Methylation in binge ethanol-induced spleen atrophy in adolescent rats

青春期大鼠暴食乙醇引起的脾萎缩的甲基化

基本信息

批准号：
10400702
负责人：
SULIE L. CHANG
金额：
$ 31.3万
依托单位：
SETON HALL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-20 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10400702
关键词：
Addictive Behavior Adolescence Adolescent Adult Aging Alcohol abuse Alcohol consumption Alcoholic Intoxication Alcoholism Alcohols Apoptosis Area Atrophic Biological Process Blood Body Weight CASP3 gene CD3 Antigens Cell Death Cell Separation Cell physiology Cells Collaborations Consumption Cytosine DNA DNA Methylation DNA Methyltransferase Inhibitor DNA Modification Methylases DNA Modification Process Data Deoxycytidine Development Doctor of Medicine Doctor of Philosophy Down-Regulation Enzymes Epigenetic Process Ethanol Ethanol Metabolism Event Extramural Activities Funding Opportunities Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Goals Hypermethylation ISGF3G protein Immune Immune System Diseases Immune response Immune system Immunity Immunophenotyping Inbred F344 Rats Individual Interferons Interleukins Intervention Investigation Lead Liver Lymphoid Male Adolescents Malpighian corpuscles Mediating Methylation Modification National Institute on Alcohol Abuse and Alcoholism Neurons Organ Physiological Population Proteins Quantitative Reverse Transcriptase PCR Rattus Regimen Regulation Research Personnel Research Project Grants Resources Role Spleen Sprague-Dawley Rats Structure Suspensions System T-Lymphocyte Testing Time Tissues Transcriptional Regulation United States National Institutes of Health Universities Water Weight adolescent alcohol abuse alcohol effect alcohol exposure alcohol research base binge drinking bisulfite sequencing chemokine receptor critical period demethylation distilled alcoholic beverage epigenetic variation experience genome wide methylation inhibitor male power analysis programs protein expression pyrosequencing research facility response splenic injury treatment strategy underage drinking whole genome

项目摘要

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.

项目摘要：该修订后的U01研究项目是对PAR-16-214的回应，其资金机会标题为“计划用于壁外/壁内研究合作”。成为塞顿霍尔大学的两个外校外调查员，而大卫·戈德曼（David Goldman，M.D。）将成为壁内 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，但完全，专业知识和经验及其研究设施和其他资源。项目目标是描述DNA甲基化参与暴饮暴食（ETOH）诱导的青少年期间脾萎缩。青少年是各种成熟的重要时期生理功能，包括免疫复杂。暴饮暴食，定义为消费在短时间内过多的酒精量导致血液ETOH浓度（BEC）至少为0.08 g/dl，在青少年中很受欢迎，可以导致加性行为，最终导致酒精中毒成年。脾脏的萎缩，一种关键的免疫器，与免疫功能障碍高度相关。我们有表明用4.8 g/kg/d etoH处理3 d的处理可降低5周旧的囊的大小青春期雄性F344大鼠，但不在成年大鼠中。脾白的区域也有所减少用EtoH处理的Sprague Dawley大鼠大鼠的纸浆和白色果肉结构的变形。表达 CD3是T细胞标记，减少了。 F344的CD3+ T细胞群也有所下降大鼠给予暴饮暴食。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纸浆和总体脾脏重量。 caspase-3的表达是细胞死亡的关键酶，与相对袖子的负相关，表明狂欢会诱导T细胞的损失导致白浆和袖萎缩的扭曲，部分原因是凋亡。 DNA甲基化是一种表观遗传事件，通过通过转录后修饰DNA进行的表观遗传事件 DNA甲基转移酶（DNMTS）调节基因表达。我们的初步研究表明 DNA甲基化的抑制剂5-aza-2'-脱氧胞丁丁（5-az）反转BENGE ETOH诱导脾萎缩。同时，我们还发现，在大鼠给予暴饮暴食。 QRT-PCR阵列数据表明6个基因被显着下调在暴饮暴食后的84个免疫和ETOH代谢相关的基因中。总的来说，我们假设DNA甲基化参与了暴饮暴食诱导的各种靶基因的调节导致T细胞凋亡，白浆变形和青少年期间脾萎缩。测试在此修订后的应用中，我们提出了以下三个目标：目标1是调查暴饮暴食对脾萎缩的影响，并分离和表征CD3+细胞的CD3+细胞目标2和3中的研究。目标2是研究甲基化在暴饮暴食中的作用使用基因组亚硫酸盐测序（WGB），然后进行全基因组甲基化分析（GWMA）。 AIM 3是确定甲基化在基因表达和通过研究甲基化基因的表达，包括已知的ETOH调节基因，机械水平（Kergs）和WGB鉴定出甲基化基因（WGIMGS）。此外，甲基化抑制剂5-az将用于确认这些已鉴定的基因的甲基化是否涉及在暴饮暴食中诱导这些基因的表达。拟议的研究将数据驱动的发现和假设驱动的研究结合在一起。我们希望在狂暴的青少年大鼠脾脏中识别全基因组甲基化的局部 EtOH方案和Kergs和Wgimgs的甲基化局部这些基因编码的蛋白质的活性。有了这些信息，我们将能够发展基于表观遗传学的干预措施，以减少青少年期间的暴饮暴食引起的脾损伤。