Functional and Imprinting Analysis in Brain of Angelman Syndrome Gene UBE3A

人类UBE 3A基因显示脑特异性部分印记，并且缺乏母系遗传的等位基因导致以神经行为异常为特征的Angelman综合征（AS）。在几种AS模型小鼠中，印迹Ube3a表达主要在海马、小脑浦肯野细胞和嗅球中检测到。因此，小鼠Ube3a的印记被认为是区域特异性的，在不同的大脑区域中，父亲Ube3a等位基因的沉默水平不同。为了确定印迹Ube3a表达的细胞类型，我们通过使用原代皮层细胞培养物分析了其在胚胎脑细胞中的印迹状态。用RT PCR和免疫荧光法检测Ube3a基因的等位基因表达。Ube3a基因在脑中编码两种RNA转录物：正义转录物和反义转录物。有义转录本在神经元中母系表达，但在胚胎脑中的神经胶质细胞中双等位基因表达，而反义转录本仅在神经元中表达，父系表达。我们的数据提出了脑细胞类型特异性印记的第一个证据，即，神经元特异性印迹，但不是在神经胶质细胞，Ube3a在原代脑细胞培养。仅在神经元中存在的正义和反义转录本的相互印记提示了与神经干细胞谱系决定相关的神经元特异性印记机制。

The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted Ube3a expression is detected predominantly in the hippocampus, cerebellar Purkinje cells, and the olfactory bulb. Therefore, imprinting of mouse Ube3a is thought to be region-specific with different levels of silencing of the paternal Ube3a allele in different brain regions. To determine cell-types of imprinted Ube3a expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT PCR and immunofluorescence were performed to determine the allelic expression of the gene The Ube3a gene encodes two RNA transcripts in the brain : sense and antisense transcripts. The sense transcript was expressed maternally in neurons but biallelically expressed in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and paternally expressed. Our data present the first evidence of brain cell-type specific imprinting, i.e., neuron-specific imprinting but not in glial cells, of Ube3a in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts presented only in neurons suggests the neuron-specific imprinting mechanism related to the lineage determination of neural stem cells.