The 2-oxoglutarate and Fe (II)-dependent ten-eleven translocation (TET) enzymes can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Thus, it was hypothesized that this could be a pathway for active DNA demethylation. However, several studies have shown that 5hmC levels accumulate especially in neurons suggesting a role as an epigenetic mark. Nevertheless, the mechanism controlling TET activity and the role of 5hmC are poorly understood. In particular, it is not known how the neuronal TET3 isoform lacking a DNA binding domain is targeted to the DNA.

In this study, the role of 5hmC during retinal maturation by genome-wide mapping of 5hmC in immature (postnatal week 2) and mature mouse retina (postnatal week 3) was studied and correlated with expression data. Furthermore, interaction partners of neu-ronal TET3, the main isoform in retinal neurons, were identified.

5hmC accumulates during retinal maturation especially in retinal and neuronal genes. Furthermore, the accumulation of 5hmC is associated with increased transcription. Among the identified proteins the transcriptional repressor REST was identified as a highly enriched TET3-specific interactor. Interestingly, REST was able to enhance TET3 hydroxylase activity. Furthermore, increased 5hmC levels were detected in REST target genes during retinal maturation and overexpression of TET3 activated transcription of REST-target genes. Moreover, NSD3 and two other histone methyltransferases were found to interact with TET3 that are able to mediate H3K36 trimethylation. Finally, it was shown that TET3 is able to enhance NSD3-mediated H3K36 trimethylation to pro-mote transcriptional activation.

In conclusion, the data suggests that 5hmC is a stable epigenetic base in retinal neurons that is involved in transcriptional activation. Furthermore, it was shown that 5hmC is generated by TET3 that is recruited to the DNA by transcriptional regulators such as REST in a context-specific manner.