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和动物中的原生动物寄生虫，可引起严重的腹泻疾病。 全世界。贾第鞭毛虫包囊通过粪便在宿主之间传播。一旦被摄取，囊肿就会转化为 定植于十二指肠上皮的滋养体--一个营养多样、氧化还原应激和免疫的区域 回应。肠道中未知的外部线索会触发滋养体包囊，然后将包囊传递给新的 主持人。据报道，贾第鞭毛虫也有一个准减数分裂阶段，称为分生孢子期，发生在胞核晚期。 其特征是囊核之间的DNA融合和交换。由于历史上缺乏分子 关于贾第鞭毛虫的遗传工具、致病机理和基础生物学等方面的研究还很少。发展转型 环境反应通常是由转录因子(TF)介导的--模块化蛋白质，通常 含有DNA结合域(DBD)。然而，在贾第虫基因组中只预测了36个患有DBDS的转铁蛋白。 相比之下，在类似大小的真核基因组中预测的约有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，这些也可以作为转录因子。只有为数不多的几个贾迪亚TF 已经进行了详细的研究；所有的都与早期到中期的作用有关。基础TF、氧化还原响应型TF、 晚期囊化/分裂因子，或与细胞周期相关的因子尚未确定。我们对此缺乏了解 贾第虫转录因子的身份和功能严重限制了我们对基因调控的理解 整个贾第鞭毛虫生命周期的网络，特别是那些与细胞分裂和致病相关的网络。 为了解决这些缺陷，我们将使用无偏见、高通量、全基因组的酵母单杂交(Y1H)筛查 识别可能代表候选转录因子的其他DNA结合蛋白(目标1)。在许多系统中，例如 筛选通常被用来定义全基因组的蛋白质-DNA相互作用。使用机器人筛选阵列 “猎物”文库，我们将筛选总共9个启动子“诱饵”(3个构成基因座；3个氧化还原压力- 相关基因座和三个囊化基因座)与三个“猎物”cdna文库(构成；氧化还原应激；晚期 加密/划线)，共27个(3x3x3)组合。预测的DBD蛋白和候选转录因子 在Y1H屏幕中识别的基因将通过CRISPRi介导的基因敲除获得额外的优先级，具有表型 囊泡生长、囊化中期和遗传标记交换缺陷的筛查(分支)和氧化还原 压力。具有强缺陷的候选TF的敲除将使用启动子-荧光素酶进一步表征 和一种新的DCAS-TF荧光素酶融合实验来评估转录激活或抑制(目标2)。 总体而言，该项目将确定贾迪亚的TFS，并将其作为未来审问贾迪亚的基础工作 整个生命周期的监管网络。