Extensive research on transcriptional mechanisms recruited during memory consolidation has established that both activation and silencing of gene expression are required for LTM formation. However, majority of these studies have primarily focused on cellular and molecular mechanisms for transcriptional gene activation while gene silencing mechanisms have been largely ignored in this context. Here, we identify an essential role for the G9a/GLP epigenetic suppressor complex in the regulation of transcriptional gene silencing in the hippocampus and in the EC during memory consolidation. We performed functional studies that establish that G9a/GLP activity blockade in the hippocampus impairs LTM formation and L-LTP at the SC-CA1 synapses. In contrast, G9a/GLP blockade in the EC resulted in enhanced LTM and normal L-LTP at the TA-CA1 synapses. In addition, molecular experiments demonstrate that G9a/GLP activity in the EC mediates H3K9me2-dependent repression of specific gene transcripts in both the EC and the hippocampus during memory consolidation. Taken together, these data implicate the G9a/GLP dimethyltransferase suppressor complex in the coordinated activation and repression of gene transcripts in the EC and the hippocampus that are necessary for the process of LTM formation.
Initial investigations into epigenetic mechanisms such as DNA methylation and histone acetylation in the adult brain have revealed that theses molecular processes are dynamic and transiently activated in the hippocampus, amygdala, and medial prefrontal cortex following fear conditioning (Levenson et al., 2004
; Lubin et al., 2008
; Guan et al., 2009
; Koshibu et al., 2009
; Gupta et al., 2010
; Monsey et al., 2011
). However, these pioneering studies still raise the question as to whether these dynamic epigenetic markings or other chromatin modifications in the adult brain can truly serve as candidate mechanisms involved in the encoding of long-lasting stable memories. Our present findings addresses this question and we found that although the transcription repressive mark H3K9me2 was transiently regulated in the EC, the transcription active mark H3K4me3 showed long-lasting alterations in the EC long after fear learning. These observations are in contrast to prior findings wherein the H3K9me2 repressive epigenetic mark in the hippocampus was found to be persistently altered following fear learning (Gupta et al., 2010
). Thus, these results are consistent with the idea of regulation of histone methylation in memory, and further suggest that HKM is an upstream regulator of chromatin structure in the adult CNS that serves to coordinate both dynamic and persistent gene expression changes in several memory-related brain regions that are critical for LTM formation and storage.
We also observed that inhibition of G9a/GLP in the EC accelerated the rate of memory extinction and that G9a/GLP suppressor activity in the EC, but not the hippocampus, affected amygdala-dependent fear learning. Together, these findings support the role of the EC in LTM extinction and amygdala-dependent fear memory formation (Phillips and LeDoux, 1992
). These results support manipulation of the G9a/GLP dimethyltransferases as a promising therapeutic avenue for the treatment of fear-related memory disorders in patients suffering from traumatic memories.
Because posttranslational histone modifications can occur in combination with each other in a cell, we examined whether G9a/GLP inhibition in the EC altered H3K9me2 regulation as well as other histone modifications in the EC and the hippocampus in response to fear learning. Analysis of G9a/GLP inhibition in the EC resulted in the down-regulation of the normally observed increases in H3K9me2 levels while further elevating H3K4me3 and H3K9ac levels in the EC during memory consolidation. Moreover, in the hippocampus, G9a/GLP blockade in the EC further increased H3K9me2 and H3K4me3 regulation. These changes in G9a/GLP-mediated histone modifications were reflected at memory-related gene promoters like Zif268, DNMT3a,
and BDNF exon IV
that correlated with altered mRNA expression within the EC and in the hippocampus. Thus, our findings are consistent with prior research demonstrating a positive correlation between histone acetylation and H3K4me3 levels (Zhang et al., 2004
) and a negative correlation with H3K9me2 levels (Gupta et al., 2010
; Warrener et al., 2010
) or G9a genetic knockdown (Plazas-Mayorca et al., 2010
). These results underscores the concept that in the CNS, histone modifications do not occur in isolation but rather their combinatorial effects mediate the transcriptional signature of genes within brain regions as well as across brain regions that are necessary for LTM formation. Furthermore, these histone modifications appear to be controlled by G9a/GLP activity in the EC.
This study also suggest that these G9a/GLP-induced histone modifications may not all be required for the transcription of some genes like cFOS
since, in BIX01294-treated rats, only H3K4me3 and H3K9ac correlate with increased gene expression. Indeed, an unexpected result from our study was that despite the presence of the repressive H3K9me2 methylation mark, cFOS
mRNA levels further increased in the EC with G9a/GLP blockade. Upon close examination of the cFOS
primers used in our experiments, BLAST genome analysis revealed that our cFOS
primer sets amplified exon 4 of the cFOS
gene and are in good agreement with prior studies suggesting that H3K9me2 enriched at gene coding regions may serve to promote active gene transcription contrary to its transcriptionally repressive role observed when present at gene promoters (Vakoc et al., 2005
). Together, these results provide insights for the role of intragenic methylation regulation specifically in the form of G9a/GLP-mediated H3K9me2 activity occurring in the EC during memory consolidation.
Cellular communication between the EC and the hippocampus are critical for relaying sensory information during memory consolidation as evidenced by synaptic plasticity studies (reviewed in Lubin et al., 2011
). However, communication in the form of cellular and molecular events exchanged between the EC and the hippocampus at the epigenetic transcriptional level has not been addressed until this study. A novel finding in our study was that G9a/GLP activity in the EC enhanced H3K9me2 levels at the promoter of the non-permissive COMT
gene and decreased COMT
mRNA levels in the hippocampus during memory consolidation. Intriguingly, COMT
is instrumental for synaptic catabolism of dopamine and a decrease in COMT
activity would result in increased synaptic dopamine levels, which is necessary for LTM (Barnett et al., 2009
Overall, these studies are exciting as they suggest for the first time that HKM may mediate cellular connectivity between brain regions (i.e. entorhinal cortex and hippocampus) during memory consolidation. Indeed, Ramon Y Cajal observed massive connections between the EC and the hippocampus, leading to the now widely accepted concept of parallel input/output cellular connectivity shared between these two brain regions (Canto et al., 2008
; Ahmed and Mehta, 2009
). However, the molecular mechanisms necessary for cellular communication between the EC and hippocampus are uncertain. In this regard, our study indicates for the first time a potential role for G9a/GLP mediated HKM regulation in the CNS that may underlie EC-hippocampus cellular connectivity in the adult nervous during LTM formation. In support of this idea, we show that G9a/GLP activity in the EC differentially regulates H3K9me2 within both the EC and the hippocampus to ultimately enhance LTM formation. Hence, this studysuggests a fascinating epigenetic mechanism mediating parallel reciprocal molecular connectivity between the EC and the hippocampus during memory consolidation, which we will further investigate in future studies.
Investigation of the role of G9a/GLP activity in mediating molecular connectivity between the EC and the hippocampus prompted additional studies at the cellular level. L-LTP is hypothesized to be the cellular correlate of LTM formation (reviewed in Lubin et al., 2011
). Additionally, cortical contributions like the EC to the hippocampus via the TA pathway, have been shown to influence hippocampal output and synaptic plasticity by directly affecting SC-CA1 spike probability (Remondes and Schuman, 2002
). Thus at the cellular level, we found that G9a/GLP inhibition resulted in normal L-LTP at the TA-CA1 synapse. These results can be interpreted in one of two ways. One possible interpretation is that G9a/GLP activity is not critical to the regulation of synaptic plasticity at the TA-CA1 synapse, and instead may function at other cortical-hippocampal cellular connections such as the perforant pathway. Another possible interpretation is that the L-LTP-induction protocol employed in these experiments resulted in maximal activation of the TA-CA1 synapse causing a ceiling-effect, and hence could not be further potentiated in the presence of G9a/GLP activity blockade. Regardless of which interpretation is correct, this study provides a plausible mechanism underlying the enhancement in LTM formation observed with inhibition of G9a/GLP activity in the EC during memory consolidation.
In contrast to TA-CA1 synaptic plasticity, G9a/GLP inhibition attenuated L-LTP at the SC-CA1 synapse, which is consistent with our behavioral findings of an attenuation of LTM formation with G9a/GLP inhibition in area CA1 of the hippocampus. The L-LTP studies performed at the TA-CA1 and the SC-CA1 synapses furthers our understanding of the differential role of HKM-dependent chromatin restructuring on synaptic plasticity in brain regions. Furthermore, these studies provide insights into whether HKM activity in the EC and the hippocampus during fear memory consolidation are a consequence or cause of synaptic plasticity underlying the process of LTM formation.
Finally, our results summarized in suggest that more than absolute values, it is the balance between G9a/GLP-mediated transcriptional gene activation and silencing in the EC that is critical for normal LTM formation. Moreover, we provide mechanistic insights into how G9a/GLP activity in the EC mediates epigenetic and transcriptional plasticity within both the EC and hippocampus during LTM formation. Our studies also suggest that G9a/GLP activity is critical for synaptic plasticity occurring within the hippocampus. In conclusion, our study is the first to implicate G9a/GLP epigenetic suppressor activity occurring across multiple brain regions recruited during the formation of a memory trace.
Model for G9a/GLP transcriptional activity in the hippocampus and EC during memory consolidation