Department of Neuroscience, Georgetown University, Washington, DC 20057, USA.
Gene. 2010 Jan 15;450(1-2):103-8. doi: 10.1016/j.gene.2009.10.008.
The expression levels of caspase-3, a major contributor to the execution of neuronal apoptosis, markedly decrease in the process of brain maturation. We have previously cloned the rat caspase-3 gene promoter and identified its essential regulatory elements. In the present study, we extended previous findings by examining transcriptional regulation of caspase-3 expression in the rat brain of two different ages, corresponding to the immature and mature brain. In particular, we determined that the rate of transcription initiation substantially declines during brain maturation. Furthermore, we established that mRNA levels of Ets1, Ets2, and Sp1 do not change in the brain with maturation, suggesting that these transcription factors do not contribute to age-dependent caspase-3 down-regulation. Hence, we examined a role of DNA methylation and histone modification in this process. Utilizing bisulfite DNA sequencing, we determined the presence of age-dependent differentially methylated fragments within the caspase-3 promoter region. Strikingly, differentially methylated CpG sites correspond to the predicted binding sites for a number of transcription factors that have been previously shown to be involved in neuronal development and differentiation. Moreover, using chromatin immunoprecipitation, we found that mature brains displayed significantly lower levels of histone 3 acetylated Lys14 and histone 4 acetylated Lys5, 8, 12, and 16. This observation is consistent with the decreased level of expression of caspase-3 in the mature brain. Together with our observation that histone deacetylase inhibitor, trichostatin A, increased the level of caspase-3 mRNA in cortical neurons in vitro, these results further indicate an important role of epigenetic factors in the regulation of caspase-3 gene expression.
半胱氨酸天冬氨酸蛋白酶-3（caspase-3）是神经元凋亡执行的主要贡献者，其表达水平在大脑成熟过程中显著降低。我们之前已经克隆了大鼠 caspase-3 基因启动子，并鉴定了其基本的调节元件。在本研究中，我们通过检查两个不同年龄（对应于不成熟和成熟大脑）的大鼠脑中 caspase-3 表达的转录调节，扩展了先前的发现。特别是，我们确定转录起始的速度在大脑成熟过程中大大降低。此外，我们确定 Ets1、Ets2 和 Sp1 的 mRNA 水平在大脑成熟过程中没有变化，这表明这些转录因子不会导致 caspase-3 的下调。因此，我们研究了 DNA 甲基化和组蛋白修饰在这个过程中的作用。利用亚硫酸氢盐 DNA 测序，我们确定了 caspase-3 启动子区域内存在与年龄相关的差异甲基化片段。引人注目的是，差异甲基化的 CpG 位点与先前显示参与神经元发育和分化的许多转录因子的预测结合位点相对应。此外，通过染色质免疫沉淀，我们发现成熟的大脑显示出显著降低的组蛋白 3 乙酰化 Lys14 和组蛋白 4 乙酰化 Lys5、8、12 和 16 的水平。这一观察结果与 caspase-3 在成熟大脑中的表达水平降低一致。与我们观察到组蛋白去乙酰化酶抑制剂 Trichostatin A 在体外增加皮质神经元中 caspase-3 mRNA 的水平一致，这些结果进一步表明表观遗传因素在 caspase-3 基因表达的调节中起着重要作用。
