Regulation of Age-Associated Heterochromatin Formation

描述（由申请人提供）：该申请的指导研究科学家发展奖是为了促进吉尔·克雷林博士过渡到衰老生物学领域的独立研究者。她的长期目标包括建立一个独立的研究项目，专注于推进我们对自然衰老过程中发生的异色变化的理解。为了实现这些目标，她将遵循结构化的职业发展和培训计划，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、会议和会议；以及在基金写作和实验室管理技能方面的培训。该计划将在主要导师John Sedivy博士的监督下实施。John Sedivy博士是衰老生物学领域的专家，在全基因组研究分析方面拥有丰富的经验。Peter Adams博士是染色质生物学领域的领军人物，他将与RNA生物学的专家Robert Reenan博士共同担任导师。此外，Charles Lawrence博士将为全基因组分析的生物信息学方法提供指导。越来越多的证据表明，在正常的衰老过程中，染色质会发生重组。这伴随着基因表达的相应变化。我们有理由认为这两个过程可能是有联系的。然而，这种广泛的染色质重排的表观遗传机制实际上是未知的。在这个应用中，我们建议研究有丝分裂后组织中与年龄相关的异染色质形成的调节机制。我们的初步数据表明，小鼠和狒狒骨骼肌中含有高度抑制性组蛋白变体macroH2A的兼性异染色质增加。最近的证据指向小非编码rna （ncRNAs）在抑制性异染色质标记沉积中的作用。许多mirna （ncrna的一个亚类）的合成受到Polycomb group （PcG）抑制的调节，我们的初步数据表明老年动物的PcG抑制减少。我们将研究ncrna在小鼠和恒河猴骨骼肌和心肌中年龄相关异染色质形成调控中的作用。具体来说，我们将把特定的ncRNA与年龄相关的异染色质形成位点联系起来，我们将研究ncRNA基因中存在的调控元件。我们的假设是：与年龄相关的PcG抑制减少导致某些miRNA基因的激活，其产物在基因组的特定位点触发与年龄相关的异染色质形成。具体目标1：我们将通过确定有丝分裂后组织中与年龄相关的特定染色质状态来开始这些研究。此外，我们将确定某些饮食干预（卡路里限制和白藜芦醇）是否可以延缓这种与年龄相关的表型的发生。具体目标2：该信息将用于设计ChIP-seq测定，以确定易受年龄相关异染色质形成影响的基因组区域。ncRNA群体将通过转录组的RNA-seq来表征，以鉴定受年龄相关表达增加影响的ncRNA。显示年龄依赖性上调的ncrna序列将与显示年龄相关性异染色质的基因组序列相关联，以确定RNA依赖性沉默标记沉积的潜在位点。具体目标3：将研究PcG抑制的标记物，以确定PcG抑制是否随着年龄的增长而减少。将分析相关miRNA基因的启动子区域，以确定是否存在可以调节表达的PcG抑制元件。最后，我们将通过培养的人类成纤维细胞的机制研究来验证我们的结果，表明在年轻细胞中，特定ncRNA的表达受到PcG调控的抑制，而这种抑制在老年细胞中被解除，导致ncRNA的表达和年龄相关的异染色质形成。总之，这个项目将扩大我们对涉及年龄相关异染色质形成调控的机制和过程的理解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: As organisms age there are changes in gene expression that contribute directly to aging phenotypes. These changes in gene expression are thought to result from alterations in the structure of the chromatin to form repressive heterochromatin. We will study the regulation of chromatin structure modification to provide insight into the mechanisms of age-associated changes in gene expression and to possibly identify interventions that delay these processes.