To elucidate the influence of 5-hmC on epigenomic dynamics in mammalian brain, we generated detailed epigenomic maps of 5-hmC expression in mouse cerebellum and hippocampus during postnatal neurodevelopment and aging. We observed a marked depletion of 5-hmC on the X chromosome and identified both stable and dynamic DhMRs during postnatal neurodevelopment and aging. In addition to the association of 5-hmC with the bodies of developmentally activated genes, we also observed developmentally regulated dynamics of 5-hmC at repetitive loci, including acquisition of 5-hmC in SINEs and LTRs during neurodevelopment. Genome-wide analyses of 5-hmC distributions in human cerebella further revealed conserved characteristics of 5-hmC in mammals. Finally, we found that the overall abundance of 5-hmC was negatively correlated with MeCP2 dosage. Notably, the loss of Mecp2 led to the specific reduction of 5-hmC signals at dynamic DhMRs. Together, these data suggest that 5-hmC is important during postnatal neurodevelopment and aging, as well as in human neurological disorders.
We found that 5-hmC markedly increased from the early postnatal stage to adulthood, suggesting a strong correlation between 5-hmC and neurodevelopment. Tet proteins (Tet1, Tet2 and Tet3) are known to oxidize 5-mC to 5-hmC2,3
. Notably, using microarray analyses, we found no significant increase in Tet family gene expression during neurodevelopment (P
> 0.05; Supplementary Fig. 12
), suggesting that the changes in genomic 5-hmC cannot be explained simply by changes in Tet expression. This indicates that, if Tet family proteins are responsible for 5-mC hydroxylation in brain, Tet activity might be modified post-transcriptionally or through additional co-factors.
Among all of the samples that we tested, analyses of DhMRs indicated that they most frequently associated with genes bodies, becoming enriched in developmentally activated genes during neurodevelopment. Gene ontology analyses on the set genes that both acquired 5-hmC and were activated in cerebellum from P7 to 6 weeks confirmed substantial enrichment of genes involved in cerebellum development and various neuronal functions (Supplementary Data Set 9
), consistent with 5-hmC having a role in neurodevelopment. Furthermore, assessment of 5-mC levels at such genes demonstrated that a substantial fraction also displayed reduced 5-mC from P7 to 6 weeks, consistent with the notion that conversion of 5-mC to 5-hmC may serve to offset the repressive effects of 5-mC. However, we also found that some such genes did not show substantial differences in 5-mC levels as measured by MeDIP. Given that dynamically regulated 5-hmC loci tended to be depleted in CpG dinucleotides (Supplementary Figs. 6d and 8b
), these data may indicate that non-CpG 5-hmC is involved in gene activation. Notably, a recent report found strand-specific non-CpG DNA methylation at 5-hmC–enriched loci in mouse ES cells, and strand-specific non-CpG methylation also correlates positively with gene expression in human ES cells9
. Together, these data could suggest the involvement of intragenic non-CpG 5-hmC in gene activation in the CNS, although further studies resolving 5-mC and 5-hmC at single base resolution will be required to fully resolve the roles of each.
Intragenically, 5-hmC associates with CGIs, but is much less frequent at CGIs associated with TSSs. Previous observations have suggested that TSS-associated 5-mC hydroxylation, via Tet family proteins, is involved in both maintaining gene expression of pluripotent associated genes and poising expression of developmentally regulated genes in ES cells2,5,12,30,40
. In ES cells, intragenic 5-hmC tends to be highest in moderately expressed genes11,12,31
. Our data suggest a more substantial enrichment of 5-hmC in gene bodies in brain relative to ES cells. In the context of current genome-wide 5-hmC datasets9-12
, the comprehensive 5-hmC maps that we generated suggest distinct regulation of 5-hmC in brain as compared with ES cells, and highlight the potentially diverse mechanisms regulating 5-hmC at TSSs and gene bodies.
The depletion of 5-hmC specifically on X chromosome in both males and females was surprising. In females, depletion of 5-hmC on the X chromosome may be expected from previous reports that found less DNA methylation on the inactive X chromosome41
. However, depletion in males is rather unexpected, as all cells must harbor an active X chromosome. These results may suggest that the maintenance of an active X chromosome, in both males and females, is generally associated with lower 5-hmC levels. Mechanistically, it is possible that the X chromosome is subjected to unique epigenetic dynamics or signature sequences that would prevent conversion of 5-mC to 5-hmC or, perhaps equally likely, promote the further modification of 5-hmC to unmethylated cytosine. Whether this phenomenon functions in cis
or requires in trans
factor(s) would need further investigation.
Our data suggest that, although 5-hmC is maintained at many specified loci, it could also be dynamically regulated during neurodevelopment and aging. It has been proposed that hydroxylation of 5-mC serves either as an intermediate to 5-mC demethylation or as a stable epigenetic modification to DNA that is distinct from 5-mC, perhaps depending on its relative stability across the genome. Our data suggest that 5-hmC can be stable across three distinct ages in two mouse brain regions: cerebellum and hippocampus. Consistent with this, we identified sets of 5-hmC–regulated loci that clearly maintained 5-hmC from 6 weeks to 1 year of age in each region tested. However, more dynamically regulated loci were also present in both cerebellum and hippocampus. Clearly, the refinement of genome-scale 5-hmC maps in specific subtypes of neuronal cells will be important in the future to give us a full understanding of the influence of 5-hmC on DNA methylation dynamics in the CNS.
Transposable elements make up a substantial portion of most eukaryotic genomes and could affect genome diversity and human disease either by insertional mutagenesis or by contributing recombination substrates, both during and long after their integration42
. Recent work suggests transposable elements may upregulate the expression of host genes and function as part of genome-wide regulatory networks43
. Somatic retrotransposition of L1-type LINEs has been seen in the human brain and may influence neurogenesis and/or affect neuronal function39
. In addition, epigenetic regulatory mechanisms have been directly linked to the regulation of transposon mobilization. Our analyses revealed that 5-hmC was also enriched in transposable elements, particularly SINE- and LTR-type repeats in mammalian brain. The enrichment of 5-hmC was dynamically regulated during neurodevelopment and aging in mouse cerebellum. At this time, however, whether 5-hmC–mediated epigenetic regulation is involved in transposon mobilization requires further investigation.
We also examined genomic 5-hmC distributions in the context of human neurological disorders. We focused on Rett syndrome, which is caused by de novo
mutations in the MECP2
. Mecp2 deficiency results in global changes in neuronal chromatin structure, and Mecp2 can dampen transcriptional noise genome-wide in a DNA methylation–dependent manner19
. Mecp2 has also been implicated in neuronal activity–dependent gene regulation44,45
. These observations indicate that Mecp2, through its affinity for 5-mC, could serve to temper transcription by modulating the conversion of 5-mC to 5-hmC. Our results indicate that the overall abundance of 5-hmC in cerebellum is negatively correlated with Mecp2 dosage and that the Mecp2-MBD may directly inhibit Tet1-CD–mediated hydroxylation of 5-mC. Consistent with this, our sequencing analyses suggest that, overall, both intragenic and transposable element–associated 5-hmC is increased in the absence of Mecp2. However, the loss of Mecp2 led to a 39% reduction of 5-hmC specifically at the dynamic, but not stable, DhMRs that we identified. This unexpected paradox might be explained by different modes of action of Mecp2 at epigenetically stable and dynamic regions. Globally, Mecp2 could dampen transcriptional noise by maintaining 5-mC and preventing conversion to 5-hmC, explaining the negative correlation between Mecp2 dosage and overall 5-hmC. Indeed, Mecp2 is expressed at near histone octamer levels and is bound genome-wide in cerebellum neurons19
. However, redundancy exists in MBDs and may function to impart robustness at a subset of loci. At epigenetically stable regions, such as those maintaining 5-hmC from 6 weeks to 1 year, redundant MBD-associated mechanisms may maintain the equilibrium between 5-mC and 5-hmC in the absence of Mecp2. However, at more dynamically modified loci, such as those that acquire 5-hmC between P7 and 6 weeks, but subsequently lose the mark at 1 year, there may be a less redundant mechanism. At such loci, Mecp2 may be the predominant MBD, and the loss of Mecp2 would lead to a change in the equilibrium between 5-mC and 5-hmC.
An intriguing possibility raised by such a Mecp2-predominant mechanism would be its involvement at developmentally activated or neuronal activity–dependent loci. On stimulation, Mecp2 may disassociate, allowing specific transcription factor(s) to activate gene expression, which could involve recruitment of Tet proteins to hydroxylate 5-mC. In the absence of Mecp2, this dynamic would be compromised, leading to a reduction in 5-hmC. Notably, such a mechanism could explain transcriptional activating affects observed at a subset of genes in MeCP2 transgenic animals. More recently, in addition to its role during neurodevelopment, MeCP2 has been shown to be required for adult neurological function46
. It is possible that these dynamic loci could be important in these processes as well; however, it remains to be determined whether the loss of MeCP2 in adulthood could alter 5-hmC modification at these loci.
To summarize, we generated detailed genome-wide maps of 5-hmC in mouse cerebellum and hippocampus during postnatal development and aging and characterized dynamic regulation of 5-hmC during neurodevelopment and aging, pointing to critical role(s) for 5-hmC in neurodevelopment. Our results could lay the groundwork for further exploration into the influence of 5-hmC on epigenomic dynamics in normal brain function and human diseases.