Study of the interplay between TET proteins and microRNAs for memory formation

Abstract

Epigenetic mechanisms are critical regulators of gene expression underlying learning and memory formation. One of the most widely studied epigenetic mechanism is the methylation of DNA, and until recently, was regarded as stable in post-mitotic cells, such as neurons. This view has, however, recently been actualized, as DNA methylation was suggested to be dynamically regulated in a locus-specific manner upon neuronal stimulation and learning. Consistently, the expression of DNA methylases also appears to be dynamically regulated in the brain. A family of DNA demethylases, Ten-eleven translocation (TET) proteins (TET 1, 2 and 3) mediate the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). There is accumulating evidence that TET proteins, and hence 5mC and 5hmC levels, are regulated by neuronal activity to control gene transcriptional regulation and memory. However, little is currently known about the mechanisms bringing about their dynamic regulation. The scope of the present work was to explore the modes of regulation of TETs in the context of neuronal activity and memory formation. Specifically, the implication of microRNAs (miRNAs), a class of short non-coding RNAs capable of modulating gene expression rapidly and reversibly, in controlling TET expression is investigated. The results indicate that Tet3 is preferentially up regulated in response to learning in vivo. Consistently, neuronal activation triggers increased Tet3 gene expression in vitro. Furthermore, targeted transcriptional analysis revealed that memory-related genes, such as Creb1, are sensitive to changes in TET3 levels. This suggests that activity-dependent TET3 regulation in the hippocampus can affect the transcriptional activity of genes related to learning and memory formation. miR-29b, a miRNA whose sequence is complementary to multiple sites in Tets 3’ untranslated transcribed regions (3’UTRs), is inversely regulated in response to learning and neuronal activation. Importantly, miR-29b binds to the 3’UTRs of all Tets and controls their expression, though, a preference towards Tet3 is observed at low concentration. Overall, these findings suggest that miRNAs play a role in the activity-dependent regulation of TETs associated with learning and memory formation. Next, SAM68, a nuclear RNA-binding protein previously known to regulate alternative splicing, was identified as an important regulator of miR-29b biogenesis at the transcriptional level. In addition, Sam68 was found to be a target of TET3, which suggests that the expression of Sam68, miR-29b, and TET3 might be intricately regulated in response to neuronal activity through a feed-back loop. In summary, this study identifies a novel molecular pathway involving the miR- 29 cluster and the RNA-binding protein SAM68 in the regulation of the DNA demethylase TET3. This regulatory mechanism may contribute to the epigenetic control of genes underlying memory formation.