Sexual Identity Maintenance in Drosophila Female Germ Cells

生殖细胞性特征的保存对生殖至关重要。针对特定性别的节目中的缺陷 导致不孕不育和生殖细胞肿瘤。尽管它对人类健康很重要，但生殖细胞的性别鉴定 是被维护的是未知的。果蝇雌性生殖系是研究这一重要性的理想模型 细胞命运决定保持不变。未能维持性认同之间的联系，反常 精子发生基因的表达和生殖细胞肿瘤的发展使基因能够被分配给 性命维系路径。这些新的研究表明，一条表观遗传调控途径 SXL是上游女性特有的调节因子，SETDB1是必需的染色质编写器，phf7是 关键下游的SETDB1靶基因，维持性别认同。SETDB1三甲基H3K9(H3K9me3)，和 组蛋白修饰与基因沉默相关。证据表明，SXL 依赖的转录因子将SETDB1定向到其精子发生靶基因，在那里它存储了一个局部的 H3K9me3标记，导致HP1a依赖的沉默。该途径的中断导致异位PHF7 表情。这种睾丸特异的蛋白质足以重新编程其目标基因的转录(包括 )，导致精子发生基因激活和生殖细胞肿瘤。这项提案中的研究将测试 这一模型并在两个目标上促进了我们对这一新发现的途径的理解。在目标1中，重点是 在SETDB1上。SETDB1通过安装基因特异性沉默生精基因的假说 通过比较染色质在其靶基因上的分布，将在全球水平上进行测试 H3K9me3及其配体HP1a在野生型和SETDB1缺失生殖细胞中的表达。由此引发的一个关键问题是 假说是SETDB1是如何招募到其精子发生靶基因的。遗传学研究已经确定 两个依赖于SXL的转录因子，可能是关键的靶向因子。结合了基因， 将使用细胞学和基因组学方法来确定这些新发现的途径成员是如何 为维护女性命运作出贡献。在目标2中，重点是PHF7。PHF7是一种染色质阅读器，可以结合 H3K4me2，一种与活跃或稳定基因相关的标记。因此，异位的PHF7可能会重编性命运 通过激活沉默但稳定的精子发生基因，和/或通过抑制 精子发生基因沉默。测试这一模型需要识别PHF7目标基因。我们将首先 建立在PHF7重新编程自身转录的证据基础上，然后采取更全球化的方法。这些 研究将导致对强迫表达如何能够扰乱女性命运的理解，并将作为一种 为这一新途径分配新基因的发现工具。生殖细胞生物学上惊人的相似之处 人和苍蝇之间的关系表明，从这项研究中获得的知识将提供 洞察人类生殖细胞如何维持他们的性身份，以及这一过程中的错误如何干扰 繁殖。

The preservation of germ cell sexual identity is essential for reproduction. Defects in sex-specific programs leads to infertility and germ cell tumors. Despite its importance to human health, how germ cell sexual identity is maintained is unknown. The Drosophila female germline is an ideal model for studying how this important cell fate decision is maintained. The connection between failures to maintain sexual identity, aberrant expression of spermatogenesis genes and germ cell tumor development enabled the assignment of genes to a sex fate maintenance pathway. These new studies demonstrate that an epigenetic regulatory pathway in which SXL is the upstream female-specific regulator, SETDB1 is the required chromatin writer and phf7 is one of the key downstream SETDB1 target genes, maintains sexual identity. SETDB1 trimethylates H3K9 (H3K9me3), an histone modification associated with gene silencing. The evidence suggests a hypothesis in which Sxl dependent transcription factors direct SETDB1 to its spermatogenesis target genes, where it deposits a local H3K9me3 mark, leading to HP1a-dependent silencing. Disruption of this pathway leads to ectopic PHF7 expression. This testis-specific protein is sufficient to reprogram transcription of its target genes (including itself), leading to spermatogenesis gene activation and a germ cell tumor. The studies in this proposal will test this model and advance our understanding of this newly identified pathway, in two Aims. In Aim 1, the focus is on SETDB1. The hypothesis that SETDB1 silences spermatogenesis genes by installing gene specific silencing chromatin on its target genes will be tested at a global level by comparing the distribution of H3K9me3 and its ligand HP1a in wild-type and SETDB1 depleted germ cells. A key question raised by this hypothesis is how SETDB1 is recruited to its spermatogenesis target genes. Genetic studies have identified two Sxl dependent transcription factors that are likely to be the critical targeting factors. Combined genetic, cytological and genomic approaches will be used to determine how these newly identified pathway members contribute to female fate maintenance. In Aim 2, the focus is on PHF7. PHF7 is a chromatin reader that binds to H3K4me2, a mark associated with active or poised genes. Thus ectopic PHF7 may reprogram sexual fate by activating silent, but poised, spermatogenesis genes, and/or by repressing active genes required for spermatogenesis gene silencing. Testing this model requires identification of PHF7 target genes. We will first build on evidence that PHF7 reprograms its own transcription, and then take a more global approach. These studies will lead to an understanding of how forced expression is able to disrupt female fate, and will serve as a discovery tool for assigning new genes to this novel pathway. The striking similarities in germ cell biology between humans and flies suggest that the knowledge gained from the studies in this proposal will provide insight into how human germ cells maintain their sexual identity, and how errors in this process interferes with reproduction.