Studying the biological functions of the TET and TDG proteins during zebrafish embryonic development

研究斑马鱼胚胎发育过程中TET和TDG蛋白的生物学功能

• 批准号: 1943438
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1943438
• 关键词: Studying; biological; functions; TET; TDG

5'-methylcytosine (5mC) is a transcriptionally repressive epigenetic mark, enriched at CpG dinucleotideswithin vertebrates. The TET proteins (TETs 1, 2 and 3) oxidize 5mC to 5'-hydroxymethylcytosine (5hmC) thensubsequently to 5'-formylcytosine (5fC) and 5'-carboxylcytosine (5caC)1. 5fC and 5caC are excised bythymine-DNA glycosylase (TDG)2. This process is known as active DNA demethylation.Active demethylation is essential during embryonic development. It prevents aberrant repression ofpluripotency factor expression and allows for the de-repression of lineage specification factors' expression -essential for regulating stem cell differentiation and organogenesis. Worth considering is that passivedemethylation (linked to cell division) may contribute significantly to embryonic development too; however theimportance relative to active demethylation is unknown.The relationship between TET expression, 5hmC presence and pluripotency varies across vertebrate species- big differences exist between mice and zebrafish (see Figs.1&2)3. This suggests a phylogenetically varieddevelopmental role for the TET proteins. Why these variations exist and the differences in theirdevelopmental functions are unknown.In mouse embryos, the knockout of individual TET genes leads to phenotypes of varying severity, with TET1causing a very mild impact upon development and viability4 and TET3 being lethal post-implantation5. TDGknockout is also lethal6. Combinatorial knockout of TETs 1&2 results in a severe phenotype4. Little reliabledata has been obtained regarding the consequences of TET1 and TET3 knockout upon zebrafish embryonicdevelopment and none for TDG. However available evidence suggests both phenotypic similarities anddifferences exist between mouse4 and zebrafish7 TET-depleted embryos.Generating CRISPR/Cas9-mediated knockout lines for TETs 1,2,3 and TDG in zebrafish is a crucial step inelucidating the functions of both the enzymes and the epigenetic marks which they deposit/process.

5'-甲基胞嘧啶 (5mC) 是一种转录抑制性表观遗传标记，在脊椎动物内的 CpG 二核苷酸处富集。 TET 蛋白（TET 1、2 和 3）将 5mC 氧化为 5'-羟甲基胞嘧啶 (5hmC)，然后氧化为 5'-甲酰胞嘧啶 (5fC) 和 5'-羧基胞嘧啶 (5caC)1。 5fC 和 5caC 被胸腺嘧啶 DNA 糖基化酶 (TDG)2 切除。这个过程被称为主动 DNA 去甲基化。主动去甲基化在胚胎发育过程中至关重要。它可以防止多能因子表达的异常抑制，并允许谱系特异性因子表达的去抑制——这对于调节干细胞分化和器官发生至关重要。值得考虑的是，被动去甲基化（与细胞分裂有关）也可能对胚胎发育做出重大贡献。然而，相对于主动去甲基化的重要性尚不清楚。TET 表达、5hmC 存在和多能性之间的关系因脊椎动物物种而异，小鼠和斑马鱼之间存在很大差异（见图 1 和 2）3。这表明 TET 蛋白在系统发育上具有不同的发育作用。为什么存在这些变异以及它们发育功能的差异尚不清楚。在小鼠胚胎中，单个 TET 基因的敲除会导致不同严重程度的表型，其中 TET1 对发育和生存能力造成非常轻微的影响，而 TET3 在植入后会致命5。 TDG 敲除也是致命的6。 TET 1 和 2 的组合敲除会导致严重的表型4。关于 TET1 和 TET3 敲除对斑马鱼胚胎发育影响的可靠数据很少，而 TDG 则没有。然而，现有证据表明，小鼠 4 和斑马鱼 7 TET 耗尽的胚胎之间存在表型相似性和差异。在斑马鱼中生成 CRISPR/Cas9 介导的 TET 1、2、3 和 TDG 敲除系，是阐明酶及其沉积/处理的表观遗传标记功能的关键步骤。