Role of a novel PGC-1α isoform in gene transcription

新型 PGC-1α 亚型在基因转录中的作用

• 批准号: 10672396
• 负责人: Xavier Rambout
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672396
• 关键词: Adhesions; Age; Aging; Alleles; Amino Acid Sequence; Amino Acids; Anti-Inflammatory Agents; Antibodies; Antisense Oligonucleotides; Area; Atherosclerosis; Binding; Biological Assay; Blood Vessels; Breeding; C-terminal; CCL2 gene; CCL7 gene; CCL8 gene; CRISPR/Cas technology; Cardiac; Cardiovascular Diseases; Cause of Death; Cellular Metabolic Process; Chemotactic Factors; Chromatin; Complementary DNA; Data; Development; Disease; Dystrophin; Endothelial Cells; Endothelium; Engineering; Exons; Female; Gene Expression Regulation; Genes; Genetic Transcription; Immunoprecipitation; In Vitro; Individual; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Knock-out; Liquid Chromatography; MAP Kinase Gene; Malignant Neoplasms; Mammalian Cell; Maps; Messenger RNA; Metabolic; Metabolism; Modeling; Monocyte Chemoattractant Proteins; Mus; Muscle; Mutate; Myoblasts; Names; Nucleotides; PPAR gamma; Phenocopy; Plasma; Play; Polymerase Chain Reaction; Premature aging syndrome; Production; Protein Isoforms; Proteins; RNA; RNA Cap-Binding Proteins; RNA Polymerase II; RNA Splicing; RNA-Binding Proteins; Regulation; Reporting; Research; Research Personnel; Resolution; Risk Factors; Role; Running; Signal Transduction; Skeletal Muscle; Small Interfering RNA; Stress; Structure; Testing; Tissues; Transactivation; Transcript; Transcription Coactivator; Transcriptional Regulation; Transfection; Variant; alpha helix; career; chemokine; coronary fibrosis; cytokine; design; functional loss; heart function; in vivo; knock-down; mRNA Precursor; male; migration; monocyte; mouse model; novel; novel therapeutics; physiologic stressor; promoter; receptor; recruit; response; sex; systemic inflammatory response; tandem mass spectrometry; transcription factor; transcriptome sequencing

Peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) is a transcriptional coactivator that is critical for the metabolic adaptation of mammalian cells to diverse physiological stresses. In addition to this well-described function in the control of cell metabolism, the number of reports describing a protective role for PGC-1α against inflammation is on the rise. I report here a novel stress-dependent pro-inflammatory PGC-1α variant that I have identified in vitro in C2C12 mouse myoblasts and in vivo in mouse skeletal muscle. My unpublished results indicate that, compared to the canonical PGC-1α isoform (also known as PGC-1α(1)), the structure of this novel 72-kDa PGC-1α isoform, which I will call PGC-1α(72), is typified by its lack of the C-terminal 15 amino acids. The deleted C-terminal sequence encompasses a 9-amino-acid alpha helix that I previously reported to be essential for PGC-1α(1) to bind to the cap-binding protein (CBP)80 and to transactivate pro-myogenic PGC-1α-dependent genes. In contrast to PGC-1α(1), PGC-1α(72) lacks the CBP80-binding motif (CBM) and, as my data indicate, activates the transcription of pro-inflammatory genes. In the first aim of my research strategy, I propose to use C2C12 myoblasts to fully characterize the sequence of this novel PGC-1α(72) isoform, identify those transcription factors and RNA-binding proteins that regulate its expression in response to stress, and comprehensively determine the transcriptional network that it activates. In the second aim, I will take advantage of my finding that functional inhibition of the PGC-1α CBM phenocopies PGC-1α(72)-dependent expression of pro-inflammatory chemoattractants such as the chemokine CCL2. Elevated CCL2 plasma levels, which are observed with aging, have been recognized as important risk factors for numerous inflammatory diseases, including cardiac fibrosis, atherosclerosis and cancers. Mechanistically, CCL2 has been found to function in monocyte recruitment onto the endothelium of blood vessels and in monocyte infiltration into inflamed tissues. I will breed our newly characterized PGC-1αCBMmut mice with the Dystrophin-deficient Dmdmdx muscle inflammatory and premature aging mouse model and use derived primary myoblasts to infer the functional consequences of PGC-1α(72) expression, in particular those attributable to its lacking the CBM, to inflammation. In particular, I will determine how PGC-1αCBMmut triggers the CCL2-dependent recruitment of monocytes onto endothelial cells, their infiltration into cardiac tissues, and development of cardiac fibrosis in aging mice as a function of sex, i.e. in males vs. females. I view demonstrating the contribution of PGC-1αCBMmut to cardiac fibrosis as a proof of concept. It will provide fertile grounds to study the function of skeletal muscle PGC-1α(72) in the development of other aging- related inflammatory diseases. I hypothesize that understanding PGC-1α(72)-dependent gene regulation and its consequences to the development of inflammatory diseases will facilitate the development of new therapeutics, such as synthetic antisense oligonucleotides regulating splicing of PGC-1α pre-mRNA.

过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）是一种转录共激活因子， 是哺乳动物细胞代谢适应不同生理压力的关键。除此之外 在控制细胞代谢方面的功能得到了充分描述，描述对细胞代谢具有保护作用的报告数量 PGC-1α抗炎症作用呈上升趋势。我在这里报告了一种新的应激依赖性促炎PGC-1α 我已经在体外C2 C12小鼠成肌细胞和体内小鼠骨骼肌中鉴定了这种变体。 我未发表的结果表明，与经典的PGC-1α亚型（也称为 PGC-1α（1）），这种新的72-kDa PGC-1α亚型的结构，我将其称为PGC-1α（72），其典型特征是其 缺少C端15个氨基酸。缺失的C-末端序列包含9个氨基酸的α-氨基酸残基。 螺旋，我以前报道过是PGC-1α（1）结合帽结合蛋白（CBP）80和 反式激活促肌生成PGC-1α依赖基因。与PGC-1α（1）相反，PGC-1α（72）缺乏 CBP 80结合基序（CBM），正如我的数据表明，激活促炎基因的转录。在 作为我研究策略的第一个目标，我建议使用C2 C12成肌细胞来全面表征 这种新的PGC-1α（72）亚型，鉴定了那些调节其表达的转录因子和RNA结合蛋白。 表达响应压力，并全面确定其激活的转录网络。 在第二个目标中，我将利用我的发现，PGC-1α CBM的功能抑制 表型PGC-1α（72）依赖性促炎趋化因子（如趋化因子）表达 CCL2。随着年龄的增长，CCL 2血浆水平升高，已被认为是重要的风险 许多炎症性疾病的因子，包括心脏纤维化，动脉粥样硬化和癌症。 在机制上，已发现CCL 2在单核细胞募集到血液内皮上中起作用 血管和单核细胞浸润到炎症组织中。我将培育我们新鉴定的PGC-1αCBMmut 具有Dystrophin缺陷的Dmdmdx肌肉炎性和过早衰老小鼠模型的小鼠及其用途 衍生的原代成肌细胞，以推断PGC-1α（72）表达的功能后果，特别是那些 由于缺乏煤层气，导致炎症。特别是，我将确定PGC-1αCBMmut如何触发 单核细胞向内皮细胞的CCL 2依赖性募集，它们向心脏组织的浸润，以及 老龄小鼠心脏纤维化的发展与性别的关系，即雄性与雌性。 我认为证明PGC-1αCBMmut对心脏纤维化的贡献是一个概念证明。它将 为研究骨骼肌PGC-1α（72）在其他衰老过程中的功能提供了肥沃的土壤- 相关的炎症性疾病。我假设，了解PGC-1α（72）依赖的基因调控及其 炎症性疾病发展的后果将促进新疗法的开发， 例如调节PGC-1α前mRNA剪接的合成反义寡核苷酸。