Specific inhibition of p300-HAT alters Global Gene Expression and Repress HIV replication. Homo sapiens

Reversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT specific small molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells and consequently inhibit the multiplication of HIV. Keywords: Response to Inhibition of p300-HAT Overall design: The total RNA was isolated from control and treated cells using TRIZOL (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer) was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturer’s instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Four arrays were used with at least two biological treatments of cells and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all four replicates.

组蛋白与非组蛋白的可逆乙酰化在细胞稳态中发挥关键调控作用。组蛋白乙酰转移酶（histone acetyltransferases, HATs）功能异常可引发多种疾病，包括癌症、神经退行性病变、哮喘、糖尿病、艾滋病（AIDS）以及心肌肥大。
本研究报道了从天然非特异性HAT抑制剂藤黄酚（garcinol）出发，合成并表征了一系列p300-HAT特异性小分子抑制剂——藤黄酚本身对细胞具有显著毒性。实验结果表明，该特异性抑制剂可在体内选择性抑制p300介导的p53乙酰化。
进一步研究发现，抑制p300-HAT会下调部分基因的表达水平，但同时也会显著上调少数关键基因的转录活性。值得注意的是，此类抑制剂对T细胞无毒性，可抑制人类免疫缺陷病毒（HIV）感染细胞的组蛋白乙酰化，进而阻断HIV的增殖。
关键词：p300-HAT抑制应答
整体实验设计：采用TRIZOL（Invitrogen公司）试剂法从对照组与处理组细胞中提取总RNA。使用Micromax直接标记试剂盒MPS502（珀金埃尔默（PerkinElmer）公司），以70 μg总RNA为模板合成标记cDNA，并将杂交后的cDNA应用于基因芯片实验。所有实验步骤均严格遵循厂商操作手册（www.perkinelmer.com/lifesciences）执行。芯片玻片通过珀金埃尔默Scanarray Gx微阵列扫描仪完成即时扫描，采用珀金埃尔默Scanarray软件对芯片图像进行网格化归一化处理。本实验共设置4张芯片，至少包含2组细胞生物学重复，并在最终分析中纳入了染料交换实验。数据通过GeneSpring GX以及班加罗尔Genotypic Technology公司开发的Biointerpreter软件进行分析。差异表达基因的判定标准为：下调基因的log₂均值≤-1，上调基因的log₂均值≥+1，且仅选取在全部4次重复实验中均可稳定重复检出的基因。