Single Cell Chromatin Profiling in Kidney Tissue

肾脏组织中的单细胞染色质分析

• 批准号: 10373426
• 负责人: MICHAEL I RAUCHMAN
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373426
• 关键词: ATAC-seq; Acute; Acute Renal Failure with Renal Papillary Necrosis; Address; Affect; American; Atlases; Biological Markers; Cells; ChIP-seq; Chromatin; Chronic Kidney Failure; Clinical; Communities; DNA Methylation; Data; Data Set; Development; Diabetes Mellitus; Dialysis procedure; Disease; Disease Progression; Disease model; Distal; End stage renal failure; Enhancers; Epigenetic Process; Epithelial Cells; Funding Opportunities; Gene Expression; Genes; Genetic Transcription; Genomics; Human; Individual; Injury; Injury to Kidney; Kidney; Kidney Diseases; Kidney Transplantation; Knowledge; Lead; Life; Localized Disease; Maps; Mediating; Metabolic; Methods; Modification; Molecular; Morbidity - disease rate; Mus; Natural regeneration; Nephrons; Neptune; Nucleic Acid Regulatory Sequences; Organoids; Pathogenesis; Population; Prevalence; Process; Property; Protocols documentation; Rare Diseases; Regulator Genes; Regulatory Element; Renal function; Research; Risk; Sampling; Signal Pathway; Techniques; Technology; Tissues; Tubular formation; Untranslated RNA; Urine; Variant; bioinformatics pipeline; bioinformatics tool; cell type; chromatin immunoprecipitation; chromatin modification; drug discovery; effective therapy; epigenome; epigenomics; functional restoration; gene repression; genetic variant; genome wide association study; genome-wide; histone modification; human disease; human model; in utero; injured; kidney allograft; kidney biopsy; kidney cell; molecular phenotype; mortality; nephrogenesis; new therapeutic target; novel; precision medicine; predictive marker; programs; promoter; repaired; response; response to injury; single-cell RNA sequencing; technological innovation; tool; transcriptomics

The function of the kidney depends on the coordinated action of multiple specialized cell types organized in a particular spatial arrangement. Progress in understanding disease pathogenesis and in developing biomarkers and therapies for kidney disease has been hindered by a lack of deep molecular phenotyping. Tissue interrogation techniques of kidney biopsies have not changed in several decades. To advance our understanding of kidney development and disease, there is a need to develop tools to define cell-type specific molecular properties in normal kidney tissue, in response to injury, and during repair and regeneration. While progress has been made in single cell transcriptomic analysis of kidney tissue, applying new genomic technologies to define the epigenome has lagged behind. This gap in knowledge is crucial because most disease variants associated with CKD map to distal regulatory elements, which are often cell-type specific genomic enhancers. Determining the function of these regulatory regions and their effect on gene expression is limited by lack of detailed information on the chromatin state of cells in specific nephron segments in normal and diseased human kidney tissue. Chromatin Immunoprecipitation with sequencing (ChIP-seq), a standard method for mapping epigenetic modifications, requires significantly more starting material than is available in a kidney biopsy. Moreover, the method has high background and lacks sensitivity to be readily used for chromatin profiling of single cells. To address these challenges, this application proposes to develop novel cutting edge technological and bioinformatic tools that have not yet been deployed in the kidney, which is the major objective of this R21 funding opportunity. We will establish a protocol for single cell chromatin profiling using a recently described approach, Cleavage Under Targets and Tagmentation (CUT & Tag). We will develop a bioinformatic pipeline to analyze single cell histone modifications, define how these epigenomic features change in specific cell types in disease states, and integrate these findings with single cell transcriptomic and ATAC-seq data. These technological innovations will have the potential to catalyze new mechanistic studies in the kidney, lead to discovery of novel therapeutic targets and identify biomarkers to predict disease progression. The technical and bioinformatic tools we develop will be broadly applicable to investigate kidney diseases in human samples, as well as disease models in mice and human organoids. Integration of genome-wide epigenetic data generated with these tools with gene expression atlases being developed for mouse and human kidneys will be extremely valuable to the research community. Because many genetic variants associated with kidney disease are localized to distal regulatory elements, the combination of transcriptomic and epigenomic data will significantly expand the power of these data sets to generate hypotheses about disease mechanisms, and make inferences about novel target genes and signaling pathways

肾脏的功能取决于以特定空间排列组织的多种专门细胞类型的协调作用。由于缺乏深入的分子表型分析，理解疾病发病机制以及开发肾脏疾病生物标志物和疗法的进展受到阻碍。 肾活检的组织询问技术几十年来没有改变。为了加深我们对肾脏发育和疾病的理解，需要开发工具来定义正常肾组织中细胞类型特定的分子特性，以响应损伤以及修复和再生过程。 虽然肾组织的单细胞转录组分析取得了进展，但应用新的基因组技术来定义表观基因组却落后了。这种知识差距至关重要，因为大多数与 CKD 相关的疾病变异都映射到远端调控元件，这些元件通常是细胞类型特异性基因组增强子。由于缺乏正常和患病人体肾组织中特定肾单位片段中细胞染色质状态的详细信息，确定这些调节区的功能及其对基因表达的影响受到限制。 染色质免疫沉淀测序 (ChIP-seq) 是一种绘制表观遗传修饰图谱的标准方法，所需的起始材料比肾活检所需的起始材料多得多。此外，该方法背景高且缺乏灵敏度，无法方便地用于单细胞染色质分析。为了应对这些挑战，该申请建议开发尚未在肾脏中部署的新型尖端技术和生物信息工具，这是本​​次 R21 资助机会的主要目标。我们将使用最近描述的方法“目标下切割和标记”（CUT & Tag）建立单细胞染色质分析方案。我们将开发一个生物信息学流程来分析单细胞组蛋白修饰，定义这些表观基因组特征在疾病状态下特定细胞类型中如何变化，并将这些发现与单细胞转录组和 ATAC-seq 数据相结合。这些技术创新将有可能促进肾脏的新机制研究，发现新的治疗靶点并识别生物标志物来预测疾病进展。 我们开发的技术和生物信息学工具将广泛适用于研究人类样本中的肾脏疾病，以及小鼠和人类类器官的疾病模型。将这些工具生成的全基因组表观遗传数据与为小鼠和人类肾脏开发的基因表达图谱整合起来，对于研究界来说将非常有价值。由于许多与肾脏疾病相关的遗传变异位于远端调控元件，转录组和表观基因组数据的结合将显着扩展这些数据集的能力，以生成有关疾病机制的假设，并对新的靶基因和信号通路做出推断