DNA Methylation and Human Endogenous Retroviruses Linking Genetic and Environmental Factors in the Pathogenesis of Psychiatric Disorders

（1）对具有脑内转录潜能的人类内源性逆转录病毒（HERV）EST数据库的综合筛选及其活性的实验验证从460万条EST中，我们筛选出44万条来源于脑相关组织和细胞系的EST，从中提取出12个可能具有脑内转录活性的HERV基因座。最后，我们通过RT-PCR和随后的克隆和测序分析鉴定了12个HERV中的6个。（2）利用HERV通读转录本研究反义RNA对邻近基因的影响在前期的研究中，我们发现了两个HERV基因座，分别属于HERV-H和HERV-K家族。HERV-H（22 q12）位于SYN 3基因下游约4kb处，反向定位。为了研究反义RNA对HERV-H通读产物的SYN 3的作用，我们构建了三个表达载体，其含有从HERV-H的3 'LTR到SYN 3的最后一个外显子的基因组序列，除了最后一个外显子被反转之外，其余基因组序列相同，并且没有外显子序列。正在研究实验条件以获得最佳结果。(3)A HERV-K甲基化的初步研究用甲基化敏感酶通过Southern印迹杂交检测HERV-K家族的甲基化状态。材料是来自三个不同个体的外周血白细胞、胎脑、胎肝和胎盘。虽然大多数位点都发生了高度甲基化，但不同组织和个体间甲基化状态存在差异。外周血白细胞甲基化程度依次为脑细胞>肝细胞>胎盘细胞。当胎盘中个体间差异为A> B> C时，肝脏中也保持这种顺序。

(1)Comprehensive screening of EST database for human endogenous retroviruses(HERVs) with transcriptional potential in the brain and experimental confirmation of their activitiesAmong 4.6-million ESTs, we selected 440 thousands that were prepared from various brain-related tissues and cell lines From the 440-thousand ESTs, we extracted 12 HERV loci that may have transcriptional activities. Finally we identified six out of the 12 HERVs by RT-PCR and subsequent cloning & sequencing analysis.(2)Investigation of an anti-sense RNA effect on nearby genes by HERV-read-through transcriptsIn a previous study, we identified two HERV loci belonging to HERV-H and HERV-K families. That HERV-H(22q12) was located about 4 kb downstream of the SYN3 gene in the reverse orientation. To investigate an anti-sense RNA effect on SYN3 of the HERV-H read-through product, we constructed three expression vectors containing a genomic sequence from the 3'LTR of the HERV-H to the last exon of SYN3, the same genomic sequence except the last exon being reversed, and no sequence of the exon. Experimental conditions are being examined to obtain the best result.(3)A preliminary study on HERV-K methylationMethylation states of the HERV-K family were examined by Southern blot hybridization using a methylation sensitive enzyme. Materials were peripheral leukocytes, fetal brain, fetal liver, and placenta from three different individuals. Although almost loci were heavily methylated, difference of methylation states was observed between tissues and between individuals. The methylation states were higher in the following order : peripheral leukocytes> brain> liver> placenta. When the difference between individuals was A> B≧C in the placentas, this order was also kept in the livers.,