An exciting new paper in Cell, links Piwi-interacting RNAs (piRNAs) to long-term memory via the epigenetic regulation of gene expression by DNA methylation.
Two different novel findings are especially important: piRNAs had been thought to be a germline specific mode of genetic control, specifically a type of genetic immunity against the mobilisation of transposable elements. Rajasethupathy et al demonstrate that in the sea hare, Aplysia, piRNAs are expressed in the CNS and other somatic tissues. Secondly, this paper demonstrates specific dynamic de novo methylation of the promoter of a gene regulating neuronal plasticity in response to neurotransmitter- mediated excitation. This provides an epigenetic mechanism by which memories can persist by molecular encoding.
To investigate microRNAs expressed in the Aplysia CNS, Rajasethupathy et al had constructed a small RNA library. Surprisingly, they found that ~20% of the sequence reads from this library were ~28nt long, and showed a bias towards having 5′ uridine residue. This fitted with them being Piwi-interacting RNAs (piRNAs) rather than microRNAs. When they were mapped to the Aplysia genome, it was clear that the piRNAs were generated from piRNA clusters (see previous introductory post). After constructing more libraries and deep sequencing, Rajasethupathy et al. identified 372 distinct Aplysia piRNA clusters. Certain piRNAs are found far more commonly than surrounding piRNAs from the same cluster, indicating that an amplification process is occurring during piRNA biogenesis. Although overall piRNA content was highest in the ovotestes (ie the germline and associated somatic tissues), various piRNAs were found to be enriched in the CNS, as well as other somatic tissues analysed.
Consistent with the presence of piRNAs in the CNS, Rajasethupathy et al. were able to clone a full-length cDNA for Piwi protein from Aplysia CNS. Using an antibody against Piwi, they were then able to co-immunoprecipitate piRNAs with Piwi protein from the CNS. By separating cell nuclei from cytoplasm and then western and northern blotting against Piwi and piRNAs respectively, they showed that both were primarily found in nuclei.
To briefly comment on the piRNA aspect of this paper; a number of outstanding questions arise. In the best characterised model systems, piRNA amplification occurs by the ‘ping-pong’ mechanism in which reciprocal recognition and cleavage reactions between sense and antisense piRNAs complexed with the (cytoplasmic) Piwi-related proteins Aubergine and AGO3 (Drosophila terminology), leads to selective amplification of piRNAs with a tell-tale 10nt offset. Neither of these proteins, nor the 10nt offset, nor the ratio between sense and antisense piRNAs are mentioned by Rajasethupathy et al. I imagine that these questions would’ve been looked into and mentioned if found, therefore it seems that the mechanism of piRNA amplification in the Aplysia CNS is potentially novel.
To explore possible functions of piRNAs in the Aplysia CNS, the researchers used a co-culture system that monitors changes in synaptic plasticity in response to stimulation by the neurotransmitter serotonin (5HT). In this assay, two sensory neurons synapse with a motor neuron. Changes in the strength of one sensory-motor synapse are monitored by electrophysiological recording from the motor neuron. This system measures long-term facilitation (LTF): ie. changes in synaptic strength in response to 5HT stimulation. LTF is considered to be a memory-related phenomenon, however, it is contentious just how well it serves as a paradigm for long-term memory.
Knockdown of Piwi (and hence of complexed piRNAs), by the injection of antisense oligonucleotides into one of the sensory neurons, significantly impaired LTF, whilst overexpression of Piwi enhanced it. To investigate how these Piwi effects on LTF were mediated, Rajasethupathy et al. looked at the expression of proteins known to be regulating synaptic plasticity in response to Piwi knockdown. Only one of the assayed proteins was responsive: CREB2, a transcriptional repressor, known to be a major inhibitory constraint on LTF, was upregulated in response to Piwi knockdown. Interestingly, an even greater increase in CREB2 mRNA was observed.
The fact that Piwi knockdown led to an increase in CREB2 mRNA, and it’s nuclear localisation, suggested that rather than acting post-transcriptionally (ie by degrading mRNAs as in Drosophila), Piwi/piRNA complexes appeared to be inhibiting CREB2 gene expression at the DNA level. It is known that in mice Piwi/piRNA complexes act to silence transcription by facilitating DNA methylation. Rajasethupathy et al therefore asked whether CREB2 regulation by Piwi occurred via DNA methylation.
The enzyme responsible for methylation of cytosine residues in CG dinucleotides, DNA methyltransferase (DNMT), was known to be expressed in the Aplysia CNS. Inhibition of DNMT activity (using a pharmacological reagent) led to a strong increase in the level of CREB2. In the normal LTF experiments, CREB2 levels are reduced 12 hours after exposure to 5HT and remain low until 48hrs. This downregulation of CREB2 was abolished when DNMT activity was inhibited, as was 5HT-dependent LTF. This led the researchers to search for CpG islands in the promoter region of CREB2 that could be sites for DNA methylation mediated transcriptional control. Indeed, they identified a CpG island in the CREB2 promoter region that normally exists in both methylated and unmethylated states. After 5HT exposure, this CpG island is almost entirely methylated, whilst in the presence of the DNMT inhibitor it becomes almost entirely unmethylated. This 5HT-dependent methylation of the CREB2 CpG island requires Piwi, as it was abolished when Piwi was inhibited. The authors then went on to search for candidate piRNAs that could be responsible for mediating this effect, by searching for those with complementarity to the CREB2 promoter. They identified 4, one of which, aca-piR-F, when knocked down caused an increse in CREB2 expression. Notably, Rajasethupathy et al. did not demonstrate the expected result that aca-piR-F knockdown would lead to demethylation of the CREB2 CpG island, although this experiment was surely attempted.
In conclusion, this paper offers a broad outline for a mechanism of memory encoding; it connects neurotransmitter synaptic stimulation with the stable epigenetic marking of the transcription state of an important regulator of neuronal plasticity, via the action of Piwi/piRNA complexes. It should be noted that ‘epigenetic’ is used in this context in a loose definition with reference only to a stable marking of cellular state. Strictly ‘epigenetic’ should refer to heritable non-genetic changes, but as neurons do not divide that is inapplicable. In this case DNA methylation is a relatively long-lasting mark. However, for instance the change in CREB2 expression with respect to 5HT-stimulated long term facilitation only lasts a couple of days – does this correspond to the level of methylation in the promoter? In which case, one gets an impression that DNA methylation and demethylation are highly dynamic processes in the Aplysia CNS. Currently there are three modes proposed to resolve the difficult question of how memories can persist for a long time, whilst the cellular components that must mediate them have a high rate of turnover: Prion-like synaptic marks, autoregulatory loops that can maintain a cell state whilst their components come and go, and epigenetic mechanisms that can alter gene expression in a long term manner. This paper shows a clear example of the latter mode, but the apparent dynamism of DNA methylation in this system suggests a lack of permanence.
Although I like the way this paper has ranged over a large terrain and connected so many disparate elements, by necessity it raises many questions and leaves many aspects of the work unmentioned. I’ve already mentioned some questions about Aplysia piRNAs; no doubt a fully annotated Aplysia genome will answer some of them. A few other questions and future directions spring to mind: The authors haven’t quite shown that DNA methylation is responsible for the transcriptional silencing at the CREB2 promoter, only correlated it. Likewise the mode by which Piwi/piRNA complexes act to promote DNA methylation is unclear. A wider question is the nature of DNA methylation in Aplysia and other invertebrates. Some invertebrates show virtually no DNA methylation (C. elegans, Drosophila) whilst the majority display mosaic patterns quite different from those found in vertebrates. This suggests functional differences, and without deeper knowledge of the role of DNA methylation in Aplysia it is difficult to guess how widely applicable these findings are in other systems. Likewise the finding that piRNAs are acting at the level of DNA methylation, previously only found in mammals, raises questions about the state of affairs in other invertebrate model systems. Also, do Aplysia piRNAs only act on DNA methylation, or post-transcriptionally aswell.? Future studies will no doubt also look at how this type of regulation corresponds to histone marks, and try to synthesise the different levels of regulation. Perhaps the most important take home message is that piRNAs are more than a germline specific immunity against tranposons. Just how widespread these other roles are is an open question.
Rajasethupathy, P., Antonov, I., Sheridan, R., Frey, S., Sander, C., Tuschl, T., & Kandel, E. (2012). A Role for Neuronal piRNAs in the Epigenetic Control of Memory-Related Synaptic Plasticity Cell, 149 (3), 693-707 DOI: 10.1016/j.cell.2012.02.057