Methylation in binge ethanol-induced spleen atrophy in adolescent rats

项目总结： 这项修订的U01研究项目是对PAR-16-214的响应，其资助机会标题为 用于壁外/壁内酒精研究合作“。张素丽博士和李明达博士将 作为西顿霍尔大学的两名校外调查人员，大卫·戈德曼医学博士将担任校内调查人员 NIAAA/NIH的调查员。对于他们的酒精研究合作，他们将分享他们独特的，但 互补性、专门知识和经验及其研究设施和其他资源。 该项目的目标是描述DNA甲基化在酒精过量(Etoh)诱导的过程中的作用 青春期脾萎缩。青春期是人的全面成熟的重要时期 生理功能，包括免疫反应。酗酒，定义为饮酒 短时间内过量饮酒导致血液乙醇浓度(BEC)至少为0.08 G/dl在青少年中很流行，会导致成瘾行为，最终导致酒精中毒。 成人期。 作为一个关键的免疫器官，脾的萎缩与免疫功能障碍密切相关。我们有 结果表明，4.8g/kg/d乙醇连续给药3d可显著减小5周龄小鼠的脾大小 青春期雄性F344大鼠，成年大鼠无此现象。脾白质的面积也减少了。 无水乙醇治疗的SD大鼠牙髓和白髓结构的扭曲。的表达 T细胞标志物CD3表达下降。在F344中CD3+T细胞数量也有不同程度的下降 大鼠大量服用乙醇。这些数据表明，T细胞的丧失可能是白细胞扭曲的部分原因 牙髓和整体的相对脾重量。细胞死亡的关键酶caspase-3的表达， 与脾相对重量呈负相关，说明暴饮乙醇可导致T细胞的丢失 导致白髓扭曲和脾萎缩，部分原因是细胞凋亡。 DNA甲基化是一种表观遗传事件，通过以下方式对DNA进行转录后修饰 DNA甲基转移酶(DNMT)调节基因表达。我们的一项初步研究表明 DNA甲基化抑制剂5-氮杂-2‘-脱氧胞苷(5-AZ)逆转乙醇诱导的狂欢 脾萎缩。同时，我们还发现DNMT1在小鼠脾组织中的表达显著降低。 大鼠大量服用乙醇。QRT-PCR阵列数据显示有6个基因显著下调 在84个与酗酒后免疫和乙醇代谢相关的基因中。总而言之，我们 假设DNA甲基化参与乙醇狂欢诱导的多种靶基因的调节 导致青春期T细胞凋亡、白髓变形、脾萎缩。为了测试 这个假设，在这个修订的应用程序中，我们提出了以下三个目标：目标1是研究 大剂量乙醇摄入对脾萎缩的影响及CD3+细胞的分离和鉴定 目标2和目标3的研究。目标2是研究甲基化在暴饮乙醇诱导的脾萎缩中的作用 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 确定甲基化在暴饮乙醇诱导的脾萎缩中的基因表达和 通过研究甲基化基因的表达，包括已知的乙醇调节基因，来实现机制水平 (KERGs)和WGBS鉴定出甲基化基因(WGIMGs)。此外，甲基化抑制剂5-AZ将 用于确认是否涉及这些已识别基因(例如，甲基化的KERGs和WGIMGs)的甲基化 在暴饮暴食中诱导了这些基因的表达。 建议的研究将数据驱动的发现和假设驱动的调查结合在一起。 我们希望在暴饮暴食的青春期大鼠的脾中识别全基因组甲基化的基因座 Etoh方案和KERGs和WGIMGs的甲基化位点，并测定其表达和 这些基因编码的蛋白质的活性。有了这些信息，我们就能够开发出 基于表观遗传学的干预措施减少青春期酗酒所致的脾损伤。

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.