Broad H3K4me3 Domains: A Discovery Tool for Regulators of Neural Stem Cell Aging

描述（由申请人提供）：本研究的目标是确定新的分子调节剂，以保持神经干细胞（NSCs）在老年人大脑中自我更新和产生新神经元的能力。成人NSCs在年轻人的学习、记忆和损伤反应中起着关键作用，但其功能随着年龄的增长而下降。在衰老过程中防止NSC功能下降的分子机制在很大程度上是未知的。我将采用一种创新的方法，以公正的方式确定这些监管机构。组蛋白H3赖氨酸4三甲基化（H3K4me3）的广泛结构域最近被提出用于标记胚胎、成人造血和皮肤干细胞中具有关键功能的分子。我的项目使用这种广泛的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，我利用染色质免疫沉淀结合高通量测序（ChIP-Seq）技术，鉴定了幼鼠成年NSCs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，我观察到在编码已知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结构域存在于长链非编码rna （lncRNAs）的基因组位点上，这令人兴奋地提出了lncRNAs可能是NSC功能的关键调节因子的可能性。我发现其中一种lncrna， Lnc15，在体内的NSC生态位中随着年龄的增长而下降，并且在体外是NSC自我更新所必需的。根据我的初步数据，我假设Lnc15的功能是保持成人NSCs的增殖和神经原性潜力，并且Lnc15的功能随着正常衰老而下降。我将检验这一观点，方法是在衰老过程中检查NSCs和分化后代中Lnc15的调控和表达，方法是结合体外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NSCs，以及对完整大脑切片进行免疫染色。我还将研究在衰老过程中调节Lnc15表达的分子机制。同时，我将在体外和体内通过病毒介导的敲低和过表达来评估Lnc15在年轻和年老NSCs增殖和分化中的功能作用和作用机制。一旦我描述了Lnc15在老化的NSCs中的作用，我将探索它在这些细胞中的作用机制。总之，这些研究将为lc15在年轻和老年NSCs中的调控和功能重要性提供新的见解，并且更普遍地强调广泛的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NSCs库的新途径至关重要，这将对减缓或预防与年龄相关的认知衰退具有重要意义。

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.