de La Fuente C, Deng L, Santiago F, Arce L, Wang L, Kashanchi F
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103, USA.
AIDS Res Hum Retroviruses. 2000 Nov 1;16(16):1695-700. doi: 10.1089/08892220050193164.
By utilizing a human cDNA expression array blot (588 genes), we have observed overexpression of various transcription factors, cell cycle regulated kinases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibitor, p21/waf1. The p21/waf1 transcription and protein is overexpressed in all HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samples. While p21/waf1 has been shown to display a selectivity for G(1)/S cyclin/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/cdk2. Functionally, the association of p21/cyclin A/cdk2 decreased the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, as well as affecting other substrates such as the C-terminus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p21/waf1 promoter in a p53-independent manner. We found that the minimal p21/waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal promoter contained two E2A transcription factor binding sites located between the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1-infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA binding site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p21/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1-infected cells.
通过使用人cDNA表达阵列印迹（588个基因），我们观察到在HTLV-1感染的T细胞中多种转录因子、细胞周期调节激酶和DNA修复基因的过表达。本研究感兴趣并重点关注的基因之一是细胞周期蛋白依赖性激酶抑制剂p21/waf1。在所有测试的HTLV-1感染细胞系以及成人T细胞白血病（ATL）和热带痉挛性截瘫（HAM/TSP）患者样本中，p21/waf1的转录和蛋白均过表达。虽然p21/waf1已被证明对G(1)/S细胞周期蛋白/细胞周期蛋白依赖性激酶（cdk）复合物具有选择性，但我们观察到p21/waf1与细胞周期蛋白A/cdk2形成复合物。在功能上，如免疫沉淀后进行激酶分析所观察到的，p21/细胞周期蛋白A/cdk2的结合在体外降低了组蛋白H1的磷酸化，并且还影响了其他底物，如参与c-Abl和组蛋白去乙酰化酶1（HDAC1）调节的视网膜母细胞瘤（Rb）蛋白的C末端。发现野生型Tax能够以不依赖p53的方式反式激活p21/waf1启动子，而Tax的突变形式（M47）则不能。我们发现最小的p21/waf1启动子（-49至+49序列）被Tax激活，并且最小启动子在TATA框和起始位点之间包含两个E2A转录因子结合位点。E2A蛋白E12和E47以及相关的螺旋-环-螺旋蛋白HEB在HTLV-1感染的T细胞中均上调。当使用凝胶迁移分析时，我们发现只有E1位点（与转录起始位点重叠）是一个功能性DNA结合位点。通过使用染色质免疫沉淀（ChIP）分析，我们观察到在体内内源性p21/waf1启动子上组蛋白H4而非组蛋白H3被乙酰化，这意味着在HTLV-1感染细胞中p21/waf1上调过程中，与E2A结合至关重要的是CBP/p300，而非SAGA复合物。
