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

描述(申请人提供)：这项研究的目标是确定新的分子调节剂，以保持神经干细胞(NSCs)自我更新的能力，并在老年人的大脑中产生新的神经元。成年神经干细胞在年轻人的学习、记忆和对损伤的反应中起着关键作用，但它们的功能随着年龄的增长而下降。在衰老过程中阻止NSC功能下降的分子机制在很大程度上是未知的。我将使用一种创新的方法，以不偏不倚的方式识别这些监管机构。组蛋白H3赖氨酸4三甲基化(H3K4me3)的广泛(广泛)结构域最近被提出用来标记在胚胎、成人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，以确定成人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)在成年幼鼠NSCs原代培养中鉴定了H3K4me3的全基因组图谱。与其他干细胞类型的工作一致，我观察到在编码已知NSC调节因子的基因组基因座上存在广泛的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(LncRNAs)的基因组位置，这增加了lncRNAs可能是NSC功能的关键调节因子的令人兴奋的可能性。我发现其中一个LncRNAs，Lnc15，在体内的NSC生态位中随着年龄的增长而下降，是NSC在体外自我更新所必需的。根据我的初步数据，我假设Lnc15的功能是保护成年神经干细胞的增殖和神经生成能力，并且随着正常年龄的增长，Lnc15的功能会下降。我将通过检测LNC15在衰老过程中的调节和表达来检验这一想法，方法是结合使用体外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NSCs以及完整脑切片上的免疫染色。我还将研究在衰老过程中调节Lnc15表达的分子机制。同时，我将通过病毒介导的体外和体内下调和过度表达来评估Lnc15在年轻和老年神经干细胞增殖和分化中的功能作用和作用机制。一旦我描述了Lnc15在衰老神经干细胞中的作用，我将探索它在这些细胞中的作用机制。综上所述，这些研究将为Lnc15在年轻和老年神经干细胞中的调节和功能重要性提供新的见解，更广泛地说，将突出广泛的H3K4M3结构域在成人干细胞老化中的重要性。了解神经干细胞保持年轻状态的机制对于寻找在衰老过程中保护或重新激活神经干细胞池的新途径至关重要，这将对减缓或防止与年龄相关的认知衰退具有重要意义。 公共卫生相关性：成人神经干细胞(NSCs)在记忆和认知方面发挥着关键作用，但随着年龄的增长，其功能急剧下降。我们发现了一系列基于染色质状态的候选新型NSC调节剂。我们对这些分子在NSC中作用的持续研究可能会发现保存NSC功能的方法，从而减缓/防止正常或疾病状态下与年龄相关的认知和感觉下降。

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. PUBLIC HEALTH RELEVANCE: Adult neural stem cells (NSCs) play pivotal roles in memory and cognition, but decline dramatically in function with age. We have discovered a list of candidate novel NSC regulators based on their chromatin state. Our continued research into the role of these molecules in NSC may uncover ways to preserve NSC function and thereby slow/prevent age-related cognitive and sensory decline in normal or disease states.