The role of ALKBH5-mediated RNA demethylation in the maintenance of genomic stability in HSPCs

ALKBH5 介导的 RNA 去甲基化在维持 HSPC 基因组稳定性中的作用

基本信息

批准号：
10476005
负责人：
Zhijian Qian
金额：
$ 10万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10476005
关键词：
Aging Apoptosis Biological Process CD34 gene CSNK1A1 gene Cell Death Cell Survival Cells Chromosomes Complex DNA Damage DNA Repair DNA lesion Databases Development Dysmyelopoietic Syndromes Engineering Fanconi&apos s Anemia Functional disorder Gene Expression Regulation Genetic Genetic Models Genetic Transcription Genome Stability Genomic Instability Goals Growth Hematological Disease Hematopoietic Hematopoietic stem cells Human Human Cell Line In Vitro Life Maintenance Malignant - descriptor Malignant Neoplasms Mediating Messenger RNA Methylation Modification Molecular Mus Mutagenesis Oncogene Activation Oncogenes Oxidative Stress Pathway interactions Patients Physiological Play Polyribosomes Post-Transcriptional Regulation Post-Translational Protein Processing Process Proteins RECQL4 gene RNA RNA methylation Reactive Oxygen Species Role Source Speed Stress Sumoylation Pathway Testing Time Transgenic Mice Up-Regulation Xenograft procedure chromosome 5 loss chromosome 5q loss crosslinking and immunoprecipitation sequencing demethylation epigenetic regulation epitranscriptome genome integrity hematopoietic stem cell self-renewal in vivo insight knock-down leukemic transformation mouse genetics mouse model novel novel therapeutic intervention overexpression prevent repaired response ribosome profiling self-renewal stem cell function stem cells transcriptome transcriptome sequencing transplant model

Aging Apoptosis Biological Process CD34 gene CSNK1A1 gene Cell Death Cell Survival Cells Chromosomes Complex DNA Damage DNA Repair DNA lesion Databases Development Dysmyelopoietic Syndromes Engineering Fanconi&apos s Anemia Functional disorder Gene Expression Regulation Genetic Genetic Models Genetic Transcription Genome Stability Genomic Instability Goals Growth Hematological Disease Hematopoietic Hematopoietic stem cells Human Human Cell Line In Vitro Life Maintenance Malignant - descriptor Malignant Neoplasms Mediating Messenger RNA Methylation Modification Molecular Mus Mutagenesis Oncogene Activation Oncogenes Oxidative Stress Pathway interactions Patients Physiological Play Polyribosomes Post-Transcriptional Regulation Post-Translational Protein Processing Process Proteins RECQL4 gene RNA RNA methylation Reactive Oxygen Species Role Source Speed Stress Sumoylation Pathway Testing Time Transgenic Mice Up-Regulation Xenograft procedure chromosome 5 loss chromosome 5q loss crosslinking and immunoprecipitation sequencing demethylation epigenetic regulation epitranscriptome genome integrity hematopoietic stem cell self-renewal in vivo insight knock-down leukemic transformation mouse genetics mouse model novel novel therapeutic intervention overexpression prevent repaired response ribosome profiling self-renewal stem cell function stem cells transcriptome transcriptome sequencing transplant model

展开

项目摘要

Abstract Myelodysplastic syndromes (MDS) are a group of diverse malignant hematological disorders that originate from hematopoietic stem cells (HSCs). Increased levels of reactive oxygen species (ROS) and DNA damage were detected in hematopoietic cells from MDS patients. An elevated level of ROS that can be generated from both endogenous and exogenous sources as well as oncogene activation, leads to loss of quiescence and self-renewal of HSCs. ROS-induced DNA damage speeds up aging process of stem cells and contributes to the mutagenesis associated with cancer development. m6A RNA methylation has significant roles in multiple biological processes by introducing another layer of post-transcriptional regulation of gene expression within cells. The goal of this project is to elucidate the role of crosstalk between RNA epigenetic regulation and DNA damage repair in the maintenance of genomic stability in hematopoietic stem/progenitor cell (HSPCs) during oxidative stress, and how deregulation of ALKBH5 contributes to promotion of leukemic transformation of HSPCs in the initiation and development of MDS. We found that ROS significantly increased global m6A RNA methylation in human cell lines, and that the elevation of m6A mRNA methylation is required for rapidly repairing ROS-induced DNA lesions and preventing cell death. Interestingly, we found that ALKBH5, the m6A RNA demethylase, is responsible for ROS-induced elevation of m6A mRNA methylation. ROS induced post- translational modification of ALKBH5, and inhibited the demethylase activity of ALKBH5. We showed that forced expression of ALKBH5 inhibited ROS-induced m6A mRNA methylation and significantly delayed repair of ROS-induced DNA damage. Thus, we hypothesize 1) that ALKBH5-mediated m6A modification has a significant role in the maintenance of genome integrity and survival of cells in response to oxidative stress in HSPCs; and 2) that deregulation of ALKBH5 may disrupt HSPC functions, thereby promoting leukemic transformation of HSPCs. In this proposal, we will determine 1) the role and underlying mechanism of Alkbh5 in the maintenance of genomic stability in HSPCs in response to oxidative stress; 2) the role of ALKBH5/Alkbh5 in the maintenance of mouse and human primary HSPCs during ROS stress in vivo; and 3) how ALKBH5/Alkbh5 contributes to the development of del(5q) MDS. We will employ both genetic murine models as well as patient-derived xeno-transplantation (PDX) models to investigate the role of ALKBH5 in the maintenance of HSPCs in vivo, and will combine transcriptome and epitranscriptome analysis to identify the key downstream targets and associated downstream pathways that mediate the role of ALKBH5 in HSPCs with or without ROS-induced stress. Our study will provide new insights into novel mechanisms underlying epitranscriptional regulation of gene expression as well as uncover novel mechanisms that regulate the activity of ALKBH5 in response to oxidative stress. This study will provide the first set of evidence to support a significant role of ALKBH5-mediated m6A mRNA demethylation in the maintenance of HSPCs.

抽象的骨髓增生综合征（MDS）是一组起源于多样的恶性血液学疾病来自造血干细胞（HSC）。活性氧（ROS）和DNA损伤的水平增加在MDS患者的造血细胞中检测到。可以从中产生的升高的ROS 内源性和外源性来源以及癌基因激活都导致静止和 HSC的自我更新。 ROS诱导的DNA损伤加快了干细胞的衰老过程，并有助于与癌症发展有关的诱变。 M6a RNA甲基化在多个生物学过程通过引入另一层的基因表达的转录后调节细胞。该项目的目的是阐明RNA表观遗传调节和DNA之间串扰的作用维持造血茎/祖细胞（HSPC）基因组稳定性的损害修复氧化应激以及ALKBH5的放松管制如何有助于促进白血病转化 HSPC在MDS的启动和开发中。我们发现ROS显着增加了全局M6A RNA 人类细胞系中的甲基化，并且需要快速需要M6a mRNA甲基化的升高修复ROS诱导的DNA病变并防止细胞死亡。有趣的是，我们发现AlkBH5，M6A RNA脱甲基酶负责ROS诱导的M6a mRNA甲基化升高。 ROS引起的后 ALKBH5的翻译修饰，并抑制ALKBH5的脱甲基酶活性。我们表明了这一点 ALKBH5强迫表达抑制ROS诱导的M6a mRNA甲基化，并显着延迟修复 ROS诱导的DNA损伤。因此，我们假设1）ALKBH5介导的M6A修饰具有在维持基因组完整性和细胞存活中对氧化应激的生存的重要作用 HSPCS; 2）ALKBH5的放松管制可能会破坏HSPC功能，从而促进白血病 HSPCS的转换。在此提案中，我们将确定1）ALKBH5的作用和潜在机制在维持HSPC中响应氧化应激的基因组稳定性方面； 2）角色在体内ROS胁迫期间，AlkBH5/ALKBH5在维持小鼠和人类原发性HSPC中； 3） ALKBH5/ALKBH5如何促进DEL（5Q）MDS的发展。我们将使用两个遗传鼠模型以及患者衍生的异种移植（PDX）模型，以研究ALKBH5在维护HSPC在体内，并将结合转录组和表演组分析以识别关键下游目标和相关的下游途径，介导ALKBH5在HSPC中的作用有或没有ROS诱导的压力。我们的研究将提供有关基础新机制的新见解基因表达的表达调节以及发现调节活性的新机制响应氧化应激的ALKBH5。这项研究将为支持 ALKBH5介导的M6a mRNA脱甲基化在HSPC中的重要作用。