High-throughput, untargeted approaches to identify and define the functions of transcription factors regulating key life cycle transitions in Giardia

高通量、无针对性的方法来识别和定义调节贾第鞭毛虫关键生命周期转变的转录因子的功能

• 批准号: 10727571
• 负责人: SCOTT C DAWSON
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727571
• 关键词: Address; Animals; Biological Assay; Biology; CRISPR interference; Categories; Cell Cycle; Cell Nucleus; Cell division; Communities; Complementary DNA; Cues; Cyst; DNA; DNA Binding; DNA Binding Domain; DNA-Binding Proteins; DNA-Protein Interaction; Defect; Development; Diet; Distal; Duodenum; Epithelium; Eukaryota; Feces; Future; Gene Expression; Genes; Genetic; Genetic Markers; Genetic Transcription; Genetic study; Genome; Giardia; Giardia lamblia; Goals; Growth; Guide RNA; Hour; Human; Hybrids; Immune; Immune response; Infection; Ingestion; Interphase; Intestinal parasite; Knock-out; Knowledge; Lead; Libraries; Life Cycle Stages; Luciferases; Mediating; Meiosis; Metabolic; Molecular Genetics; Nutrient; Oxidants; Oxidation-Reduction; Parasites; Pathogenesis; Periodicals; Phenotype; Protein Binding Domain; Proteins; Regulator Genes; Reporting; Research; Resources; Robotics; Role; Severities; Small Intestines; Stress; System; Testing; Transcription Coactivator; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Yeasts; bZIP Domain; biological adaptation to stress; cDNA Library; cell motility; density; diarrheal disease; druggable target; excystation; extracellular; gene repression; genome-wide; global health; gut colonization; host colonization; in vivo; knock-down; mutant; novel; prevent; promoter; response; screening; tool; transcription factor; transcription regulatory network; transmission process

Giardia lamblia is a widespread protozoan parasite of humans and animals causing significant diarrheal disease worldwide. Giardia cysts are transmitted between hosts through feces. Once ingested, cysts transform into trophozoites that colonize the duodenal epithelium – a region of varied nutrients, redox stress, and immune responses. Unknown external cues in the gut trigger trophozoites to encyst, and then cysts are passed to new hosts. Giardia is also reported to have a quasi-meiotic stage, termed diplomixis, that occurs in late encystation and is characterized by fusing and exchange of DNA between cyst nuclei. Due to a historical lack of molecular genetic tools, the pathogenesis and basic biology of Giardia is grossly understudied. Developmental transitions and environmental responses are commonly mediated by transcription factors (TFs) – modular proteins that often contain DNA binding domains (DBDs). However, only 36 TFs with DBDs are predicted in the Giardia genome as compared to about 200 predicted in similar sized eukaryotic genomes. Over 500 proteins have more general motifs such as basic leucine-zipper domains, and these can also function as TFs. Only a handful of Giardia TFs have been studied in any detail; all are associated with early to mid-encystation. Basal TFs, redox responsive TFs, late encystation/diplomixis TFs, or cell cycle related TFs have not yet been identified. Our lack of knowledge of the identities and functions of Giardia TFs has severely limited our understanding of genetic regulatory networks throughout the Giardia life cycle, particularly those associated with cell division and pathogenesis. To address these deficits, we will use an unbiased, high-throughput, genome-wide yeast one-hybrid (Y1H) screen to identify additional DNA binding proteins likely representing candidate TFs (Aim 1). In many systems, such screens are commonly used to define genome-wide protein-DNA interactions. Using robotic screening of arrayed “prey” libraries, we will screen a total of nine promoter “baits” (three constitutive loci; three redox stress- associated loci, and three encystation loci) against three “prey” cDNA libraries (constitutive; redox stress; late encystation/diplomixis) for a total of 27 (3 x 3 x 3) combinations. Both predicted DBD proteins and candidate TFs identified in the Y1H screen will be additionally prioritized by CRISPRi mediated knockdown, with phenotypic screening for defects in growth, mid-encystation and genetic marker exchange in cysts (diplomixis), and redox stress. Knockdowns of candidate TFs with strong defects will be further characterized using promoter-luciferase assays and a novel dCas-TF luciferase fusion assay to evaluate transcriptional activation or repression (Aim 2). Overall, this project will define and prioritize Giardia TFs as groundwork for future interrogation of Giardia regulatory networks throughout the life cycle.

蓝氏贾第鞭毛虫是一种广泛存在于人类和动物体内的原生寄生虫， 国际吧贾第虫包囊通过粪便在宿主之间传播。一旦被摄入，囊肿会转化为 营养体定植在十二指肠上皮-一个不同营养物质，氧化还原应激和免疫反应的区域 应答肠道中未知的外部线索触发滋养体成囊，然后包囊被传递给新的 hosts.贾第鞭毛虫也被报道有一个准减数分裂阶段，称为核融合，发生在后期成囊 其特征是胞核之间DNA的融合和交换。由于历史上缺乏分子 然而，由于贾第鞭毛虫的遗传工具、致病机理和基本生物学还没有得到充分研究。发展转型 和环境反应通常是由转录因子（TF）介导的-模块蛋白， 含有DNA结合结构域（DBD）。然而，在贾第虫基因组中预测只有36个具有DBD的TF 相比之下，在类似大小的真核生物基因组中预测约有200个。超过500种蛋白质具有更普遍的 基序，如碱性亮氨酸拉链结构域，这些也可以作为TF。只有少数贾第虫TF 所有这些都与早期到中期的包囊有关。基础TF，氧化还原响应TF， 晚期包囊形成/包囊形成TF或细胞周期相关TF尚未被鉴定。我们缺乏知识， 贾第虫转铁蛋白的特性和功能严重限制了我们对基因调控的理解， 在贾第虫的整个生命周期，特别是那些与细胞分裂和发病机制。 为了解决这些缺陷，我们将使用无偏见，高通量，全基因组酵母单杂交（Y1 H）筛选 以鉴定可能代表候选TF的另外的DNA结合蛋白（目的1）。在许多系统中， 筛选通常用于定义全基因组蛋白质-DNA相互作用。使用机器人筛选阵列 “猎物”文库中，我们将筛选总共九个启动子“诱饵”（三个组成型基因座;三个氧化还原应激- 相关基因座和三个包囊化基因座）针对三个“猎物”cDNA文库（组成型;氧化还原应激;晚期 包囊形成/包囊形成），总共27种（3 × 3 × 3）组合。预测的DBD蛋白和候选TF 将通过CRISPRi介导的敲低另外优先考虑在Y1 H筛选中鉴定的具有表型 筛选生长缺陷、包囊中期和包囊中的遗传标记交换（包囊融合），以及氧化还原 应力具有强缺陷的候选TF的敲低将使用启动子-荧光素酶进一步表征 用新的dCas-TF荧光素酶融合测定和新的dCas-TF荧光素酶融合测定来评估转录激活或抑制（Aim 2）。 总体而言，本项目将定义贾第虫TF并将其作为未来贾第虫审讯的基础 整个生命周期的监管网络。