Department of Pediatrics, Division of Neonatology and Developmental Biology, Neonatal Research Center, David Geffen School of Medicine University of California LosAngeles, Los Angeles, California 90095-1752, USA.
Endocrinology. 2013 Apr;154(4):1598-611. doi: 10.1210/en.2012-2076. Epub 2013 Mar 14.
The murine neuronal facilitative glucose transporter isoform 3 (Glut3) is developmentally regulated, peaking in expression at postnatal day (PN)14. In the present study, we characterized a canonical CpG island spanning the 5'-flanking region of the glut3 gene. Methylation-specific PCR and bisulfite sequencing identified methylation of this CpG ((m)CpG) island of the glut3 gene, frequency of methylation increasing 2.5-fold with a 1.6-fold increase in DNA methyl transferase 3a concentrations noted with advancing postnatal age (PN14 vs PN3). 5'-flanking region of glut3-luciferase reporter transient transfection in HT22 hippocampal neurons demonstrated that (m)CpGs inhibit glut3 transcription. Contrary to this biological function, glut3 expression rises synchronously with (m)CpGs in PN14 vs PN3 neurons. Chromatin immunoprecipitation (IP) revealed that methyl-CpG binding protein 2 (Mecp2) bound the glut3-(m)CpGs. Depending on association with specific coregulators, Mecp2, a dual regulator of gene transcription, may repress or activate a downstream gene. Sequential chromatin IP uncovered the glut3-(m)CpGs to bind Mecp2 exponentially upon recruitment of Creb1 rather than histone deacetylase 1. Co-IP and coimmunolocalization confirmed that Creb1 associated with Mecp2 and cotransfection with glut3-(m)CpG in HT22 cells enhanced glut3 transcription. Separate 5-aza-2'-deoxycytidine pretreatment or in combination with trichostatin A reduced (m)CpG and specific small interference RNAs targeting Mecp2 and Creb1 separately or together depleting Mecp2 and/or Creb1 binding of glut3-(m)CpGs reduced glut3 expression in HT22 cells. We conclude that Glut3 is a methylation-sensitive neuronal gene that recruits Mecp2. Recruitment of Creb1-Mecp2 by glut3-(m)CpG contributes towards transactivation, formulating an escape from (m)CpG-induced gene suppression, and thereby promoting developmental neuronal glut3 gene transcription and expression.
鼠神经元易化葡萄糖转运体 3 型（Glut3）在发育过程中受到调节，在出生后第 14 天（PN）达到表达高峰。在本研究中，我们对横跨 glut3 基因 5'侧翼区的典型 CpG 岛进行了特征描述。甲基化特异性 PCR 和亚硫酸氢盐测序鉴定了 glut3 基因该 CpG（mCpG）岛的甲基化，随着出生后年龄的增加（PN14 与 PN3），该岛的甲基化频率增加了 2.5 倍，DNA 甲基转移酶 3a 浓度增加了 1.6 倍。在 HT22 海马神经元中转染 glut3-荧光素酶报告基因的 5'侧翼区显示，（m）CpG 抑制 glut3 转录。与这种生物学功能相反，glut3 在 PN14 与 PN3 神经元中的表达与（m）CpG 同步上升。染色质免疫沉淀（ChIP）显示，甲基-CpG 结合蛋白 2（Mecp2）结合 glut3-（m）CpG。根据与特定共激活因子的关联，Mecp2 作为基因转录的双重调节剂，可能抑制或激活下游基因。顺序染色质 ChIP 揭示，glut3-（m）CpG 结合 Mecp2 的指数募集 Creb1 而不是组蛋白去乙酰化酶 1。Co-IP 和共免疫定位证实，Creb1 与 Mecp2 结合，并且在 HT22 细胞中与 glut3-（m）CpG 共转染增强了 glut3 转录。单独的 5-氮杂-2'-脱氧胞苷预处理或与 Trichostatin A 联合处理降低了（m）CpG，并分别或联合使用针对 Mecp2 和 Creb1 的特定小干扰 RNA 耗尽 Mecp2 和/或 Creb1 对 glut3-（m）CpGs 的结合降低了 HT22 细胞中的 glut3 表达。我们得出结论，Glut3 是一种对甲基化敏感的神经元基因，它募集 Mecp2。glut3-（m）CpG 募集 Creb1-Mecp2 有助于转录激活，形成逃避（m）CpG 诱导的基因抑制，并由此促进发育中的神经元 glut3 基因转录和表达。
