A TET Homologue Protein from <i>Coprinopsis cinerea</i> (CcTET) That Biochemically Converts 5‑Methylcytosine to 5‑Hydroxymethylcytosine, 5‑Formylcytosine, and 5‑Carboxylcytosine

DNA methylation (5-methylcytosine, 5mC) plays critical biological functions in mammals and plants as a vital epigenetic marker. The Ten-Eleven translocation dioxygenases (TET1, 2, and 3) have been found to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in mammalian cells. We report herein three mushroom TET homologues from <i>Coprinopsis cinerea</i> that can mediate 5mC oxidation. Specifically, one homologue (CC1G_05589, CcTET) shows similar activity to its mammalian TET homologues. Biochemically, CcTET actively converts 5mC to 5hmC, 5fC, and 5caC under natural conditions (pH 7.0). Interestingly, CcTET also converts the majority of 5mC to 5fC under slightly acidic (pH 5.8) and neutral conditions. Kinetics analyses of the oxidation by CcTET under neutral conditions indicate that conversion of 5mC to 5hmC and 5hmC to 5fC are faster than that of 5fC to 5caC, respectively. Our results provide an example of a TET homologue in a non-mammalian organism that exhibits full 5mC-to-5caC oxidation activity and a slight preference to producing 5fC. The preferential accumulation of 5fC in the <i>in vitro</i> oxidation reactions under both neutral and acidic conditions may have biological implications for 5mC oxidation in fungi species

The t⁶A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in <i>Streptococcus mutans</i>

<p>Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNA^Lys _UUU in the anticodon loop. <i>In vitro</i> studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated <i>in vivo</i>. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. <i>Streptococcus mutans</i> is one of the few bacteria where the t⁶A synthesis gene <i>tsaE</i> (<i>brpB</i>) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the <i>S. mutans tsaE</i> mutant was devoid of t⁶A. However, we describe here a novel and a more sensitive hybridization-based t⁶A detection method (compared to HPLC) that showed t⁶A was still present in the <i>S. mutans</i> Δ<i>tsaE</i>, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other <i>S. mutans</i> t⁶A synthesis genes (<i>tsaB</i> and <i>tsaC</i>) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t⁶A modification ratios and of t⁶A synthesis genes mRNA levels in <i>S. mutans</i> suggest they may be regulated by growth phase.</p

Novel and previously identified BMI and WHR_adjBMI loci at <i>P</i> < 5×10⁻⁸ in African ancestry discovery and replication samples, and European ancestry replication samples.

<p>Novel and previously identified BMI and WHR_adjBMI loci at <i>P</i> < 5×10⁻⁸ in African ancestry discovery and replication samples, and European ancestry replication samples.</p

Additional novel BMI and WHR_adjBMI loci at <i>P</i> < 5×10⁻⁸ in sex-stratified analyses of African ancestry discovery and replication samples.

<p>Additional novel BMI and WHR_adjBMI loci at <i>P</i> < 5×10⁻⁸ in sex-stratified analyses of African ancestry discovery and replication samples.</p