Hassig C A, Tong J K, Fleischer T C, Owa T, Grable P G, Ayer D E, Schreiber S L
Howard Hughes Medical Institute, Department of Chemistry, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3519-24. doi: 10.1073/pnas.95.7.3519.
Treatment of mammalian cells with small molecule histone deacetylase (HDAC) inhibitors induces changes in the transcription of specific genes. These changes correlate directly with an increase in the acetylation levels of all four core histones in vivo. Antibodies directed against endogenous HDAC1, HDAC2, or HDAC3 immunoprecipitate histone deacetylase activity that is inhibited in vitro by the small molecule trapoxin (TPX), and all three HDACs are retained by a TPX-affinity matrix. HDAC1 and HDAC2 are associated in HeLa cells in a complex that is predominantly separate from an HDAC3 immune complex. Both Jurkat HDAC1 and HeLa HDAC1/2 immune complexes deacetylate all four core histones and recombinant HDAC1 deacetylates free and nucleosomal histones in vitro. Purified recombinant HDAC1 deacetylates core histones in the absence of protein cofactors. Site-directed mutagenesis was used to identify residues required for the enzymatic and structural integrity of HDAC1. Mutation of any one of four conserved residues causes deleterious effects on deacetylase activity and a reduced ability to bind a TPX-affinity matrix. A subset of these mutations also cause a decreased interaction with the HDAC1-associated proteins RbAp48 and mSin3A. Disruption of histone deacetylase activity either by TPX or by direct mutation of a histidine presumed to be in the active site abrogates HDAC1-mediated transcriptional repression of a targeted reporter gene in vivo.
用小分子组蛋白去乙酰化酶（HDAC）抑制剂处理哺乳动物细胞会诱导特定基因转录的变化。这些变化与体内所有四种核心组蛋白乙酰化水平的增加直接相关。针对内源性HDAC1、HDAC2或HDAC3的抗体免疫沉淀出在体外被小分子曲古抑菌素（TPX）抑制的组蛋白去乙酰化酶活性，并且所有三种HDAC都被TPX亲和基质保留。HDAC1和HDAC2在HeLa细胞中以一种复合物形式存在，该复合物主要与HDAC3免疫复合物分开。Jurkat HDAC1和HeLa HDAC1/2免疫复合物都能使所有四种核心组蛋白去乙酰化，并且重组HDAC1在体外能使游离和核小体组蛋白去乙酰化。纯化的重组HDAC1在没有蛋白质辅因子的情况下能使核心组蛋白去乙酰化。使用定点诱变来鉴定HDAC1酶活性和结构完整性所需的残基。四个保守残基中任何一个的突变都会对去乙酰化酶活性产生有害影响，并降低与TPX亲和基质结合的能力。这些突变中的一部分还会导致与HDAC1相关蛋白RbAp48和mSin3A的相互作用减少。通过TPX或通过直接突变假定位于活性位点的组氨酸来破坏组蛋白去乙酰化酶活性，会消除HDAC1在体内对靶向报告基因的转录抑制作用。
