Fzr调控家蚕丝腺细胞核内复制的新机制

The cells of silkworm silk gland and fruit fly salivary gland enter into endoreplication during larval period and undergo multiple rounds of DNA replication without cell division. Previous reports in the fruit fly have been demonstrated that Fzr regulates the entry and maintenance of the endoreplication by mediating the ubiquitin ligase to target on CycB and other proteins for their ubiquitin-mediated degradation. But, we also found that Fzr RNAi in fruit fly salivary gland can promote the transcription of the CycB gene and inhibit the transcription of the Mcm genes involved in DNA replication; we further observed the continuous expression of the Fzr gene but the lack of CycB expression in silkworm silk gland at mRNA level. Given that Fzr is not a transcription factor, we speculate that Fzr may be involved in the transcriptional regulation of CycB and other genes involved in endoreplication. In this study, using the silkworm and fruit fly as excellent models and combining the approaches of knock out, RNAi, transgenic overexpression, RNA-seq, Co-IP, mass spectrometry, luciferase reporter assay, EMSA and ChIP, we are planning to systematically investigate the roles of Fzr in endorelication, to indentify the downstream potential transcription factor targets of Fzr, and to decipher the mechanisms underlying the response of transcription factor targets to Fzr and the regulation of Fzr on the transcription of genes involved in endoreplication. We hope that the results from this study not only provide new insights into the Fzr regulation on endoreplication at transcriptional level, but also help us to get new cues for genetically improving silk protein synthesis and the yields of silk production in the silkworm.

家蚕丝腺和果蝇唾液腺细胞在幼虫期发生核内复制，进行多轮DNA复制而不分裂。果蝇中的研究表明，Fzr通过蛋白互作直接介导泛素连接酶对CycB等靶蛋白的泛素化降解来调控核内复制。但是，我们发现果蝇Fzr的RNAi引起CycB转录并抑制DNA复制相关基因Mcm的表达，家蚕丝腺中Fzr持续表达但没有CycB的mRNA表达；鉴于Fzr并非转录因子，我们推测Fzr还可能间接调控CycB等核内复制相关基因转录。本项目拟以家蚕和果蝇为材料，综合应用基因敲除、RNAi、转基因过表达、转录组测序、Co-IP、质谱分析、荧光素酶报告基因分析、EMSA和ChIP等方法，深入分析Fzr在核内复制中的功能并鉴定Fzr的下游转录因子靶标，解析Fzr下游转录因子对Fzr的应答机制及其对核内复制相关基因转录的调控机制。相关结果有助于建立Fzr在转录水平调控核内复制的新机制，并可为家蚕丝蛋白合成及丝产量的分子改良提供新线索。

家蚕丝腺是丝蛋白合成与分泌的场所，其细胞在幼虫阶段进入核内复制细胞周期，只进行DNA复制而不能发生胞质分裂，从而形成多倍体细胞。果蝇唾液腺等类似器官中的研究显示，支架蛋白Fzr是核内复制细胞周期进入与维持的关键调控分子。本研究通过在家蚕丝腺和果蝇唾液腺中的联合系统研究，建立了Fzr调控细胞核内复制的新机制，并证实唾液腺对系统性生长的调控作用。主要研究发现如下：（1）建立了调控细胞核内复制的Fzr-H2Bub-Myc信号级联途径：证实转录因子Myc是Fzr的下游信号分子，Fzr表达降低所导致的DNA复制停滞及细胞生长受阻等异常表型能被Myc过表达回救；Fzr与组蛋白H2B发生互作，进而增强Myc基因启动子区H2B的泛素化（H2Bub）水平并促进Myc转录；Myc对细胞周期蛋白基因CycB和DNA复制过程所必需的微小染色体维持蛋白（MCM）家族成员MCM6的转录分别具有直接抑制和促进作用；Fzr-H2Bub-Myc级联途径在家蚕、果蝇及人细胞中高度保守。（2）揭示了Fzr敲除突变对丝腺细胞核内复制及丝蛋白合成的影响：利用CRISPR/Cas9系统在家蚕后部丝腺中特异敲除Fzr，导致丝腺发育及丝腺细胞核内复制进程受阻，丝蛋白合成降低。蛋白质组学分析发现，Fzr敲除突变抑制了核糖体生成途径，上调了蛋白质降解途径。（3）证实了Myc对家蚕丝腺细胞核内复制的调控作用：利用家蚕FibH基因启动子驱动Myc在家蚕后部丝腺特异过表达，导致MCM家族成员的表达上调，并促进后部丝腺细胞中的DNA复制过程，提高了丝蛋白合成。（4）发现了果蝇唾液腺分泌的参与调控个体系统性生长的Sgsf蛋白：唾液腺特异表达的Sgsf蛋白可以分泌至血淋巴，进而通过内分泌方式调控个体系统性生长。该研究不仅揭示了昆虫细胞核内复制调控新机制，也为提高家蚕丝腺细胞中DNA复制及丝蛋白合成的遗传改造提供了新思路。

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