Hu Zhilian, Holzschuh Jochen, Driever Wolfgang
Developmental Biology, Institute Biology I, Faculty of Biology, Albert-Ludwigs-University Freiburg, Hauptstrasse 1, 79104, Freiburg, Germany; Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, 48109-5646, United States of America.
Developmental Biology, Institute Biology I, Faculty of Biology, Albert-Ludwigs-University Freiburg, Hauptstrasse 1, 79104, Freiburg, Germany.
PLoS One. 2015 Jul 30;10(7):e0134299. doi: 10.1371/journal.pone.0134299. eCollection 2015.
DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.
DNA损伤结合蛋白1(DDB1)是异二聚体DDB复合物的大亚基,可识别DNA损伤并启动核苷酸切除修复过程。DDB1也是CUL4 E3连接酶复合物的一个组成部分,通过对关键调节因子进行靶向泛素化参与广泛的细胞过程。DDB1在发育中的功能已在几种模式生物中得到研究,然而,迄今为止尚未完全了解。在此,我们报告了在斑马鱼(Danio rerio)中鉴定出的一种ENU诱导的突变ddb1等位基因(ddb1m863),并分析了其对发育的影响。斑马鱼ddb1在母源和合子阶段均广泛表达,在增殖区表达增强。(ddb1m863)突变等位基因影响外显子20的剪接受体位点,导致剪接缺陷,致使1140个氨基酸的蛋白质在第800位残基后截短,缺失部分β-螺旋桨结构域BPC和C端螺旋结构域CTD。ddb1m863合子突变胚胎具有多效性表型,包括脑、头部骨骼、眼睛、颌骨和鳃弓较小且形状异常,以及多巴胺能神经元群减少。然而,早期形成的组织在ddb1m863合子突变胚胎中发育正常,这可能是由于母源拯救。在ddb1m863突变胚胎中,表达增殖细胞核抗原(PCNA)的增殖细胞群体减少,同时细胞凋亡增加。我们还观察到在增殖组织中肿瘤抑制因子p53(tp53)和细胞周期抑制剂cdkn1a(p21a/bCIP1/WAF1)的转录本同时强烈上调。此外,在ddb1m863突变体中,细胞周期蛋白基因ccna2和ccnd1的转录失调。通过反义吗啉代寡核苷酸降低p53活性可减轻ddb1m863突变体中的凋亡表型。这些结果表明,Ddb1可能通过调节参与细胞周期控制和细胞存活的基因的转录机制,参与维持发育过程中分裂细胞的正常细胞周期进程和活力。