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

描述（由申请人提供）：本研究的目的是鉴定新的分子调节剂，其保留神经干细胞（NSC）自我更新的能力，并在老年人的大脑中产生新的神经元。成人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是未知的。我将使用一种创新的方法，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（H3 K4 me 3）的广泛（广泛）结构域最近已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。我的项目使用这个广泛的H3 K4 me 3域签名作为发现工具，以确定成人NSC功能的新调节器。在初步实验中，我确定了H3 K4 me 3的基因组范围内的景观成年原代培养的神经干细胞从年轻的小鼠使用染色质免疫沉淀结合高通量测序（ChIP-Seq）。与在其他干细胞类型中的工作一致，我观察到在编码已知NSC调节子的基因组位点存在广泛的H3 K4 me 3结构域。有趣的是，我发现最广泛的H3 K4 me 3结构域存在于长非编码RNA（lncRNA）的基因组位点，这提高了lncRNA可能是NSC功能的关键调节因子的可能性。我发现这些lncRNA之一，Lnc 15，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Lnc 15的功能，以保持成人神经干细胞的增殖和神经原性的潜力，Lnc 15的功能下降与正常老化。我将测试这一想法，通过检查的调控和表达的Lnc 15在神经干细胞和分化的后代在老化过程中，使用体外神经干细胞培养的组合，新鲜纯化的神经干细胞从大脑中的荧光激活细胞分选（FACS），和完整的脑切片的免疫染色。我还将研究在衰老过程中调节Lnc 15表达的分子机制。与此同时，我将评估的功能作用和作用机制Lnc 15在年轻和老年神经干细胞的增殖和分化的病毒介导的敲低和过表达在体外和体内。一旦我描述了Lnc 15在衰老神经干细胞中的作用，我将探索其在这些细胞中的作用机制。这些研究将为Lnc 15在年轻和老年NSC中的调节和功能重要性提供新的见解，并且更普遍地将突出广泛的H3 K4 m3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期间保持或重新激活NSC池的新途径至关重要，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.