Transgenerational inheritance of impaired larval T cell development in zebrafish

The transgenerational inheritance of epigenetic information is an attractive mechanism by which the phenotypic consequences of exposure of parents to certain environmental conditions could be transmitted to the next generation. Although this phenomenon is well described in yeast and plants, and in some invertebrates, such as the D. melanogaster and C. elegans, the evidence for transgenerational inheritance in vertebrates is scarce. Among the mechanisms that are known or suspected to be involved in mediating the propagation of epigenetic information to subsequent generations, the process of enzymatic DNA methylation is of particular interest. During DNA replication, the methylation pattern of the parental strand is faithfully copied onto the daughter strand by the maintenance methylase DNMT1, although methylation patterns can be changed through the action of de novo methylases and mechanisms of active and passive demethylation. In the context of epigenetic inheritance, the induction of aberrant methylation patterns in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. Surprisingly, homozygous mutants present with an essentially normal phenotype, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. Male but not female mutants are fertile. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by about 50% of fish that are genotypically wildtype. Whole genome bisulfite sequencing of sperm DNA of transmitting and non-transmitting males identified about 200 differentially methylated regions, including hypermethylated sites associated with runx3 and rptor genes. Knock-down of these two genes leads to impaired larval T cell development. Our results raise the possibility of an epigenetic origin of certain immunodeficiency syndromes in animals and humans. Overall design: Our forward genetic screen identified a missense mutation in maintenance DNA methylase Dnmt1 specifically affecting lymphoid development. To examine the molecular consequences and transgenerational inheritance, whole genome methylome analyses were conducted for zebrafish Dnmt1 mutant.

表观遗传信息的跨代遗传（transgenerational inheritance）是一种极具吸引力的机制，可将亲本暴露于特定环境条件所产生的表型后果传递给子代。尽管该现象在酵母、植物以及部分无脊椎动物（如黑腹果蝇Drosophila melanogaster和秀丽隐杆线虫Caenorhabditis elegans）中已有充分描述，但脊椎动物中跨代遗传的相关证据仍较为匮乏。在已知或被推测参与介导表观遗传信息向后代传递的机制中，酶促DNA甲基化（enzymatic DNA methylation）尤为引人关注。在DNA复制过程中，维持性甲基转移酶DNMT1（maintenance methylase DNMT1）可将母链的甲基化模式精准复制到子链，不过甲基化模式也可通过从头甲基转移酶的作用以及主动、被动去甲基化机制发生改变。在表观遗传遗传的背景下，配子中异常甲基化模式的诱导可能为向未来世代的传递奠定基础。本研究描述了斑马鱼中一种具有生存能力的dnmt1低功能等位基因（hypomorphic allele），该等位基因会导致精子与体细胞组织中CpG二核苷酸（CpG dinucleotides）发生广泛的去甲基化。令人意外的是，纯合突变体除了淋巴细胞生成严重受损（影响T细胞发育的幼虫期与成虫期阶段）外，表型基本正常。雄性突变体可育，雌性则不然。幼虫期（而非成虫期）T细胞发育受损的表型可通过约50%基因型为野生型的鱼类传递给后代。对具有传递能力与无传递能力的雄性个体的精子DNA进行全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing），共鉴定出约200个差异甲基化区域（differentially methylated regions），其中包括与runx3和rptor基因相关的高甲基化位点。对这两个基因进行敲低（knock-down）会导致幼虫期T细胞发育受损。本研究结果提示，动物与人类的某些免疫缺陷综合征（immunodeficiency syndromes）可能存在表观遗传起源。整体实验设计：本研究通过正向遗传筛选（forward genetic screen）鉴定出了维持性DNA甲基转移酶Dnmt1（maintenance DNA methylase Dnmt1）上一个特异性影响淋巴发育的错义突变（missense mutation）。为探究其分子后果与跨代遗传特性，本研究对斑马鱼Dnmt1突变体开展了全基因组甲基化组（methylome）分析。