Kobayashi Hanako, Tan Er Mei, Fleming Sharon E
Department of Nutritional Science and Toxicology, University of California, Berkeley, CA 94720, USA.
Int J Cancer. 2004 Mar 20;109(2):207-13. doi: 10.1002/ijc.11697.
Butyric acid is well recognized as a histone deacetylase (HDAC) inhibitor, and changes in histone acetylation are thought to alter gene expression. The mechanism by which sodium butyrate (NaB) induces p21WAF1/CIP1, a critical gene involved in the antiproliferative effect of NaB, was studied at the chromatin level. Using chromatin immunoprecipitation (ChIP) assay, acetylation of histone H3 was observed at the proximal region of the promoter within 30 min of NaB exposure and this extended to the distal region within 2 hr. By contrast, histone H4 was acetylated both at the proximal and the distal regions of the promoter within 30 min. NaB did not influence other histone modifications. NaB stimulated recruitment of the transcription factors ZBP89 and Sp1 as well as GCN5, but did not influence recruitment of Sp3, HDAC1, p300, or CBP. As recruitment of HDAC1 to the promoter appeared not to account for NaB-induced changes in histone acetylation, we aimed to influence HDAC activity by altering its phosphorylation status. The kinase inhibitor, H7, suppressed p21WAF1/CIP1 mRNA in both the absence and the presence of NaB without influencing the butyrate-induced hyperacetylation of H3 and H4 associated with the p21WAF1/CIP1 promoter. These results suggest that acetylation of histones at the p21WAF1/CIP1 promoter is not sufficient for NaB to exert antiproliferative effects via transcription of the p21WAF1/CIP1 gene. Induction of p21WAF1/CIP1 transcription by the phosphatase inhibitor, okadaic acid, in the absence of changes in association of acetylated histones with the p21WAF1/CIP1 promoter provides further evidence of the importance of phosphorylation to p21WAF1/CIP1 transcription.
丁酸是一种广为人知的组蛋白去乙酰化酶（HDAC）抑制剂，人们认为组蛋白乙酰化的变化会改变基因表达。本文在染色质水平上研究了丁酸钠（NaB）诱导p21WAF1/CIP1的机制，p21WAF1/CIP1是一个与NaB抗增殖作用相关的关键基因。使用染色质免疫沉淀（ChIP）分析，在NaB暴露30分钟内，在启动子近端区域观察到组蛋白H3的乙酰化，2小时内这种乙酰化扩展到远端区域。相比之下，组蛋白H4在30分钟内就在启动子的近端和远端区域发生了乙酰化。NaB不影响其他组蛋白修饰。NaB刺激了转录因子ZBP89、Sp1以及GCN5的募集，但不影响Sp3、HDAC1、p300或CBP的募集。由于HDAC1募集到启动子似乎不能解释NaB诱导的组蛋白乙酰化变化，我们旨在通过改变其磷酸化状态来影响HDAC活性。激酶抑制剂H7在有无NaB的情况下均抑制p21WAF1/CIP1 mRNA，且不影响丁酸盐诱导的与p21WAF1/CIP1启动子相关的H3和H4的超乙酰化。这些结果表明，p21WAF1/CIP1启动子处组蛋白的乙酰化不足以使NaB通过p21WAF1/CIP1基因的转录发挥抗增殖作用。在乙酰化组蛋白与p21WAF1/CIP1启动子的结合没有变化的情况下，磷酸酶抑制剂冈田酸诱导p21WAF1/CIP1转录，这进一步证明了磷酸化对p21WAF1/CIP1转录的重要性。
