Dong E, Guidotti A, Grayson D R, Costa E
Psychiatric Institute, Department of Psychiatry, University of Illinois, Chicago, IL 60612, USA.
Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4676-81. doi: 10.1073/pnas.0700529104. Epub 2007 Mar 7.
Reelin and glutamic acid decarboxylase 67 (GAD(67)) expression down-regulation in GABAergic interneurons of mice exposed to protracted treatment with l-methionine (MET) is attributed to RELN and GAD(67) promoter cytosine-5-hypermethylation. This process recruits various transcription repressor proteins [methyl-CpG binding protein (MeCP2) and histone deacetylases (HDACs)] leading to formation of transcriptionally inactive chromatin. Here, we tested the hypothesis that RELN and GAD(67) promoter cytosine-5-hypermethylation induced by a protracted MET treatment is reversible and that repeated administration of HDAC inhibitors influences this process by an activation of DNA-cytosine-5-demethylation. In the frontal cortices of mice receiving MET (5.2 mmol/kg twice a day for 7 days) and killed at 1, 2, 3, 6, and 9 days during MET washout, we measured RELN (base pairs -414 to -242) and GAD(67) (base pairs -1133 to -942) promoter methylation and MeCP2 bound to methylated cytosines of RELN (base pairs -520 to -198) and GAD(67) (base pairs -446 to -760) promoters. Levels of RELN and GAD(67) promoter hypermethylation induced by 7 days of MET treatment declines by approximately 50% after 6 days of MET withdrawal. When valproate (VPA) (2 mmol/kg) or MS-275 (0.015-0.12 mmol/kg), two structurally unrelated HDAC inhibitors, was given after MET treatment termination, VPA and MS-275 dramatically accelerated RELN and GAD(67) promoter demethylation in 48-72 h. At these doses, VPA and MS-275 effectively increased the binding of acetylhistone-3 to RELN and GAD(67) promoters, suggesting that histone-3 covalent modifications modulate DNA demethylation in terminally differentiated neurons, supporting the view that, directly or indirectly, HDAC inhibitors may facilitate DNA demethylation.
长期接受L-蛋氨酸（MET）治疗的小鼠，其γ-氨基丁酸能中间神经元中Reelin和谷氨酸脱羧酶67（GAD(67)）表达下调，这归因于RELN和GAD(67)启动子的胞嘧啶-5-超甲基化。这一过程招募了各种转录抑制蛋白[甲基化CpG结合蛋白（MeCP2）和组蛋白脱乙酰基酶（HDACs）]，导致形成转录无活性的染色质。在此，我们检验了以下假设：长期MET治疗诱导的RELN和GAD(67)启动子胞嘧啶-5-超甲基化是可逆的，并且重复给予HDAC抑制剂通过激活DNA-胞嘧啶-5-去甲基化来影响这一过程。在接受MET（5.2 mmol/kg，每天两次，共7天）并在MET洗脱期的第1、2、3、6和9天处死的小鼠额叶皮质中，我们测量了RELN（碱基对-414至-242）和GAD(67)（碱基对-1133至-942）启动子的甲基化以及与RELN（碱基对-520至-198）和GAD(67)（碱基对-446至-760）启动子甲基化胞嘧啶结合的MeCP2。MET治疗7天诱导的RELN和GAD(67)启动子超甲基化水平在MET撤药6天后下降约50%。在MET治疗终止后给予丙戊酸（VPA）（2 mmol/kg）或MS-275（0.015 - 0.12 mmol/kg）这两种结构不相关的HDAC抑制剂后，VPA和MS-275在48 - 72小时内显著加速了RELN和GAD(67)启动子的去甲基化。在这些剂量下，VPA和MS-275有效地增加了乙酰化组蛋白-3与RELN和GAD(67)启动子的结合，表明组蛋白-3共价修饰调节终末分化神经元中的DNA去甲基化，支持了HDAC抑制剂可能直接或间接促进DNA去甲基化的观点。
