Investigating the role of SIRT1 in hematopoetic stem cell maintenance

研究 SIRT1 在造血干细胞维持中的作用

• 批准号: 8349505
• 负责人: Philipp Oberdoerffer
• 金额: $ 32.11万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349505
• 关键词: Ablation; Acute; Acute Myelocytic Leukemia; Address; Affect; Age; Animal Model; Animals; Binding; Biological Assay; Bone Marrow; Bone Marrow Transplantation; Candidate Disease Gene; Cell Maintenance; Cell Proliferation; Cell physiology; Cells; Characteristics; Chromatin; Chronic Myeloid Leukemia; Complex; DNA; DNA Damage; Data; Development; Developmental Gene; Epigenetic Process; Exhibits; Financial compensation; Frequencies; Future; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genomics; Genotoxic Stress; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Histone Deacetylase; Investigation; Knock-out; Light; Link; Longevity; Maintenance; Malignant - descriptor; Malignant Neoplasms; Mediating; Mediator of activation protein; Metabolic stress; Microarray Analysis; Molecular; Mus; Oncogene Deregulation; Ontology; Pathway interactions; Perinatal; Phase; Physiological; Play; Polycomb; Population; Process; Proliferating; RNA Interference; Regulation; Role; Signal Transduction; Site; Sorting - Cell Movement; Stem cells; System; Testing; Tissues; Transcriptional Regulation; Undifferentiated; Work; age related; base; chromatin immunoprecipitation; design; embryonic stem cell; epigenomics; exhaustion; functional decline; mature animal; mouse model; normal aging; novel; precursor cell; programs; promoter; reconstitution; research study; response; self-renewal; stem; stem cell fate

BACKGROUND: DNA damage is emerging to be a key mediator of age-associated chromatin reorganization, promoting many of the epigenetic changes that correlate with, or mimic, transcriptional deregulation observed with age11. The (histone) deacetylase SIRT1 was shown to be critically involved in several aspects of the DNA damage response as well as in the transcriptional regulation of a variety of key developmental regulators, placing it at the crossroads of DNA damage and epigenetic gene regulation. Consistent with this notion, we have shown previously that SIRT1 redistributes on chromatin from certain promoters and repetitive DNA to sites of DNA breaks, resulting in gene expression changes at ostensibly undamaged genomic loci that are normally regulated by SIRT112. While the resulting chromatin reorganization may at least in part explain age-associated gene deregulation, functional consequences of this process remain to be indentified. To study the role of SIRT1 in epigenomic maintenance, we thus turned to a physiological system that displays a well-established link between DNA damage and epigenomic changes, the hematopoietic stem and precursor cell (HSPC) compartment. Work from Weisman and colleagues showed that various types of DNA damage could severely impair stem cell maintenance, predominantly by reducing self-renewal capacity. Moreover, HSPCs accumulate DNA damage during normal aging and harbor characteristic gene expression changes that may ultimately affect HSPC function. However, it is unclear how DNA damage triggers HSPC exhaustion and if the observed gene expression changes are functionally related. Supporting a role for SIRT1 in the epigenetic (de)regulation of stem cell fate, SIRT1 was shown to be part of a novel polycomb repressive complex, PRC4, which has distinctive chromatin modifying activity and is expressed specifically in undifferentiated cells including embryonic stem cells. Polycomb Group (PcG)-mediated gene regulation is known to play a critical role in HSPC maintenance as loss of the PcG component Bmi-1 ablates HSPC development. Together these observations call for an in depth investigation of SIRT1 function in hematopoietic stem cells. RESULTS AND FUTURE DIRCTIONS: To test the hypothesis that SIRT1 is a critical (epigenetic) mediator of HSPC maintenance and function, we generated a mouse model that allows for the inducible ablation of SIRT1 gene expression in adult animals, thereby avoiding perinatal lethality and/or possible compensation observed in SIRT1-deficient mice. We found that, upon inactivation of SIRT1, the HSPC compartment undergoes an initial phase of proliferative expansion, followed by increased DNA damage and an eventual dramatic depletion in the long-term hematopoietic stem cell compartment. These data demonstrate an important role for SIRT1 in the maintenance of HSPC quiescence as well as the regulation of DNA damage accumulation. We then sought to investigate the mechanism by which SIRT1 mediates these effects, focusing on SIRT1-dependent changes in maintenance of genomic integrity and gene expression. Using microarray analysis in sorted HSPCs, we found that, out of a group of 11 Gene Ontology annotated genes associated with definitive hematopoiesis, a single candidate gene was strongly upregulated in the absence of SIRT1. This gene was previously shown to promote HSPC expansion and has further been linked to acute myelogenous leukemia (AML), a cancer derived from aberrantly proliferating hematopoietic progenitor cells. Using chromatin immunoprecipitation (ChIP) we found that Sirt1 binds to the this gene at the DNA level and that Sirt1 enrichment is correlated with the polycomb-group (PcG) repressive mark H3K27 (3Me), provinding the first evidence that Sirt1 may modulate PcG associated developmental genes to promote the maintenance of stem cells. Future work will dissect the molecular basis for this observation using depletion of this SIRT1 target gene by RNAi as well as a detailed analysis of theo role of PRC4 in this process. To address the functional consequences of HSPC expansion/exhaustion associated with loss of SIRT1, we are currently performing both competitive and (serial) non-competitive bone marrow reconstitution experiments. IMPLICATIONS: Our preliminary data suggest an important role for SIRT1 in HSPC maintenance, where loss of SIRT1 promotes HSPC expansion and eventually exhaustion corroborating the importance of SIRT1 as a regulator of developmental programs. The tight regulation of HSPC proliferation is a critical aspect of mammalian development and does not only affect the generation of normal hematopoietic cells but is closely linked to tumors of the hematopoietic lineage. Both acute and chronic myeloid leukemia arise from aberrant proliferation of HSPCs and a better understanding of the underlying regulators is critical to design targeted therapy.

背景：DNA损伤已成为与年龄相关的染色质重组的关键介体，促进了与与年龄相关的或模仿转录失调的许多表观遗传变化。 （组蛋白）脱乙酰基酶SIRT1被证明与DNA损伤响应的多个方面以及各种关键发育调节剂的转录调控有关，将其置于DNA损伤和表观遗传基因调控的十字架上。与这个概念一致，我们先前已经证明，SIRT1从某些启动子和重复的DNA上重新分布到DNA断裂部位，从而导致表面上通常受SIRT112调节的基因表达变化。尽管所得的染色质重组至少可能部分解释了与年龄相关的基因放松管制，但该过程的功能后果仍有待凹陷。为了研究SIRT1在表观基因组维持中的作用，我们转向了一个生理系统，该系统显示了DNA损伤与表观基因组变化，造血茎和前体细胞（HSPC）隔室之间建立良好的联系。 Weisman及其同事的工作表明，各种DNA损伤可能严重损害干细胞维持，主要通过降低自我更新能力。此外，HSPC在正常衰老期间会累积DNA损伤，并且最终可能影响HSPC功能的基因表达变化。但是，目前尚不清楚DNA损伤如何触发HSPC的衰竭以及观察到的基因表达变化是否在功能上相关。 SIRT1支持SIRT1在干细胞命运表观遗传（DE）调节中的作用，SIRT1被证明是一种新型PolyComb抑制性复合物PRC4的一部分，该复合物具有独特的染色质修饰活性，并且在包括胚胎干细胞在内的未分化细胞中特异性表达。已知PolyComb组（PCG）介导的基因调节在HSPC维持中起关键作用，因为PCG组件BMI-1消失了HSPC的发展。这些观察结果共同进行了对造血干细胞中SIRT1功能的深入研究。结果和未来的障碍：为了测试SIRT1是HSPC维持和功能的关键（表观遗传）介体的假设，我们生成了一种小鼠模型，该模型允许在成年动物中诱导SIRT1基因表达的诱导消融，从而避免围产期致死性和/或在SIRT1缺乏的小鼠中观察到围产期致死性和/或可能的补偿。我们发现，在SIRT1失活后，HSPC室经历了增生膨胀的初始阶段，然后在长期造血干细胞室中增加了DNA损伤和最终的急剧耗竭。这些数据证明了SIRT1在维持HSPC静止以及DNA损伤积累的调节中的重要作用。然后，我们试图研究SIRT1介导这些作用的机制，重点是依赖于SIRT1的基因组完整性和基因表达的变化。使用分类的HSPC中的微阵列分析，我们发现，在与确定造血相关的11个基因本体学注释基因中，在没有SIRT1的情况下，单个候选基因被强烈上调。先前已显示该基因可促进HSPC扩张，并与急性骨髓性白血病（AML）进一步有关，这是一种来自异常增殖的造血祖细胞的癌症。使用染色质免疫沉淀（CHIP），我们发现SIRT1在DNA水平上与该基因结合，并且SIRT1富集与PolyComb-group（PCG）抑制性Mark H3K27（3ME）相关，证明SIRT1 SIRT1可以调节PCG相关的发育基因以促进Stemencence of Stemencence of Stemencence of Stemencence of Stemencence of Stemencence of Stemencence of Stemencence of Stemencence。未来的工作将使用RNAi对该SIRT1靶基因的耗竭以及对PRC4在此过程中的Theo作用进行详细分析来剖析这种观察的分子基础。为了解决与SIRT1丢失相关的HSPC扩展/精疲力尽的功能后果，我们目前正在执行竞争性和（串行）非竞争性骨髓重建实验。 含义：我们的初步数据表明，SIRT1在HSPC维护中的重要作用，在HSPC维护中，SIRT1的损失促进了HSPC的扩展，并最终耗尽了SIRT1作为发展计划的调节者的重要性。 HSPC增殖的严格调节是哺乳动物发育的关键方面，不仅会影响正常造血细胞的产生，而且与造血谱系的肿瘤密切相关。急性和慢性髓样白血病都来自HSPC的异常增殖，并且对潜在调节剂的更好了解对于设计有针对性的治疗至关重要。