Regulation of protamine incorporation and nuclear reorganization during Drosophila spermatogenesis

果蝇精子发生过程中鱼精蛋白掺入和核重组的调节

• 批准号: 10462097
• 负责人: Jun Il Park
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462097
• 关键词: Alleles; Architecture; Attention; Biological Models; Biological Process; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Chromatin; Cytology; DNA; DNA-Directed RNA Polymerase; Data; Defect; Development; Dominant-Negative Mutation; Drosophila genus; Drosophila melanogaster; Failure; Fertility; Fertilization; Gene Expression; Genes; Genetic Transcription; Genome; Growth; Histones; Homologous Gene; Human; Lead; Literature; Male Infertility; Mediating; Molecular Biology; Morphogenesis; Nuclear; Ontology; Phenotype; Process; Protamines; Protein Isoforms; Protein Sequence Analysis; Protein Splicing; Proteins; RNA; RNA Splicing; Regulation; Role; Somatic Cell; Sperm Maturation; Spermatids; Spermatocytes; Spermatogenesis; Spermiogenesis; Sterility; Testing; Testis; Y Chromosome; base; chromatin remodeling; clinically relevant; genome integrity; insight; interest; mutant; novel; nucleolin; protein expression; protein protein interaction; sperm cell; transcription factor; transcriptome sequencing

ABSTRACT In many species, sperm maturation requires a process of nuclear compaction, which occurs via dramatic chromatin reorganization from histone to protamine-based architecture. Nuclear compaction is essential for sperm fertility, functioning to enhance hydrodynamics and genomic integrity. While several genes have been identified to be involved in this process, it is unknown how these genes are regulated. Here, we have utilized the Drosophila testis model system to identify Modulo, the Drosophila homolog of Nucleolin, as a key player in sperm nuclear reorganization. In somatic cells, modulo has been shown to be involved in chromatin remodeling, cell proliferation, and morphogenesis. Viable modulo mutant allele that specifically influences testis-specific isoform has been known to be sterile, but the reason underlying this sterility remained unknown. We find that modulo mutants display nuclear compaction defects during late spermatogenesis alongside decreased protamine protein incorporation. Importantly, modulo’s unique phenotype cannot be explained simply by lack of protamine incorporation: compound mutant that lacks protamines Prot A/B and Mst77F causes only mild nuclear deformation rather than complete nuclear decompaction as observed in modulo mutant. Indeed, in addition to decreased incorporation of main protamines in mutant, we have detected increased expression of Mst77Y, a lesser-studied protamine gene with numerous duplications on the Y-chromosome. Thus, we hypothesize that modulo mediates sperm nuclei compaction by coordinating expression of multiple genes that are specifically involved in nuclear compaction during spermiogenesis. To test this hypothesis, we will conclusively establish the cytological defects of modulo mutant by inducing expression of mst77Y in wild-type nuclei and assessing protamine protein expression in whole testis vs. in spermatid nuclei. Additionally, we will investigate transcription and/or splicing as modulo’s mechanism for regulating its target genes. Previous literature has identified modulo to interact with testis-specific transcription factors while protein sequence analysis and gene ontology have predicted modulo as being involved in splicing. Therefore, we further hypothesize that modulo regulates its target genes at the RNA level either by functioning during transcription or splicing. If our hypothesis is true, it would result in the identification of a previously uncharacterized regulatory network governing nuclear reorganization in late stage spermatogenesis and have implications for male infertility.

摘要 在许多物种中，精子成熟需要一个核致密过程，这一过程通过 染色质从组蛋白到鱼精蛋白的戏剧性重组。核子 紧凑化对精子受精率至关重要，可以增强流体力学和基因组的功能。 正直。虽然已经确定有几个基因参与了这一过程，但尚不清楚。 这些基因是如何被调控的。在这里，我们利用果蝇睾丸模型系统来 确认核仁的果蝇同源物modulo是精子核中的关键角色 重组。在体细胞中，模数已被证明与染色质重塑有关， 细胞增殖和形态发生。活的模数突变的等位基因，特别影响 已知睾丸特异性亚型是不育的，但这种不育背后的原因是 仍然不为人所知。我们发现，模变异体在后期表现出核压实缺陷 精子发生伴随鱼精蛋白掺入减少。重要的是，模数的 独特的表型不能简单地用缺乏鱼精蛋白掺入来解释：化合物 缺少精蛋白A/B和Mst77F的突变体只引起轻微的核变形，而不是 而不是像在模变异体中观察到的那样完全核分解。事实上，除了 突变体中主要鱼精蛋白的掺入减少，我们检测到表达增加 Mst77Y是一种研究较少的鱼精蛋白基因，在Y染色体上有大量重复。 因此，我们假设，模数通过协调表达来调节精子核的紧凑。 在精子发生过程中与核紧凑相关的多个基因。至 检验这一假说，我们将最终确定模变异体的细胞学缺陷 Mst77Y在野生型细胞核中的诱导表达及鱼精蛋白的表达 整个睾丸与精子细胞核。此外，我们还将研究转录和/或剪接 作为模数调节其目标基因的机制。以前的文献已经证明了模数 在蛋白质序列分析和基因分析时与睾丸特异性转录因子相互作用 本体论预测，模数参与了剪接。因此，我们进一步假设 该模块在RNA水平上调节其目标基因，或者通过在转录过程中发挥作用 或者拼接。如果我们的假设是真的，它将导致识别之前的 后期管理核重组的未定性监管网络 精子发生，并对男性不育有影响。