Regulation of Age-Associated Heterochromatin Formation

描述(由申请者提供)：本次申请导师研究科学家发展奖是为了促进吉尔·克莱林博士向衰老生物学领域的独立研究人员的过渡。她的长期目标包括建立一个独立的研究计划，专注于促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，她将遵循有系统的职业发展和培训计划，其中包括培训实用实验室技术和生物信息分析；大学和校外课程；参加研讨会、会议和会议；以及赠款撰写和实验室管理技能培训。这项计划将在主要导师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，在全基因组研究的分析方面有相当丰富的经验。染色质生物学领域的领军人物彼得·亚当斯博士将与专门研究RNA生物学的罗伯特·里南博士一起担任共同导师。此外，查尔斯·劳伦斯博士将为全基因组分析提供生物信息学方法的指导。越来越多的证据表明，染色质在正常衰老过程中会发生重组。这伴随着基因表达的相应变化。有理由认为这两个过程可能是相互关联的。然而，这种广泛的染色质重排的表观遗传机制几乎是未知的。在这个应用中，我们建议研究在有丝分裂后组织中调节与年龄相关的异染色质形成的机制。我们的初步数据表明，在小鼠和狒狒骨骼肌中，兼性异染色质与高度抑制组蛋白变体宏H_2A的结合增加。最近的证据表明，小的非编码RNA(NcRNAs)在抑制性异染色质标记的沉积中起到了作用。许多miRNAs(ncRNAs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ncRNAs在调节小鼠和恒河猴骨骼肌和心肌中与年龄相关的异染色质形成中的作用。具体地说，我们将把特定的ncRNA与年龄相关的异染色质形成部位联系起来，并研究ncRNA基因中存在的调节元件。我们的假设是：与年龄相关的PcG抑制减少会导致某些miRNA基因的激活，其产物会在基因组上的特定位置触发与年龄相关的异染色质的形成。具体目标1：我们将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来开始这些研究。此外，我们将确定某些饮食干预措施(卡路里限制和白藜芦醇)是否可以推迟这种与年龄相关的表型的出现。具体目标2：这些信息将被用于设计芯片序列分析，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来表征，以识别受年龄相关表达增加影响的ncRNA。显示年龄相关上调的ncRNAs序列将与显示年龄相关异染色质的基因组序列相关联，以确定依赖RNA沉积沉默标记的潜在位置。具体目标3：将调查PcG抑制的标志物，以确定PcG抑制是否随着年龄的增长而减少。相关miRNA基因的启动子区域将被分析，以确定是否存在可能调节表达的PcG抑制元件。最后，我们将用培养的人成纤维细胞中的机制研究来验证我们的结果，表明年轻细胞中特定的ncRNAs的表达受到PcG调控的抑制，而在老年细胞中这种抑制被解除，导致ncRNA的表达和与年龄相关的异染色质的形成。综上所述，该项目将扩大我们对AGE相关异染色质形成调控机制和过程的理解。 公共卫生相关性：随着生物体年龄的增长，基因表达的变化直接导致了衰老表型。基因表达的这些变化被认为是染色质结构改变形成抑制性异染色质的结果。我们将研究染色质结构修饰的调节，以深入了解与年龄相关的基因表达变化的机制，并可能确定延缓这些过程的干预措施。

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.