Gene expression data from mouse HDAC4 KO pups, postnatal day 3

Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue-specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation, we employed the unbiased AcetylScan proteomic screen. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only CamK4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain. mRNA expression analysis was performed by microarray in 3-day-old HDAC4 KO pups and WT littermates. Ten samples were analysed for each genotype. Microarray quality control was performed using the software package provided on RACE (http://race.unil.ch).蛋白质可逆乙酰化是调控基因表达与细胞信号转导的核心机制，其由两类功能相互拮抗的酶家族协同调控：组蛋白乙酰转移酶（histone acetyltransferases，HATs）与组蛋白去乙酰化酶（histone deacetylases，HDACs）。HDAC4与其他IIa类家族成员类似，可通过其N端结构域与组织特异性转录因子结合，发挥强效的转录抑制作用。尽管IIa类组蛋白去乙酰化酶的赖氨酸去乙酰化酶活性远弱于I类酶，但已有研究表明HDAC4可通过与HDAC3的相互作用参与蛋白质去乙酰化过程。为探究HDAC4对蛋白质乙酰化的影响，我们采用了无偏倚的AcetylScan蛋白质组筛选技术。我们鉴定出多种已知存在乙酰化修饰的蛋白质，但实验结果显示，HDAC4的缺失并不会改变小鼠新生脑组织的乙酰化修饰谱。这与生化研究结论一致——后者提示HDAC4可能并不具备赖氨酸去乙酰化酶活性，但本研究的体内数据并不支持此前“HDAC4与HDAC3的相互作用可调控后者酶活性”的报道。为补充本研究的发现，我们使用Affymetrix基因芯片探究了HDAC4敲除（knock-out，KO）对小鼠出生后脑组织转录谱的影响。实验未观察到整体转录水平的显著变化，这与组蛋白乙酰化修饰谱无差异的结果相吻合。通过TaqMan定量聚合酶链反应（Taq-man qPCR）对芯片数据进行验证后发现，仅鱼精蛋白1（protamine 1）与胰岛素样生长因子结合蛋白6（Igfbp6）的mRNA水平上调超过1倍，仅钙/钙调蛋白依赖性蛋白激酶4（CamK4）的mRNA水平出现下调。HDAC4在P3小鼠脑组织中定位于细胞质，这与转录谱未受显著影响的实验结果一致。本研究通过微阵列分析了3日龄HDAC4敲除幼鼠及其野生型（wild type，WT）同窝仔鼠的mRNA表达水平，每种基因型均分析了10个样本。微阵列的质量控制采用RACE平台（http://race.unil.ch）提供的软件包完成。