Regulation of Age-Associated Heterochromatin Formation

本申请的指导研究科学家发展奖是为了促进吉尔Kreiling博士的 转变为衰老生物学领域的独立研究者。长期目标包括建立 一个独立的研究项目，专注于推进我们对异染色质变化的理解， 发生在自然衰老过程中。为了实现这些目标，她将遵循结构化的职业生涯 制定和培训计划，包括培训实用实验室技术和生物信息学 分析;大学和校外课程;参加研讨会、会议和大会; 申请书撰写和实验室管理技能。该计划将在联合国监督下实施。 主要导师，约翰Sedivy博士，谁是在衰老生物学领域的专家，并有相当大的 全基因组研究分析的经验。染色质领域的领军人物彼得亚当斯博士 生物学，将作为一个共同的导师，沿着博士罗伯特Reenan谁专门从事RNA生物学。此外，本发明还提供了一种方法， 博士查尔斯·劳伦斯将提供指导与生物信息学方法全基因组分析。那里 越来越多的证据表明，在正常衰老过程中，染色质会发生重组。这是 伴随着基因表达的相应变化。有理由相信，这两个 进程可以连接。然而，这种广泛的重组的表观遗传机制， 染色质几乎是未知的。在这个应用程序中，我们建议研究调节年龄的机制- 有丝分裂后组织中相关的异染色质形成。我们的初步数据显示， 在小鼠中并入高度抑制性组蛋白变体macroH 2A的兼性异染色质， 狒狒骨骼肌最近的证据表明，小的非编码RNA（ncRNA）在细胞凋亡中的作用。 抑制性异染色质标记的沉积。许多miRNAs（ncRNAs的一个亚类）的合成是由 受Polycomb组（PcG）抑制的调节，我们的初步数据表明PcG减少 对老年动物的压抑我们将研究ncRNA在调节年龄相关的 小鼠和恒河猴骨骼肌和心肌中异染色质形成。具体来说，我们将 将特定的ncRNA与年龄相关的异染色质形成位点相关联，我们将研究 存在于ncRNA基因中的调控元件。我们的假设是：与年龄相关的PcG减少 抑制导致某些miRNA基因的激活，其产物触发与年龄相关的 在基因组的特定位点形成异染色质。具体目标1：我们将开始这些研究 通过鉴定有丝分裂后组织中与年龄相关的特定染色质状态。此外，我们将 确定某些饮食干预（卡路里限制和白藜芦醇）是否可以延迟这个年龄的开始- 相关表型具体目标2：该信息将用于设计ChIP-seq测定，以确定 基因组中易受年龄相关异染色质形成影响的区域。ncRNA群体 将通过转录组的RNA-seq进行表征，以鉴定受年龄相关增加影响的ncRNA 在表达上。显示年龄依赖性上调的ncRNA序列将与以下因素相关： 显示年龄相关异染色质的基因组序列，以确定RNA的潜在位点- 沉默标记的依赖性沉积。具体目标3：将研究PcG抑制的标志物， 确定PcG抑制是否随着年龄的增长而减少。相关miRNA的启动子区域 将分析基因以确定是否存在可以调节表达的PcG抑制元件。 最后，我们将用培养的人成纤维细胞的机制研究来验证我们的结果，以表明 在年轻细胞中，特异性ncRNA的表达受到PcG调控的抑制，而这种抑制在年轻细胞中被解除。 导致ncRNA表达和年龄相关异染色质形成的老年细胞。两者合计 这个项目将扩大我们对年龄调节机制和过程的理解， 相关的异染色质形成。

This application for a Mentored Research Scientist Development Award is to facilitate Dr. Jill Kreiling's transition to an independent investigator in the field of aging biology. Her long-term goals include establishing an independent research program focused on advancing our understanding of heterochromatic changes that occur during the natural aging process. To achieve these goals she will follow a structured career development and training plan that includes training practical laboratory techniques and in bioinformatic analyses; university and extramural courses; attending seminars, meetings and conferences; and training in grant writing and laboratory management skills. This plan will be implemented under the supervision of the primary mentor, Dr. John Sedivy, who is an expert in the field of aging biology and has considerable experience with the analysis of genome-wide studies. Dr. Peter Adams, a leader in the field of chromatin biology, will serve as a co-mentor, along with Dr. Robert Reenan who specializes in RNA biology. In addition, Dr. Charles Lawrence will provide guidance with the bioinformatic approach to genome-wide analyses. There is a growing body of evidence that chromatin undergoes reorganization during normal aging. This is accompanied by a corresponding change in gene expression. It is reasonable to expect that these two processes may be connected. However, the epigenetic mechanisms of this extensive rearrangement of the chromatin are virtually unknown. In this application we propose to study the mechanisms that regulate age- associated heterochromatin formation in post-mitotic tissues. Our preliminary data suggest an increase in facultative heterochromatin incorporating the highly repressive histone variant macroH2A in mouse and baboon skeletal muscle. Recent evidence points towards a role of small non-coding RNAs (ncRNAs) in the deposition of repressive heterochromatin marks. The synthesis of many miRNAs (a subclass of ncRNAs) is regulated by Polycomb group (PcG) repression, and our preliminary data suggest a decrease in PcG repression in old animals. We will study the role of ncRNAs in the regulation of age-associated heterochromatin formation in skeletal and cardiac muscle from mice and rhesus monkeys. Specifically we will correlate specific ncRNAs to sites of age associated heterochromatin formation and we will investigate the regulatory elements present in the ncRNA genes. Our hypothesis is: An age-associated reduction in PcG repression leads to activation of certain miRNA genes, whose products trigger age-associated heterochromatin formation at specific sites on the genome. Specific Aim 1: We will begin these studies by identifying the specific chromatin states that correlate with age in post-mitotic tissues. In addition, we will determine if certain dietary interventions (calorie restriction and resveratrol) can delay the onset of this age- associated phenotype. Specific Aim 2: This information will be used to design ChIP-seq assays to determine the regions of the genome susceptible to age-associated heterochromatin formation. The ncRNA populations will be characterized by RNA-seq of the transcriptome to identify ncRNAs subject to age associated increases in expression. The sequences of the ncRNAs showing an age-dependent up-regulation will be correlated with sequences of the genome showing age-associated heterochromatin to determine potential sites of RNA- dependent deposition of silencing marks. Specific Aim 3: Markers of PcG repression will be investigated to determine if there is a decrease in PcG repression with age. The promoter regions of the associated miRNA genes will be analyzed to determine if PcG repressive elements are present that could regulate expression. Finally, we will verify our results with mechanistic studies in cultured human fibroblasts to show that the expression of specific ncRNAs are repressed by PcG regulation in young cells and this repression is lifted in older cells leading to expression of the ncRNA and age-associated heterochromatin formation. Taken together this project will broaden our understanding of the mechanisms and processes involved in the regulation of age- associated heterochromatin formation.