Identification of stage-and tissue-specific endogenous tick promoters

阶段和组织特异性内源蜱启动子的鉴定

• 批准号: 10648720
• 负责人: Monika Gulia-Nuss
• 金额: $ 20.11万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648720
• 关键词: ATAC-seq; Adult; Anaplasma; Arthropods; Babesia; Binding; Bioinformatics; Biological Assay; Biology; Black-legged Tick; Blood; Borrelia burgdorferi; CMV promoter; CRISPR/Cas technology; Cell Line; Centers for Disease Control and Prevention (U.S.); Chromatin; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Development; Embryo; Eukaryotic Cell; Eye Color; Future; Gene Combinations; Gene Expression; Genes; Genomics; Goals; Heat shock proteins; High-Throughput Nucleotide Sequencing; Individual; Injections; Insecta; Life; Lyme Disease; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Mutation; Nuclear; Order Spirochaetales; Organism; Pattern; Phenotype; Promoter Regions; Proteins; Protocols documentation; Reporter; Research; Research Personnel; Rickettsia; Risk Taking; Screening procedure; Serine Protease; System; Technology; Temperature; Testing; Ticks; Tissues; Transcript; Transposase; United States; Validation; Vector-transmitted infectious disease; Vertebrates; Viral; Virus; Vitellogenins; Work; differential expression; experience; feeding; functional genomics; gene function; genetic regulatory protein; genome editing; genome sequencing; genomic locus; genomic tools; improved; in vivo evaluation; knockout gene; mutant; pathogen; promoter; protocol development; screening; sex; success; tool; transcriptome; transcriptome sequencing; transcriptomics; transmission process; vector

SUMMARY The lack of a promoter-reporter system in ticks makes functional genomics studies challenging. A few exogenous promoters that appear to work in tick cell lines are viral (i.e. CAAG/CMV promoter) and are constitutively expressing. However, not all research questions can be approached by constitutively expressing a gene. It is useful to gain temporal control over gene expression. In eukaryotic cells, nuclear DNA and proteins combine to form chromatin, which then undergoes complex and orderly folding to form chromosomes. For genes to be expressed, chromatin must be in an open conformation. Open chromatin allows regulatory proteins to bind to DNA and regulate DNA function. The assay for transposase-accessible chromatin with high- throughput sequencing (ATAC-seq) enables high-throughput sequencing of open chromatin regions with the help of transposases. ATAC-seq detects chromatin accessibility of related genes and indicates their regulatory mechanisms. Thus, genes with chromatin accessibility in promoters are more likely to be differentially expressed at the mRNA level. Therefore, by combining the power of ATAC-seq to analyze chromatin accessibility in the promoter regions of whole genes in ticks’ tissues and life stages and then screening differentially expressed genes (DEGs) at the mRNA level by transcriptome sequencing technology (RNA-seq), we will obtain temporally and spatially expressed genes and their promoters. Therefore, our hypothesis/ objective is that by combining gene expression (RNAseq) and open chromatin regions using ATACseq (Aim 1), and promoter assays (Aim 2), we will identify constitutive and stage/sex/tissue-specific promoters for gene expression in ticks. Our pioneering work in tick genome sequencing and CRISPR-Cas9-based genome editing has now made tick gene-editing possible and this proposal will further help us improve the gene-editing protocol by developing the promoter-reporter systems that will allow the screening of mutants without the need to sequence every individual.

概括 蜱虫缺乏启动子-报告基因系统使得功能基因组学研究具有挑战性。 一些似乎在蜱细胞系中起作用的外源启动子是病毒性的（即 CAAG/CMV 启动子）并组成型表达。然而，并不是所有的研究问题都可以 通过组成型表达基因来实现。获得对基因的时间控制是有用的 表达。在真核细胞中，核 DNA 和蛋白质结合形成染色质， 然后经过复杂有序的折叠形成染色体。为了表达基因， 染色质必须处于开放构象。开放的染色质允许调节蛋白结合 DNA 并调节 DNA 功能。转座酶可及染色质的测定具有高 高通量测序 (ATAC-seq) 可实现开放染色质的高通量测序 区域在转座酶的帮助下。 ATAC-seq 检测相关染色质的可及性 基因并指出其调控机制。因此，具有染色质可及性的基因 启动子更有可能在 mRNA 水平上差异表达。因此，通过 结合 ATAC-seq 的能力来分析启动子区域的染色质可及性 蜱组织和生命阶段的全基因，然后筛选差异表达基因 通过转录组测序技术（RNA-seq）在mRNA水平上（DEGs），我们将获得 时间和空间表达的基因及其启动子。因此，我们的假设/ 目标是通过使用组合基因表达（RNAseq）和开放染色质区域 ATACseq（目标 1）和启动子测定（目标 2），我们将鉴定组成型和 用于蜱中基因表达的阶段/性别/组织特异性启动子。我们在蜱虫领域的开创性工作 基因组测序和基于 CRISPR-Cas9 的基因组编辑现已使基因编辑成为可能 可能，该提案将通过开发进一步帮助我们改进基因编辑协议 启动子-报告基因系统将允许筛选突变体而无需 对每个人进行排序。