In the studies presented here, we investigated the potential role of histone lysine methylation in memory formation and established several findings. First, we found that fear conditioning triggers changes in H3K4 tri-methylation (a transcriptional active marker) and H3K9 di-methylation (a transcriptional repressive marker) in area CA1 of the hippocampus. Second, we observed that H3K4-specific methyltransferase (Mll) deficient mice have a deficit in long-term memory formation. Third, treatment of animals with the HDAC inhibitor NaB altered histone methylation levels in hippocampus during memory consolidation, suggesting that altered histone methylation is coupled to HDAC inhibition. Finally, H3K4 tri-methylation significantly increased at the promoter of two activated gene targets (Zif268 and bdnf) during memory consolidation. Together, these findings support the role of histone methylation changes in the consolidation of long-term memory formation.
Covalent post-translational modifications of histones serve as an important mechanism for transcriptional regulation during consolidation of long-term memories. For example, increases in histone H3 phosphorylation and acetylation in hippocampus have been shown to be regulated during long-term memory formation (Levenson et al., 2004a
; Chwang et al., 2006
; Fischer et al., 2007
). Additionally, regulation of histone modifications including histone methylation have been implicated in mental disorders including schizophrenia (Deutsch et al., 2008
; Akbarian and Huang, 2009
). However, investigation of the role of histone methylation in the process of long-term memory formation had not been explored prior to the current studies. Encouragingly, studies on abnormal gene expression in postmortem brain have revealed that histone methylation may be a viable avenue for early detection for some cases of schizophrenia (Akbarian and Huang, 2009
). Therefore, the study of histone methylation in the regulation of memory formation is intriguing and will provide further insights into the epigenetic mechanisms that may be dysregulated in cognitive impairments.
We investigated the contribution of hippocampal histone methylation in the consolidation of long-term memory in a rodent model of fear conditioning. We were particularly interested in studying this form of histone modification because it is unique in that various specific sites of methylation of histones can have opposite roles in gene regulation; that is, methylation of H3K4 is associated with active transcription whereas methylation of H3K9 is associated with transcriptional silencing (Strahl et al., 1999
; Lachner and Jenuwein, 2002
; Schotta et al., 2002
). Indeed, the process of storing stable long-term memories is likely to involve coordinated changes in both transcriptional activation and silencing of genes (Ressler et al., 2002
; Levenson et al., 2004b
). Now with the results presented here, we provide the first evidence for histone methylation changes in hippocampus in response to contextual learning, in both episodic learning and associative contextual fear conditioning. Furthermore, these results support the hypothesis that histone methylation plays a role in the regulation of gene expression changes that are permissive for memory formation.
One fascinating finding from our studies is that HDAC inhibition significantly altered histone methylation levels. Specifically, treatment with the non-selective class I HDAC inhibitor NaB significantly attenuated H3K9 di-methylation in hippocampus after fear conditioning. This suggests the new idea that a possible mechanism for the permissive actions of inhibiting HDACs, resulting in memory enhancement, is through the negative regulation of hippocampal H3K9 di-methylation. However, the specific HDAC isoform (s) inhibited by the HDAC inhibitor NaB to mediate this effect on H3K9 di-methylation is unknown and remains an intriguing focus of ongoing studies. Promisingly, the HDAC2 isoform has recently been identified as a modulator of dendritic spine density, synapse number, and synaptic plasticity which negatively regulates memory formation (Guan et al., 2009
). Although very speculative at this point, an intriguing hypothesis is that inhibition of HDAC2 negatively regulates H3K9 di-methylation to allow memory enhancement. Whether this scenario is true or not, our present study is consistent with the idea that inhibition of HDAC isoforms promotes active gene transcription for stable formation of enhanced long-term memories.
Another remarkable finding from these studies is that changes in H3K4 tri-methylation were associated with increased DNA methylation at the Zif268
promoter region, while Zif268
gene expression was active during memory consolidation. In addition, these changes in Zif268
DNA methylation were transient returning to baseline levels 24 h later long after the consolidation period. These observations are in sharp contrast to findings from developmental studies that suggest that DNA methylation is primarily associated with the repression of gene transcription. However, a recent study by Chahrour et al. (2008)
indicates that methyl-CpG binding proteins, such as MeCP2, bind to DNA methylation sites with the transcriptional activator cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein 1 to actively regulate gene transcription. Indeed, our results suggest that MeCP2 levels increased at the Zif268
promoter region in association with increased DNA methylation resulting in transcriptional activation of the Zif268
gene following fear conditioning. Correspondingly, we found a cAMP response element (CRE; TCACGTCA) binding site for the transcription factor CREB downstream of the CpG island 1 region and upstream from the transcriptional start site of the Zif268
gene (). Thus, based on previous studies by Chahrour et al. (2008)
and our present data, we propose that dynamic changes in DNA methylation may serve as both a repressive or active marker of transcription in hippocampus during long-term memory formation. This new concept raises several intriguing possibilities for future research. For example, in our study we demonstrated that contextual learning induces global changes in histone methylation as well as altered DNA methylation. However, it remains to be determined what specific cell-types are involved in these observed epigenetic mechanisms.
In summary, our results continue to support a role for epigenetic mechanisms in the process of stable formation of long-term memories. Specifically, our findings demonstrate considerable histone methylation changes in hippocampus in association with memory formation and furthermore suggest that this process is necessary for long-term memory formation. In addition our results provide the first evidence for an association between differential histone methylation and DNA methylation regulation at the Zif268 gene promoter in the adult brain and implicate a possible role for these processes in the formation of long-term memories. Finally, as epigenetic mechanisms continue to be linked to cognitive dysfunctions a better understanding of the complex molecular interaction and regulation of these processes will need to be further refined.
The importance of continued investigation of such mechanisms is underscored by several prior studies. For example, similar to DNA methylation, histone methylation was once considered to be a non-plastic process. The concept of the irreversibility of histone methylation arose from the belief that histone demethylases did not exist. However, this view has recently been challenged with the identification of several histone H3 demethylases including LSD1 and JHDM1 (Shi et al., 2004
; Tsukada et al., 2006
; Whetstine et al., 2006
; Tahiliani et al., 2007
) which can specifically demethylate lysine-4 within histone H3. Thus, the discovery of these enzymes may lead to more selective therapeutic interventions that include the use of histone demethylase activators or inhibitors as well as HDAC inhibitors for treatment of cognitive impairments associated with neurological disorders.