domain of GAL4 was cotransfected into COS cells with a chloramphenicol acetyltransferase (CAT) reporter gene containing five GAL4-binding sites and a simian virus 40 enhancer region. The GAL4-Sp140 fusion protein increased the expression of the reporter gene. In contrast, Sp100 fused to the GAL4 DNA-binding domain inhibited CAT activity in transfected mammalian cells. The results of this study demonstrate that Sp140 associates with a subset of PML-Sp100 nuclear bodies in IFN-gamma-treated cells and that Sp140 may activate gene transcription. Taken together, these observations suggest that the nuclear bodies within a cell may be heterogeneous with respect to both composition and function.
核体是一种细胞结构，似乎与急性早幼粒细胞白血病的发病机制及病毒感染有关。此外，核体是自身免疫性疾病原发性胆汁性肝硬化患者自身抗体的靶标。尽管核体在正常细胞生物学中的精确功能尚不清楚，但该结构可能在基因转录调控中发挥作用。在先前的一项研究中，我们鉴定出一种白细胞特异性、γ干扰素（IFN-γ）诱导的自身抗原，命名为Sp140。本研究的目的是研究Sp140相对于核体成分PML和Sp100的细胞定位，并探讨Sp140在基因转录调控中的潜在作用。我们利用腺病毒介导的基因转移在人细胞中表达Sp140，观察到该蛋白在静息细胞中与PML和Sp100共定位，并在有丝分裂期间与含有PML的结构相关联。在用表达Sp140的腺病毒感染并与IFN-γ孵育的细胞中，每个细胞中PML-Sp100核体的数量增加，但免疫反应性Sp140在核体中分布不均。Sp140与IFN-γ诱导的一部分PML-Sp100核体相关联。为了研究Sp140对基因转录的潜在影响，将一个编码与GAL4 DNA结合结构域融合的Sp140的质粒与一个含有五个GAL4结合位点和一个猿猴病毒40增强子区域的氯霉素乙酰转移酶（CAT）报告基因共转染到COS细胞中。GAL4-Sp140融合蛋白增加了报告基因的表达。相反，与GAL4 DNA结合结构域融合的Sp100在转染的哺乳动物细胞中抑制CAT活性。本研究结果表明，在IFN-γ处理的细胞中，Sp140与一部分PML-Sp100核体相关联，且Sp140可能激活基因转录。综上所述，这些观察结果表明，细胞内的核体在组成和功能方面可能是异质性的。
