Regulation of endogenous genes by sexually dimorphic piRNA expression during germline development in C. elegans

线虫种系发育过程中性二态性 piRNA 表达对内源基因的调节

基本信息

批准号：
10389801
负责人：
Margaret Starostik
金额：
$ 4.68万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10389801
关键词：
Algorithms Animals Base Pairing Binding Biogenesis Biological Biological Assay Biological Process CRISPR/Cas technology Caenorhabditis elegans ChIP-seq Complex Consensus Sequence DNA Transposable Elements DNA-Binding Proteins Data Databases Defect Development Disease Drosophila genus Elements Embryonic Development Epigenetic Process Female Fertility Fertility Disorders Gametogenesis Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Genes Genetic Transcription Human Individual Infertility Knowledge Literature Mammals Mediating Men&apos s Role Modeling Modification Molecular Mus Mutagenesis Mutate Mutation Nucleotides Oogenesis Pathway interactions Pattern Physiology Process Production RNA RNA Interference Regulation Regulatory Element Reporter Reporting Research Resolution Role Site Site-Directed Mutagenesis Small Interfering RNA Small RNA Specific qualifier value Sperm Maturation Spermatogenesis Testing Therapeutic Trans-Activators Translations Untranslated RNA base comparative computerized tools density design differential expression experimental study fly genome integrity human disease improved insight mRNA Stability mRNA sequencing male male fertility mutant novel nuclease preservation promoter selective expression sex sexual dimorphism sperm cell transcription factor transcriptome sequencing

项目摘要

PROJECT ABSTRACT Small RNAs – short, noncoding RNAs – are critical regulators in animal physiology and disease that silence gene expression by complementary base pairing interactions to control mRNA stability/translation and epigenetic modifications. Small RNA-mediated silencing pathways are evolutionarily conserved, and the largest class of small RNAs comprise Piwi-interacting RNAs (piRNAs). Extensive studies in fly established that piRNAs silence transposons and are expressed in a sex-specific manner; piRNAs are essential for genome integrity and germline development. However, most piRNAs in mammals and worm do not map to transposons, and instead are predicted to regulate germline-expressed genes. Although novel associations of individual piRNAs with endogenous genes have been reported, endogenous gene targets of piRNAs remain largely unknown. In C. elegans, each of the ~15,000 piRNAs is autonomously transcribed and contains an upstream cis- regulatory element, the Ruby motif. We found piRNAs are differentially expressed during spermatogenesis and oogenesis, and male piRNAs have a strong bias for the 5’C nucleotide in the Ruby motif. Furthermore, we identified SNPC-1.3 as a sex-specific transcription factor critical for male fertility. SNPC-1.3 depends on a known core piRNA biogenesis factor, SNPC-4, to drive male piRNA expression during spermatogenesis. Loss of snpc- 1.3 during spermatogenesis results in global depletion of male piRNAs and sperm maturation defects. Other piRNA biogenesis factors have been identified, but how these trans-acting factors interact with each other and the Ruby motif is poorly understood. In this proposed research, I hypothesize that sex-specific regulatory mechanisms underlie piRNA expression to regulate endogenous genes critical for proper germline development. To test my hypothesis in the context of spermatogenesis, in Aim 1 I will use a recently developed strategy Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to characterize protein-DNA binding profiles of 5 piRNA biogenesis trans- acting factors at the Ruby motif cis-regulatory element. Furthermore, I will determine if the 5’ C nucleotide in the Ruby motif acts as a male specific element for SNPC-1.3. I will also use computational tools to identify putative, novel cis-regulatory elements for male and female piRNAs. In Aim 2, I will computationally identify endogenous gene targets of male piRNAs from small RNA-seq and mRNA-seq in wild-type and snpc-1.3(-) animals as well as piRTarBase, a database of computationally predicted and experimentally identified piRNA targeting sites. I will experimentally validate male piRNAs and their predicted endogenous gene targets using reporter assays for endogenous and synthetic piRNAs and applying CRISPR/Cas9-mediated mutagenesis of piRNAs or their targets. Collectively, this research will strengthen our understanding of how sex-specific piRNA expression is transcriptionally regulated and provide new insights into biological roles of piRNAs beyond transposon silencing.

项目摘要小 RNA（短的非编码 RNA）是动物生理学和疾病的关键调节因子，可抑制通过互补碱基配对相互作用来控制 mRNA 稳定性/翻译和表观遗传的基因表达小RNA介导的沉默途径在进化上是保守的，并且是最大的一类。小 RNA 包括 Piwi 相互作用 RNA (piRNA)，对果蝇的广泛研究证实 piRNA 沉默。转座子并以性别特异性方式表达；piRNA 对于基因组完整性至关重要；然而，哺乳动物和蠕虫中的大多数 piRNA 并不映射到转座子，而是映射到转座子。尽管单个 piRNA 与种系表达基因之间存在新的关联，但预计它们会调节种系表达的基因。内源基因已有报道，但 piRNA 的内源基因靶点仍然很大程度上未知。在秀丽隐杆线虫中，大约 15,000 个 piRNA 中的每一个都是自主转录的，并且包含一个上游顺式- 我们发现 piRNA 在精子发生过程中表达差异。卵子发生过程中，雄性 piRNA 对 Ruby 基序中的 5'C 核苷酸有很强的偏向性。此外，我们确定 SNPC-1.3 是对男性生育力至关重要的性别特异性转录因子 SNPC-1.3 取决于已知的已知因素。核心 piRNA 生物发生因子 SNPC-4，在精子发生过程中驱动雄性 piRNA 表达。 1.3 精子发生过程中导致男性 piRNA 整体耗尽和精子成熟缺陷。 piRNA 生物合成因子已被鉴定，但这些反式作用因子如何相互作用以及人们对 Ruby 主题知之甚少。在这项拟议的研究中，我发现 piRNA 背后的性别特异性调控机制表达来调节对正常种系发育至关重要的内源基因，以检验我的假设。在精子发生的背景下，在目标 1 中，我将使用最近开发的策略 Cleavage Under Targets 和使用核酸酶 (CUT&RUN) 表征 5 个 piRNA 生物发生反式的蛋白质-DNA 结合谱此外，我将确定 Ruby 基序顺式调控元件中的 5'C 核苷酸是否起作用。 Ruby 主题充当 SNPC-1.3 的男性特定元素，我还将使用计算工具来识别假定的、男性和女性 piRNA 的新型顺式调控元件在目标 2 中，我将通过计算识别内源性。野生型和 snpc-1.3(-) 动物中小 RNA-seq 和 mRNA-seq 的雄性 piRNA 的基因靶标作为 piRTarBase，一个通过计算预测和实验确定的 piRNA 靶向位点的数据库。将使用报告基因分析实验验证雄性 piRNA 及其预测的内源基因靶标内源和合成的 piRNA 以及应用 CRISPR/Cas9 介导的 piRNA 或其诱变总的来说，这项研究将加深我们对性别特异性 piRNA 表达的理解。转录调控，并为 piRNA 除了转座子沉默之外的生物学作用提供了新的见解。