DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.

No abstract

SummaryBread wheat is a leading cereal crop worldwide. Limited amount of superior allele loci restricted the progress of molecular improvement in wheat breeding. Here, we revealed new allelic variation distribution for 13 yield‐related traits in series of genome‐wide association studies (GWAS) using the wheat 90K genotyping assay, characterized in 163 bread wheat cultivars. Agronomic traits were investigated in 14 environments at three locations over 3 years. After filtering SNP data sets, GWAS using 20 689 high‐quality SNPs associated 1769 significant loci that explained, on average, ~20% of the phenotypic variation, both detected already reported loci and new promising genomic regions. Of these, repetitive and pleiotropic SNPs on chromosomes 6AS, 6AL, 6BS, 5BL and 7AS were significantly linked to thousand kernel weight, for example BS00021705_51 on 6BS and wsnp_Ex_c32624_41252144 on 6AS, with phenotypic variation explained (PVE) of ~24%, consistently identified in 12 and 13 of the 14 environments, respectively. Kernel length‐related SNPs were mainly identified on chromosomes 7BS, 6AS, 5AL and 5BL. Plant height‐related SNPs on chromosomes 4DS, 6DL, 2DS and 1BL were, respectively, identified in more than 11 environments, with averaged PVE of ~55%. Four SNPs were confirmed to be important genetic loci in two RIL populations. Based on repetivity and PVE, a total of 41 SNP loci possibly played the key role in modulating yield‐related traits of the cultivars surveyed. Distribution of superior alleles at the 41 SNP loci indicated that superior alleles were getting popular with time and modern cultivars had integrated many superior alleles, especially for peduncle length‐ and plant height‐related superior alleles. However, there were still 19 SNP loci showing less than percentages of 50% in modern cultivars, suggesting they should be paid more attention to improve yield‐related traits of cultivars in the Yellow and Huai wheat region. This study could provide useful information for dissection of yield‐related traits and valuable genetic loci for marker‐assisted selection in Chinese wheat breeding programme.