Post-transcriptional mechanisms underlying age related expression of cytokine mRNAs in hematopoietic stem cells

造血干细胞中细胞因子 mRNA 年龄相关表达的转录后机制

• 批准号: 10471675
• 负责人: Emmanouil Maragkakis
• 金额: $ 33.32万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 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/10471675
• 关键词: ATAC-seq; Aging; Binding; Biological Assay; Cells; Chromatin; Chronic; Coupled; DNA; Data; Development; Disease; Epigenetic Process; Event; Genetic Transcription; Genome; Goals; Hematopoietic stem cells; Human Cell Line; Hyperactivity; Inflammatory; Interferon Gamma Receptor Complex; Interferon Type II; Investigation; Measures; Messenger RNA; Microglia; Mouse Strains; Mus; Myelogenous; Physiological; Population; Promoter Regions; Protocols documentation; RNA; RNA Decay; RNA Degradation; Regulator Genes; Reporter; Role; Signal Transduction; Sorting - Cell Movement; Techniques; Tn5 transposase; Transposase; Work; age related; aged; aging brain; cell type; computational pipelines; cytokine; epigenomics; experimental study; interest; mRNA Decay; mRNA Expression; macrophage; neuroinflammation; novel; transcription factor; transcriptome sequencing; transcriptomics

In this study we aim to identify novel regulatory mechanisms that may contribute to the chronically elevated inflammatory cytokines in physiological aging, using the newly developed experimental techniques such as direct RNA Sequencing, Assay for Transposase Accessible Chromatin assay (ATAC-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag). Fluorescent reporter mice for the endogenous expression of IFN-gamma have been aging and are now available for isolation of HSCs and microglia. The reporter mice allow for accurate flow sorting of the cytokine-high and cytokine-low cells from each mouse strain. We will compare 9 mice 72 weeks old or older with 9 mice 20-30 weeks old. We have successfully applied direct RNA-Seq, which will be used to simultaneously profile the abundance and decay of RNAs, in human cell-lines and we have established an efficient processing computational pipeline. We are working on further optimizations on the protocol to support sequencing from primary cells. Direct RNA-Seq results will determine key gene regulatory features in the aged HSCs and microglia such as whether RNA degradation increases/decreases during aging and whether RNA degradation varies between the cytokine-high and cytokine-low populations allowing for a selective survival advantage of one population over another. To identify the chromatin mechanisms contributing to RNA differences in the cell populations of interest, we will perform ATAC-seq with a hyperactive Tn5 transposase to make cuts in accessible regions of the genome. ATAC-seq data will be further searched for regions with depletion of cleavage events due to transcription factor binding and underlying motifs will be used to create a list of candidate transcription factors. To maximize the yield of our DNA and RNA extraction experiments, in this fiscal year we have worked towards isolating DNA and RNA from the same cells. Transcription factors that bind the promoter regions of RNAs most altered in RNA expression analysis will be prioritized for further investigation by CUT&Tag.

在这项研究中，我们的目标是确定新的调节机制，可能有助于慢性升高的炎症细胞因子在生理老化，使用新开发的实验技术，如直接RNA测序，转座酶可降解染色质测定（ATAC-seq）和切割下的目标和标签化（CUT&Tag）。用于内源性表达IFN-γ的荧光报告小鼠已经老化，现在可用于分离HSC和小胶质细胞。 报告小鼠允许对来自每个小鼠品系的高精氨酸和低精氨酸细胞进行准确的流式分选。我们将比较9只72周龄或更大的小鼠与9只20-30周龄的小鼠。我们已经成功地应用了直接RNA-Seq，这将用于同时分析RNA的丰度和衰变，在人类细胞系中，我们已经建立了一个有效的处理计算管道。我们正在进一步优化协议，以支持原代细胞测序。 直接RNA-Seq结果将确定衰老HSC和小胶质细胞中的关键基因调控特征，例如RNA降解在衰老过程中是否增加/减少，以及RNA降解是否在高精氨酸和低精氨酸群体之间变化，从而允许一个群体相对于另一个群体的选择性生存优势。为了确定导致感兴趣的细胞群体中RNA差异的染色质机制，我们将使用高度活跃的Tn 5转座酶进行ATAC-seq，以在基因组的可访问区域进行切割。将进一步搜索ATAC-seq数据以寻找由于转录因子结合而具有切割事件耗尽的区域，并且将使用基础基序来创建候选转录因子的列表。为了最大限度地提高我们的DNA和RNA提取实验的产量，在本财政年度，我们致力于从同一细胞中分离DNA和RNA。在RNA表达分析中，结合RNA启动子区域的转录因子将优先用于CUT&Tag的进一步研究。