Broad Domains of H3K4me3: A Discovery Tool for Novel Regulators of Adult Neural S

H3K4me3 的广泛领域：成人神经元新型调节器的发现工具

• 批准号: 8723727
• 负责人: Elizabeth A Pollina
• 金额: $ 3.16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8723727
• 关键词: Address; Adult; Age; Age-associated memory impairment; Aging; Aging-Related Process; Binding; Brain; Cell Aging; Cell Culture Techniques; Cell Cycle; Cell physiology; Cells; Chromatin; Chromosomes, Human, Pair 15; Cognition; Cognitive; Coupled; Cues; Data; Disease; Embryo; Epigenetic Process; Fluorescence-Activated Cell Sorting; Functional RNA; Gene Expression; Genomics; Goals; Hematopoietic; High-Throughput Nucleotide Sequencing; Histone H3; Homeostasis; In Situ Hybridization; In Vitro; Individual; Induction of Apoptosis; Injection of therapeutic agent; Learning; Longevity; Lysine; Mediating; Memory; Molecular; Mus; Neuronal Differentiation; Northern Blotting; Nucleic Acid Regulatory Sequences; Parabiosis; Pattern; Play; Population; Regulation; Research; Role; Sensory; Skin; Stem cells; Testing; Tissues; Viral; Virus; Work; adult stem cell; age related; aged; aging brain; base; cell age; cell type; chromatin immunoprecipitation; embryonic stem cell; environmental intervention; genome wide association study; genome-wide; immunocytochemistry; in vivo; innovation; insight; lentiviral-mediated; nerve stem cell; neuroblast; neurogenesis; normal aging; novel; overexpression; prevent; relating to nervous system; research study; response; response to injury; self-renewal; small hairpin RNA; stem; stem cell differentiation; stem cell niche; tool; transcription factor; young adult

DESCRIPTION (provided by applicant): The goal of this study is to identify novel molecular regulators that preserve the ability of neural stem cells (NSCs) to self-renew and produce new neurons in the brains of aged individuals. Adult NSCs play key roles in learning, memory, and response to injury in young adults, but their function declines with age. The molecular mechanisms that prevent the decline in NSC function during aging are largely unknown. I will use an innovative approach to identify such regulators in an unbiased manner. Widespread (broad) domains of histone H3 lysine 4 trimethylation (H3K4me3) have been recently proposed to mark molecules with key functions in embryonic, adult hematopoietic and skin stem cells. My project uses this broad H3K4me3 domain signature as a discovery tool to identify new regulators of adult NSC function. In preliminary experiments, I identified the genome-wide landscape of H3K4me3 in adult primary cultures of NSCs from young mice using chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq). Consistent with work in other stem cell types, I observed the presence of broad H3K4me3 domains at the genomic loci encoding known NSC regulators. Interestingly, I found that the broadest H3K4me3 domains are present at the genomic loci of long non-coding RNAs (lncRNAs), raising the exciting possibility that lncRNAs could be key regulators of NSC function. I found that one of these lncRNAs, Lnc15, declines with age in the NSC niche in vivo and is required for NSC self-renewal in vitro. Based on my preliminary data, I hypothesize that Lnc15 functions to preserve the proliferative and neurogenic potential of adult NSCs and that Lnc15 function declines with normal aging. I will test this idea by examining the regulation and expression of Lnc15 in NSCs and differentiated progeny during aging, using a combination of in vitro NSC cell culture, NSCs freshly purified from the brain by fluorescence-activated cells sorting (FACS), and immunostaining on intact brain sections. I will also examine the molecular mechanisms that regulate Lnc15 expression during aging. In parallel, I will assess the functional role and mechanism of action of Lnc15 in proliferation and differentiation of young and old NSCs by viral-mediated knockdown and overexpression in vitro and in vivo. Once I delineate the role of Lnc15 in aging NSCs, I will explore its mechanism of action in these cells. Together these studies will provide new insight into the regulation and functional importance of Lnc15 in young and old NSCs, and, more generally will highlight the importance of broad H3K4m3 domains in adult stem cell aging. Understanding the mechanisms that preserve youthful NSC state be critical for identifying new avenues for preserving or reactivating the pool of NSCs during aging, which will have important implications for slowing or preventing age-related cognitive decline.

描述（由申请人提供）：本研究的目标是确定新型分子调节剂，以保持神经干细胞（NSC）自我更新并在老年人大脑中产生新神经元的能力。成人神经干细胞在年轻人的学习、记忆和损伤反应中发挥着关键作用，但其功能随着年龄的增长而下降。防止衰老过程中 NSC 功能下降的分子机制在很大程度上尚不清楚。我将使用创新的方法以公正的方式识别此类监管机构。最近提出了组蛋白 H3 赖氨酸 4 三甲基化 (H3K4me3) 的广泛（广泛）结构域来标记在胚胎、成体造血和皮肤干细胞中具有关键功能的分子。我的项目使用这个广泛的 H3K4me3 域签名作为发现工具来识别成人 NSC 功能的新调节因子。在初步实验中，我使用染色质免疫沉淀结合高通量测序 (ChIP-Seq) 技术，鉴定了幼鼠 NSC 成年原代培养物中 H3K4me3 的全基因组图谱。与其他干细胞类型的研究一致，我观察到编码已知 NSC 调节因子的基因组位点上存在广泛的 H3K4me3 结构域。有趣的是，我发现最广泛的 H3K4me3 结构域存在于长非编码 RNA (lncRNA) 的基因组位点，这提出了令人兴奋的可能性，即 lncRNA 可能是 NSC 功能的关键调节因子。我发现其中一种 lncRNA Lnc15 在体内 NSC 生态位中随着年龄的增长而下降，并且是体外 NSC 自我更新所必需的。根据我的初步数据，我假设 Lnc15 的功能是保护成年 NSC 的增殖和神经源性潜力，并且 Lnc15 功能会随着正常衰老而下降。我将通过结合体外 NSC 细胞培养、通过荧光激活细胞分选 (FACS) 从大脑中新鲜纯化的 NSC 以及完整脑切片的免疫染色，检查衰老过程中 NSC 和分化后代中 Lnc15 的调节和表达，来检验这一想法。我还将研究衰老过程中调节 Lnc15 表达的分子机制。与此同时，我将通过病毒介导的体外和体内敲低和过度表达来评估 Lnc15 在年轻和老年 NSC 增殖和分化中的功能作用和作用机制。一旦我描述了 Lnc15 在衰老 NSC 中的作用，我将探索它在这些细胞中的作用机制。这些研究将为年轻和老年 NSC 中 Lnc15 的调节和功能重要性提供新的见解，并且更广泛地强调广泛的 H3K4m3 结构域在成体干细胞衰老中的重要性。了解保持年轻 NSC 状态的机制对于确定在衰老过程中保存或重新激活 NSC 库的新途径至关重要，这对于减缓或预防与年龄相关的认知衰退具有重要意义。