Neuronal Tet3-mediated gene expression in the retina

Abstract

Purpose: The retina is a complex neural network dedicated to detection, processing and transfer of visual information to higher brain regions. This retinal network is established in a precisely timed and organized fashion during development. In our study, we analyzed the role of epigenetic mechanisms involving DNA hydroxymethylation in orchestrating this process. 5-hydroxymethylcytosine (5hmC) is particularly enriched in neurons were it has evolved as a novel epigenetic mark involved in the regulation of gene expression during neuronal differentiation. Members of the ten-eleven translocation (Tet) hydroxylase family (Tet1, Tet2 and Tet3) oxidize 5-methylcytosine (5mC) to 5hmC. How Tet hydroxylase activity is controlled and directed to specific neuronal genes for context-specific generation of 5hmC is not known. In particular, it is not known how neuronal Tet3, the major Tet isoform in neurons, which lacks a CXXC DNA binding domain is targeted to the DNA.

Methods: We used the mouse retina as a model system to study the role of neuronal Tet3 and 5hmC during neuronal differentiation and maturation of neuronal circuits. To identify factors interacting with neuronal Tet3 we combined affinity purification of retinal nuclear proteins with mass spectrometry. Selected interacting proteins were subsequently tested for their effect on Tet3 hydroxylase activity. Functional interactions were further studied using a proxymity ligation assay (PLA).

Results: We used the mouse retina as a model system to study the role of neuronal Tet3 and 5hmC during neuronal differentiation and maturation of neuronal circuits. To identify factors interacting with neuronal Tet3 we combined affinity purification of retinal nuclear proteins with mass spectrometry. Selected interacting proteins were subsequently tested for their effect on Tet3 hydroxylase activity. Functional interactions were further studied using a proxymity ligation assay (PLA).

Conclusions: We propose a mechanism in which Rest and other transcriptional regulators target Tet3 to the DNA for directed generation of 5hmC, allowing for chromatin remodeling and facilitation of gene expression in neurons.