Methylation in binge ethanol-induced spleen atrophy in adolescent rats

项目摘要： 这个修订后的U 01研究项目是对PAR-16-214的回应，其资助机会标题为“计划 为校外/校内酒精研究合作”。苏利湖Chang博士和Ming D.李博士将 塞顿霍尔大学的两名校外调查员，以及医学博士大卫戈德曼。将是校内 NIAAA/NIH的研究人员。对于他们的酒精研究合作，他们将分享他们独特的，但 它们的研究设施和其他资源具有互补性、专门知识和经验。 该项目的目标是描述DNA甲基化参与酒精（EtOH）引起的 青春期脾脏萎缩。青春期是各种人成熟的重要时期， 生理功能，包括免疫反应。酗酒，定义为消费 在短时间内过量饮酒，导致血液EtOH浓度（BEC）至少为0.08 g/dl，在青少年中很受欢迎，并可能导致成瘾行为，最终导致酒精中毒。 成年 脾脏是重要的免疫器官，其萎缩与免疫功能紊乱密切相关。我们有 结果表明，用4.8g/kg/d乙醇处理3d，可使5周龄的脾脏大小有差异地减小 青春期雄性F344大鼠，但在成年大鼠中没有。脾白色区面积也减少 牙髓和白色髓结构的扭曲在Sprague道利大鼠用EtOH狂欢处理。表达 T细胞标志物CD 3降低。在F344中，CD 3 + T细胞群也有差异性降低， 给大鼠大量乙醇这些数据表明，T细胞的损失可能部分地解释了白色的扭曲。 果肉和整体相对脾脏重量。细胞死亡的关键酶caspase-3的表达， 与相对脾脏重量呈负相关，表明过量乙醇诱导T细胞损失 导致部分由于细胞凋亡引起的白色髓的变形和脾萎缩。 DNA甲基化是一种表观遗传事件，其通过DNA的转录后修饰进行操作， DNA甲基转移酶（DNMT）调节基因表达。我们的一项初步研究表明， 用DNA甲基化抑制剂5-氮杂-2 ′-脱氧胞苷（5-AZ）处理， 脾脏萎缩与此同时，我们还发现，DNMT 1表达的显着下降，在脾脏中， 给大鼠大量乙醇qRT-PCR阵列数据显示6个基因表达显著下调 84个免疫和乙醇代谢相关基因。总之，我们 假设DNA甲基化参与了乙醇过量诱导多种靶基因调控 导致青春期T细胞凋亡、白色髓变形和脾脏萎缩。测试 这个假设，在这个修改后的申请，我们提出了以下三个目标：目标1是调查 过量EtOH消耗对脾萎缩的影响，并分离和表征CD 3+细胞， 研究目标2和3。目的二是探讨甲基化在乙醇中毒所致脾萎缩中的作用 使用基因组亚硫酸氢盐测序（WGBS），随后进行全基因组甲基化分析（GWMA）。目标3是 为了确定甲基化在过量EtOH诱导的脾萎缩中的基因表达的参与， 通过研究甲基化基因的表达，包括已知的EtOH调节基因， （KERG）和WGBS鉴定甲基化基因（WGIMG）。此外，甲基化抑制剂5-AZ将 用于确认这些鉴定的基因的甲基化（例如，甲基化KERG和WGIMG）参与 在binge-EtOH诱导这些基因的表达。 这些研究将数据驱动的发现和假设驱动的调查结合在一起。 我们期望在经历过暴饮暴食的青春期大鼠的脾脏中鉴定出全基因组甲基化位点 EtOH方案和KERG和WGIMG的甲基化位点，并确定KERG和WGIMG的表达和 这些基因编码的蛋白质的活性。有了这些信息，我们将能够开发 基于表观遗传学的干预措施，以减少青春期酗酒引起的脾损伤。

Project Summary: This revised U01 research project is in response to PAR-16-214 with the Funding Opportunity Title, “Program for Extramural/Intramural Alcohol Research Collaborations”. Sulie L. Chang, Ph.D. and Ming D. Li, Ph.D. will be the two extramural investigators at Seton Hall University, and David Goldman, M.D. will be the intramural investigator at NIAAA/NIH. For their alcohol research collaborations, they will share their unique, yet complementary, expertise and experiences as well as their research facilities and other resources. The project goal is to delineate the involvement of DNA methylation in binge ethanol (EtOH)-induced spleen atrophy during adolescence. Adolescence is an important period for maturation of various physiological functions, including immune responses. Binge alcohol drinking, defined as consumption of excessive amounts of alcohol in a short time resulting in a blood EtOH concentration (BEC) of at least 0.08 g/dl, is popular among adolescents and can lead to addictive behaviors and eventually alcoholism in adulthood. Atrophy of the spleen, a key immune organ, is highly associated with immune dysfunction. We have shown that treatment with 4.8 g/kg/d EtOH for 3 d differentially decreases the size of the spleen in 5 wk old adolescent male F344 rats, but not in adult rats. There was also a decrease in the area of the splenic white pulp and a distortion of white pulp structure in Sprague Dawley rats binge treated with EtOH. Expression of CD3, a T cell marker, was decreased. The CD3+ T cell population was also differentially decreased in F344 rats given binge EtOH. These data showed that loss of T cells might partially, account for distortions of white pulp and the overall relative spleen weight. The expression of caspase-3, a key enzyme for cell death, negatively correlated with the relative spleen weight, indicating that binge EtOH induces loss of T cells leading to distortions of the white pulp and spleen atrophy, in part, due to apoptosis. DNA methylation is an epigenetic event that operates through post-transcriptional modification of DNA by DNA methyltransferases (DNMTs) to regulate gene expressions. One of our preliminary studies showed that treatment with 5-Aza-2′-deoxycytidine (5-AZ), an inhibitor of DNA methylation, reversed binge EtOH-induced spleen atrophy. In parallel, we also found that significant decreases of DNMT1 expression in the spleens of rats given binge EtOH. The qRT-PCR array data showed that 6 genes were significantly downregulated among 84 immunity and EtOH metabolism related genes after binge drinking. Taken together, we hypothesize that DNA methylation is involved in binge EtOH-induced regulation of various target genes leading to apoptosis of T cells, distortion of white pulp, and spleen atrophy during adolescence. To test this hypothesis, in this revised application, we propose the following three aims: Aim 1 is to investigate the effects of binge EtOH consumption on spleen atrophy, and to isolate and characterize CD3+ cells for the studies in Aims 2 and 3. Aim 2 is to investigate the role of methylation in binge EtOH-induced spleen atrophy using genome bisulfite sequencing (WGBS), followed by genome-wide methylation analysis (GWMA). Aim 3 is to determine the involvement of methylation in binge EtOH-induced spleen atrophy at the gene expression and mechanistic levels by studying the expression of methylated genes, including the known EtOH regulated genes (KERGs) and WGBS identified methylated genes (WGIMGs). In addition, the methylation inhibitor, 5-AZ, will be used to confirm if methylation of these identified genes (e.g.,methylated KERGs and WGIMGs) is involved in binge-EtOH induced expression of these genes. The proposed studies have combined data-driven discovery and hypothesis-driven investigation together. We expect to identify whole-genome methylated loci in the spleens of adolescent rats subjected to a binge EtOH regimen and the methylation loci of the KERGs and WGIMGs and determine the expression and activities of the proteins encoded by these genes. With these information, we will be able to develop epigenetic-based interventions to curtail binge alcohol-induced spleen injury during adolescence.