Evolutionary Origin And Function Of Genomic Imprinting In Vertebrates

Genomic imprinting is an epigenetic process that results in diploid cells expressing a small subset of genes from only their maternal-or paternal-inherited chromosome. In mammals, genome imprinting causes functional nonequivalence and complement between the parental genomes, which prevents parthenogenesis. Genomic imprinting is found exclusively in higher plants and mammals, and no obvious imprinting has been seen in other vertebrates. In this case, the evolutionary origin of genomic imprinting has attracted great attention for researchers. However, the evolutionary mechanisms and biological functions of genomic imprinting are still not clear.Previous studies in artificial gynogenetic fishes indicate that a certain extent of functional nonequivalence and complement exist between the parental genomes of diploid fish, and transgenes in zebrafish showed parent-specific methylation, suggesting that imprinting-like process might exist in lower vertebrate. Goldfish includes both subspecies of bisexual diploid (C. auratus) and unisexual polyploid (C. auratus, pengze), which serves as a suitable experimental system for studying the evolution of genomic imprinting.In this study, we analyzed the methylation status of a developmental decisive gene no tail (ntl) in germ cells and early developmental embryos from fishes with different reproduction style and chromosome ploidy, and then we further analysed the effects of DNA methylation on gene expression, and at last, we investigated the roles of cis-element played in DNA methylation. The main results are listed as follows:1) BSP analysis in goldfish germ cells revealed that CpG island at ntl promoter was hypermethylated in the egg genome from diploid goldfish, while not menthylated in sperm from diploid goldfish and egg from polyploid goldfish.2) BSP analysis in early goldfish embryos showed that CpG island at ntl promoter was first passively demethylated and was completely non-methylated at mid-blastula stage, and then it was re-methylated since late-gastrulation stage and was hypermethylated when at4days after fertilization. Similarly, ntl CpG island in artificial gynogenetic diploid goldfish embryos which contained only maternal genome also undergone the demethylation-remethylation progress. However, no methylation was detected in early embryos from polyploid goldfish.3) Using identified SNP sites as the marker to distinguish maternal and paternal transcripts, it was observed directly that the expression of maternally methylated allele of ntl was inhibited in early embryos of bisexual diploid goldfish.4) CpG island at ntl promoter was also germ cell-specifically methylated in bisexual diploid zebrafish, which also showed epigenetic asymmetry and transcriptional nonequivalence of the parental alleles during early embryogenesis.5) More than2K bp of ntl promoter was cloned from both bisexual diploid and unisexual polyploid goldfish by four rounds of genome walker, and sequence alignment showed a significant difference in an imperfect TG tandem repeat. The similar TG repeat also exist in zebrafish ntl promoter, and the length of which is almost the same with that in bisexual diploid goldfish, while exceed that of unisexual polyploid goldfish with about110bp.6) To further study the roles of this TG repeat, we tried to modify this sequence in zebrafish genome using engineered TAL effector nuclease (TALEN), and analysis of the mutants showed that TALEN could indeed induce desired mutation in TG repeat.Germ cell-specific DNA methylation is one of the fundamental mechanisms of genome imprinting, and the results of this study suggest that genome imprinting might originate from lower vertebrate with genome duplication and diploidization, where it could play a role in inhibiting gynogenesis.