UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development.
UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development.
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DOI：
10.1016/j.ydbio.2016.01.036
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发表时间：
2016-04-01
影响因子：
2.7
通讯作者：
Sadler KC
中科院分区：
文献类型：
--
作者：
Kent B;Magnani E;Walsh MJ;Sadler KC
具有里程碑意义的表观遗传事件是早期胚胎发育的基础，但如何调控表观遗传修饰物以实现快速的表观基因组重构尚不清楚。已知uhrf1和DNA甲基转移酶1(Dnmt1)在很大程度上介导DNA甲基化，并且uhrf1对Dnmt1的定位和稳定性也是必需的。在这里，我们研究这两个关键的表观遗传修饰物如何调节斑马鱼的早期发育，并表征破坏它们的动态平衡关系的发育后果。与uhrf1基因敲除导致原肠形成之前的发育停滞和死亡不同，过表达人uhrf1(WT-uhrf1)会导致不对称的外胚层、低效的原肠形成和多系统缺陷。先前证明uhrf1的磷酸化是斑马鱼胚胎发生所必需的，我们发现在注射编码非磷酸化的uhrf1(UHRF1S661a)的胚胎中，不对称外膜表型的外显率显著增加。令人惊讶的是，WT-uhrf1和UHRF1S661a的过表达都导致了DNA低甲基化。然而，由于其他导致同等程度的DNA低甲基化的方法并没有引起不对称的表型，我们得出结论，大量DNA甲基化不是主要的机制。相反，UHRF1S661a的过表达导致Dnmt1蛋白的积累，WT和催化失活的Dnmt1的过表达都出现在WT-uhrf1过表达的胚胎上。DNMT1基因敲除可抑制UHRF1S661A过表达引起的表型改变，而uhrf1基因敲除可抑制DNMT1过表达所致的表型改变。因此，我们得出结论，这两种蛋白质之间的相互作用是导致观察到的发育缺陷的机制。这表明Dnmt1的稳定需要uhrf1的磷酸化，蛋白质之间的串扰对于这两个重要的表观遗传调节因子在原肠形成过程中的功能是必不可少的。
Landmark epigenetic events underlie early embryonic development, yet how epigenetic modifiers are regulated to achieve rapid epigenome re-patterning is not known. Uhrf1 and DNA methyltransferase 1 (Dnmt1) are known to largely mediate maintenance DNA methylation and Uhrf1 is also required for both Dnmt1 localization and stability. Here, we investigate how these two key epigenetic modifiers regulate early zebrafish development and characterize the developmental consequences of disrupting their homeostatic relationship. Unlike Uhrf1 knockdown, which causes developmental arrest and death prior to gastrulation, overexpression of human UHRF1 (WT-UHRF1) caused asymmetric epiboly, inefficient gastrulation and multi-systemic defects. UHRF1 phosphorylation was previously demonstrated as essential for zebrafish embryogenesis, and we found that penetrance of the asymmetric epiboly phenotype was significantly increased in embryos injected with mRNA encoding non-phosphorylatable UHRF1 (UHRF1S661A). Surprisingly, both WT-UHRF1 and UHRF1S661A overexpression caused DNA hypomethylation. However, since other approaches that caused an equivalent degree of DNA hypomethylation did not cause the asymmetric epiboly phenotype, we conclude that bulk DNA methylation is not the primary mechanism. Instead, UHRF1S661A overexpression resulted in accumulation of Dnmt1 protein and the overexpression of both WT and a catalytically inactive Dnmt1 phenocopied the WT-UHRF1 overexpressing embryos. Dnmt1 knockdown suppressed the phenotype caused by UHRF1S661A overexpression, and Uhrf1 knockdown suppressed the effect of Dnmt1 overexpression. Therefore, we conclude that the interaction between these two proteins is the mechanism underlying the observed developmental defects. This indicates that Dnmt1 stability requires UHRF1 phosphorylation and that crosstalk between the proteins is essential for the function of these two important epigenetic regulators during gastrulation.