Characterizing 5-hydroxymethylcytosine in human prefrontal cortex at single base resolution
© Gross et al. 2015
Received: 15 July 2015
Accepted: 24 August 2015
Published: 3 September 2015
The recent discovery that methylated cytosines are converted to 5-hydroxymethylated cytosines (5hmC) by the family of ten-eleven translocation enzymes has sparked significant interest on the genomic location, the abundance in different tissues, the putative functions, and the stability of this epigenetic mark. 5hmC plays a key role in the brain, where it is particularly abundant and dynamic during development.
Here, we comprehensively characterize 5hmC in the prefrontal cortices of 24 subjects. We show that, although there is inter-individual variability in 5hmC content among unrelated individuals, approximately 8 % of all CpGs on autosomal chromosomes contain 5hmC, while sex chromosomes contain far less. Our data also provide evidence suggesting that 5hmC has transcriptional regulatory properties, as the density of 5hmC was highest in enhancer regions and within exons. Furthermore, we link increased 5hmC density to histone modification binding sites, to the gene bodies of actively transcribed genes, and to exon-intron boundaries. Finally, we provide several genomic regions of interest that contain gender-specific 5hmC.
Collectively, these results present an important reference for the growing number of studies that are interested in the investigation of the role of 5hmC in brain and mental disorders.
Keywords5-hydroxymethylcytosine Prefrontal cortex Human Epigenetics Brain
In mammalian cells, 5-methylcytosine (5mC) has been the most widely studied epigenetic mark, and has long been regarded as a stable DNA modification. Recently, however, research has shown that 5mC can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) family of proteins [1–3]. 5hmC has been shown to be most abundant in brain tissue [4–6] and to influence transcriptional regulatory activity [7–10].
Although its role is not yet completely understood, 5hmC has been increasingly investigated in neuropsychiatric phenotypes, likely due to its involvement in neuronal development and enrichment in brain tissue [11, 12]. In humans, global levels of 5hmC have been shown to be reduced in Alzheimer’s disease , while site-specific differences have been demonstrated in autism spectrum disorder , Huntington’s disease , and psychosis . In mouse models, genome-wide alterations have also been observed in Huntington’s disease , as well as in fragile X-associated tremor/ataxia syndrome . Taken together, these observations suggest a significant role of 5hmC in the etiology of neuropsychiatric diseases.
To date, however, investigations of 5hmC in the human brain have either been in very small samples or have utilized low-throughput techniques. Here, we use a sample with good power and sequencing resolution to provide insight into genomic characteristics of 5hmC in the brain. Although we observe sizable variability of 5hmC between subjects, lending to the hypothesis of a dynamic cytosine-modifying pathway, we also demonstrate a strong association between stable 5hmC and epigenetic properties. In its entirety, the descriptive data presented here provide a map of 5hmC at single base resolution, which is of interest to future studies of 5hmC in the cortex of the human brain.
Genome-wide inter-individual differences of 5hmC
A major focus of this study was to determine whether 5hmC sites were present at similar genomic locations in brain tissue from different individuals. We first investigated what proportion of all 5hmC sites were common to multiple individuals. We identified a total of 17,368,538 unique 5hmC sites across 19 individuals, of which 81.3 % were also identified in a previous study examining 5hmC content in the prefrontal cortex using Tet-assisted bisulfite sequencing (TAB-Seq) . We found that 41.7 % of all 5hmC sites observed were found in at least 5 individuals, whereas 12.2 % of all 5hmC sites were common to at least 10 individuals, and only 1.3 % were common to at least 15 individuals (Additional file 2). As a point of comparison, we investigated the proportion of 5mC sites common to multiple individuals by whole genome bisulfite sequencing (WGBS). 5mC showed considerable stability across the 19 subjects investigated when considering 5, 10, or 15 individuals, however we observed an abrupt decrease in the percentage of sites common to all 19 subjects (Additional file 3). Although these data indicated that 5hmC is considerably more variable than 5mC, they also suggested a relative degree of stability of 5hmC sites in brain tissue from different individuals.
We next assessed the probability of detecting similar 5hmC sites between any two individuals. To do so, we selected two representative samples with respect to sequencing depth and number of unique 5hmC sites. We subsequently calculated the hypergeometric probability of detecting the same 5hmCs in sequencing reads across independent samples. First, we estimated the total number of 5hmC sites in the human brain genome to be around 27 million, based on previously published data . We observed 6,793,582 and 3,818,749 total 5hmC sites in each of the representative samples considered in this analysis, of which 1,999,493 5hmC sites were common to both subjects (P < 3.34e−178), suggesting that the pattern of common 5hmC sites observed between samples was not random.
Chromsomal distribution of 5hmC in human prefrontal cortex
The total number of 5hmC sites analyzed in the intermediate stringency model was 2,121,951, which was slightly lower than the average number of 5hmC sites observed in any single individual (4,277,308 ± 319,846). Furthermore, 94.75 % of sites in the intermediate stringency were also present in a recent TAB-Seq study of the prefrontal cortex of one individual human female . For these reasons, we focused further analyses on the intermediate stringency model.
5hmC densities across chromosomal features
The density of 5hmC across chromosomal features is of interest as it may provide insight into its potential functional properties. Genomic features were defined based on GRCh37/hg19 genomic annotations downloaded from the UCSC browser and Ensembl release 75. Our data showed an increase in 5hmC density in both poised and active enhancers, as well as in exons (Fig. 3b). The increase in 5hmC in these regions has been previously observed in human embryonic stem cells [20, 21] and both fetal and adult mouse brain . In contrast, 5hmC density appeared to be relatively depleted in transcription start sites (TSS), CpG shores, and, most strikingly, CpG islands and both long and short interspersed elements (LINEs and SINEs, respectively).
Potential functional properties of 5hmC
The observed increase in 5hmC density in enhancer regions of the genome led to the hypothesis that 5hmC may also be associated with histone marks on an individual basis. Using data from chromatin maps in the frontal lobe , we calculated the mean 5hmC levels in contiguous 100 bp bins flanking the proposed histone modification binding site. Consistent with the results showing increased density of 5hmC in poised enhancer regions (Fig. 3b), we also observed an increase in 5hmC density at the H3K4me1 ChIP-Seq peak (Fig. 3c). In addition, opposite results were seen at both the H3K4me3 and H3K27ac ChIP-Seq peaks, as the density of 5hmC decreased at the midpoint for both histone marks (Fig. 3c).
Along with the putative function of 5hmC in regulating gene transcription, two previous reports have also suggested an association between 5hmC and exon-intron boundaries [9, 24]. We looked at 5hmC content across 20 bp flanking the 5’ and 3’ ends of 144,157 internal exons spanning 20,745 genes. We observed patterns of increased 5hmC at exon-intron boundaries, specifically at the 5’ splice site and four bases downstream (Fig. 4b). This spike in 5hmC was not strand-specific, as similar results were obtained on both the sense and anti-sense strands (Fig. 4c). The present results may be indicative of a potential splicing mechanism, perhaps through the association of 5hmC and the CCCTC-binding factor (CTCF). Not only is CTCF a transcription factor that has been linked to alternative splicing by regulating the activity of RNA polymerase II , but it has also been shown to interact with 5hmC and TET enzymes in both mouse and human embryonic stem cells [21, 26, 27]. Taken together, these results provide an interesting avenue for further research into alternative splicing mechanisms.
Brain 5hmC clusters are predominant in genes associated with neurodevelopment
GO terms related to neurological, epigenetic, and developmental processes from cluster analysis in intermediate stringency
Neurological system process
Generation of neurons
Cell morphogenesis involved in neuron differentiation
Neuron projection morphogenesis
Glial cell differentiation
Regulation of neuron differentiation
Regulation of neurogenesis
Protein amino acid methylation
Lysine N-methyltransferase activity
Protein-lysine N-methyltransferase activity
Histone-lysine N-methyltransferase activity
Histone H2A acetylation
Sensory perception of smell
Sensory perception of chemical stimulus
Anatomical structure development
Multicellular organismal development
Nervous system development
Regulation of developmental process
Positive regulation of developmental process
Central nervous system development
Neuron projection development
Sensory organ development
Regulation of nervous system development
Regulation of neuron projection development
Sex differences in 5hmC in the human prefrontal cortex
GO terms related to sexual differentiation and development in 5hmC clusters unique to females
Anatomical structure morphogenesis
Anatomical structure development
Anatomical structure formation involved in morphogenesis
Regulation of anatomical structure morphogenesis
Using the publicly available RNA-Seq data , we investigated genes showing differential expression between genders (Additional file 8). This data showed the X-linked zinc finger protein (ZFX) gene on the X chromosome to be one of the most significantly differentially expressed genes in females (log2 FC = 0.653; FDR-corrected P < 0.0001). Of the 5hmC clusters unique to females, two are located within the ZFX intergenic region (Fig. 6b). ZFX, which has been shown to escape X-inactivation, is analogous to the Y-linked zinc finger protein (ZFY) on the Y chromosome, and both have been linked to sex determination [30–33]. Similar to AMH, none of the clusters, using all sites common in at least 10 males, were found in the ZFX gene.
Genes escaping X-inactivation show greater gene body 5hmC density
Following the results showing gender-specific differentially hydroxymethylated regions (DhmRs) in genes related to sexual anatomical development, we hypothesized that 5hmC may play a role in additional gender related developmental processes. 5mC plays an important role in X-inactivation, and previous literature showed that genes escaping X-inactivation have a decrease in promoter CG methylation and an increase in intragenic CH methylation . Previous studies have separated genes on a scale from 1 to 9, with 1 and 9 respectively indicating genes least or most likely to escape inactivation. Using all sites found in our female subjects, our results indicated that these same genes that escape X-inactivation also show a significant increase in gene body 5hmC compared to genes that remain inactivated (Fig. 6c). Furthermore, genes that are most likely to escape X-inactivation showed even greater intergenic 5hmC density. Although this is only an initial characterization, it provides an interesting avenue for future studies investigating the potential etiology of X-linked diseases or disorders.
Discussion and conclusions
In this study, we provide a deep characterization of the genomic locations of 5hmC in the human prefrontal cortex in a large sample. Using AbaSI-Seq, a high-throughput technique, we confirm previously published work using either low-throughput techniques or single samples. Furthermore, we extend the current knowledge of the role of 5hmC in the brain by linking stable 5hmC sites to enhancer regions and exon-intron junctions, both of which are involved in gene transcription. We also show the existence of regions of the genome that contain gender-specific 5hmC patterns, in addition to providing a putative mechanism for how certain genes escape X-inactivation. These data are of interest, as genomic mapping of 5hmC in the prefrontal cortex will likely be of reference for future studies investigating brain and mental disorders.
Inter-individual variability is common across many fields of study. This is especially true in epigenetics, where both the environment and the genetic landscape are contributing factors in establishing epigenetic features [34–37]. It is, therefore, not surprising to see varying degrees of 5hmC across the chromosomes and specific genomic features. These findings provide a basis for a dynamic DNA demethylation pathway. More importantly, our data show regions of the genome that are consistently hydroxymethylated in many individuals. These results confirm the stability of 5hmC in the human genome and provide a foundation for furthering our understanding of the epigenetic properties of 5hmC. Similarly, we show that, in order to increase our understanding of 5hmC, future research will have to be performed on large samples. Furthermore, increased stringency of bioinformatics analyses will reduce levels of random variability, thereby enhancing the power of studies to infer biological relevance from the 5hmC sequencing data.
In this study, we show that an intermediate level of stringency, defined as 5hmC sites present in at least half the total sample, was appropriate in discovering regions with a consistently high density of 5hmC across subjects. Our results provide reliable evidence that 5hmC is associated with gene regulatory features, such as enhancer regions and histone marks that associate with active gene transcription. Furthermore, we add to the available data in mammalian cells and tissues showing a clear correlation between gene body 5hmC and highly expressed genes. Finally, our cluster analyses show an increased density of 5hmC in genes associated with many developmental processes, specifically those related to neurogenesis and nervous system development. These results are of interest considering the many publications that have linked 5hmC to neurodevelopment, neurodegenerative, and neuropsychiatric phenotypes, and are consistent with the role of 5hmC and 5mC in tissue-specific gene regulation [7, 38], thus supporting the internal validity of our findings. Although the exact function of 5hmC remains unknown, our findings lend to the idea that 5hmC may act as an epigenetic mark by regulating gene transcription.
In addition to characterizing 5hmC in a large sample, we also provide evidence to suggest that 5hmC may be an important factor in anatomical development. Not only do GO analyses of unique DhmRs in females show clusters of 5hmC on genes associated with gender-specific properties and functions, but our results also indicate that genes escaping X-inactivation have increased intragenic 5hmC density. Early research into 5hmC and TET proteins showed specific properties of each based on developmental stages. For example, TET1 and TET2 are more highly expressed in primordial germ cells, while TET3 is primary found in zygotes [39, 40]. Similarly, levels of 5hmC tend to be high in zygotes, decrease rapidly during cell division, and are reestablished in the blastocyst . Interestingly, TET protein knockdown and knockout experiments show that TET proteins appear to have redundant and compensatory roles in establishing 5hmC patterns [40, 42, 43]. As such, although the exact function of 5hmC in development remains unclear, the presence of gender-specific DhmRs highlights the putative roles of 5hmC and TET proteins in embryonic and adult development.
Whether 5hmC is solely an intermediate molecule in the process of DNA demethylation or whether this 6th DNA base may have additional regulatory properties is a question that remains at the forefront of 5hmC research. Recent literature has shown that the oxidation of 5mC to 5hmC occurs on the parental strand after replication, and in the presence of a stable pool of 5hmC in multiple tissues from adult mice . Furthermore, several studies have shown the existence of 5hmC in RNA, from archaeal species to mammalian cells, that occurs through the TET-mediated oxidation of 5mC [45, 46]. Certainly, although 5hmC marks may continue through the oxidation pathway and ultimately lead to a demethylated cytosine, mounting evidence, including those presented here, all suggest that 5hmC does regulate gene transcription. Its mechanism of action, however, remains to be elucidated.
In addition to providing characterizations of 5hmC genomic location and function, we also confirm the efficacy of a high-throughput method to appropriately detect 5hmC sites at single base resolution. AbaSI-Seq, which has been used previously in embryonic stem cells, bases itself on a 5hmC-sensitive restriction enzyme digestion. This technique is valuable as it avoids the biases caused by antibody-based approaches  and does not cause DNA damage like other methods that employ bisulfite conversion . Furthermore, AbaSI has independently been shown to recognize both glucosylated and non-glucosylated 5hmC , thereby increasing its validity as an effective tool in studying 5hmC.
In summary, this manuscript provides a comprehensive assessment of 5hmC in human prefrontal cortex using a high-throughput technique. Moreover, we provide a reference for comparison of 5hmC in other mammalian species, and shed light on potential avenues of interest for further research in determining the functional relevance of 5hmC.
Post-mortem human brain tissue from the prefrontal cortex was obtained from the Douglas – Bell Canada Brain Bank. The prefrontal cortex was chosen given its diverse functions relative to human cognition and of importance to neuropsychiatric phenotypes. Additionally, the prefrontal cortex has been widely implicated in a variety of mental illnesses through dysregulation of epigenetic mechanisms [50–56]. Brain tissue used in this study was dissected at 4 °C, snap-frozen in liquid nitrogen, and stored at −80 °C following standard procedures. The Quebec Coroner’s office assessed the cause of death for each subject, and subsequently, we obtained information on the subjects’ mental health using psychological autopsies using the Structured Clinical Interviews for DSM-IV Axis 1 . In addition, brain tissue samples from all subjects were assessed for absence of pathological processes by a neuropathologist. Subjects died from either natural (n = 11) or accidental (n = 13) causes, and all were psychiatrically and neurologically healthy. All subjects were French-Canadian. Mean age was 43.33 ± 4.46 years. Written informed consent was obtained from next-of-kin for all subjects, and the Douglas Institute Research Ethics Board approved this study.
Genomic DNA was extracted from the prefrontal cortex using QIAGEN’s QIAamp DNA Mini Kit (QIAGEN, cat. # 51304). Concentrations of genomic DNA were assessed using the Thermo Scientific Nanodrop 1000 spectrophotometer and each sample had a 260/280 ratio greater than 1.8. AbaSI-Seq library construction was performed as described previously . Briefly, DNA was glucosylated by using UDP-glucose and T4-β-glucosyltransferase (New England Biolabs, cat. #M0357L). The DNA was then digested using AbaSI (New England Biolabs, cat. #R0665S) and custom biotinylated adaptors were ligated to DNA ends. The DNA was then sheared using the Covaris S220 Focused-Ultrasonicator (peak incident power = 140; duty % = 10; cycles per burst = 200; time = 120 s). Sheared DNA was captured using Dynabeads® MyOne™ Streptavidin C1 beads (Life Technologies, cat. # 65001). NEBNext™ End Repair Module (New England Biolabs, cat. #E6050L) and NEBNext™ dA-Tailing Module (New England Biolabs, cat. #E6053L) were used to generate blunt ended fragments and create an adenosine tail, respectively. A second set of custom adaptors were ligated to the DNA, followed by PCR amplification using NEBNext™ High-Fidelity 2x PCR Master Mix (New England Biolabs, cat. #E6013AA). Illumina TruSeq indices 1 through 12 were used to barcode samples. 50 cycle, single-read sequencing was performed on Illumina’s HiSeq 2000. A minimum of 60,000,000 sequencing reads was obtained per subject. Sequencing quality control metrics can be found in Additional file 1.
FASTQ reads were filtered based on a quality score greater than 30 and were then aligned to the human reference genome (UCSC Hg19) using Bowtie 2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), with the preset parameters of very-sensitive (−D 20 -R 3 -N 0 -L 20 -i S,1,0.50). Duplicates were removed using Picard (http://broadinstitute.github.io/picard/) and realignment was performed using the GATK alignment procedures from the Broad Institute (https://www.broadinstitute.org/gatk/). Subsequent filtering of aligned reads was based on a quality score of greater or equal to 10. 5hmC sites were identified using a custom PERL script  that detected potential 5hmC sites based on their expected distance from the AbaSI enzymatic cleavage site. A combination of BEDtools, R packages, and custom scripts were used for downstream analyses. Exact specifications are found below.
All 5hmC sites in the intermediate stringency were used to assess chromosomal 5hmC density. Density was defined as the total number of 5hmC sites per chromosome, corrected for the length of the chromosome and the total number of CGs on the chromosome.
5hmC sites in the intermediate stringency category were plotted against genomic regions and corrected for the length of the region and the number of CGs within the region. All genomic features were defined based on the GRCh37/hg19 genomic annotation downloaded from the UCSC database. Different genic elements, including transcription start sites (TSS), exons, introns, and transcription end sites (TES), were defined based on the Ensembl (release 75). Since genes can have multiple transcripts, we selected the 5’-most TSS on the positive strand as the single TSS associated with each gene. The reverse (3’ most TSS) was done for genes on the negative strand. We limited downstream analysis to protein-coding genes, resulting in 20,745 TSSs in total. Similarly, annotations for retro-elements (i.e., LINEs and SINEs) and CpG islands were acquired from the UCSC database. CpG shores were defined as the 2 kb flanking a CpG island. Coordinates of predicted of promoter and enhancer regions were obtained from recently published genome-wide maps of chromatin states in the adult brain midfrontal lobe , including H3K4me3, H3K4me1 and H3K27ac. Two types of enhancers were distinguished: active enhancers that were simultaneously marked by distal H3K4me1 and H3K27ac, and poised enhancers that were solely marked by distal H3K4me1 [9, 58].
To plot 5hmC profiles around ChIP-Seq peaks, the mean 5hmC was calculated for each contiguous 100 bp bin from 3 kb upstream to 3 kb downstream of the central position of the peak.
Gene expression counts were obtained from RNA-seq data from the preferontal cortex of 11 controls subjects from previously published work . Genes were then classified into quartiles based on their basal gene expression levels: 1st quartile is lowest and 4th is highest. Gene bodies and 20 kb regions upstream and downstream were each divided into 50 intervals. We gathered hydroxymethylation data from windows within each of these intervals and plotted the mean hydroxymethylation level for all windows overlapping each position.
A total of 144,157 internal exons representing 20,745 genes were retrieved from the Ensembl database, with exclusion of all first and last exons and single-exon genes. 5hmC count was plotted against the 20 bp flanking the 5’ and 3’ exon-intron boundaries on both the sense and anti-sense strands.
Cluster analyses and gene ontology (GO)
Cluster analyses were performed using online software. Briefly, a region was deemed to have a cluster of 5hmC if there were at least three 5hmCs each within 200 bp of each other. 5hmC clusters was located within a gene body were assigned to that gene, otherwise 5hmC cluster were assigned to the closest TSS from the center position of the 5hmC cluster. GeneTrail  was used to test for enrichment of functional annotations among genes nearby 5hmC clusters (<250 kb), using the set of all Ensembl genes as a background. Analysis was done with default parameters and results corrected for multiple testing by the method of Benjamini and Hochberg to control the False Discovery Rate (FDR). GO terms were deemed significant if they had an FDR-corrected P ≤ 0.05. The background set included all protein-coding genes.
Cluster analyses for sites unique to females
5hmC sites in all 5 females were compared to 5hmC sites detected in all 19 males. Cluster analyses, as described above, were performed using only the 5hmC sites unique to females.
Cluster analyses for sites unique to males
5 randomly selected sets of 5 males were used to determine male-specific 5hmC clusters. Briefly, all 5hmC sites in each set, separately, were compared to 5hmC sites in all 5 females. Cluster analyses, as described above, were performed in each set using only the 5hmC sites unique to the males in the respective set. Only the clusters that were common in all of the 5 sets of males were deemed to be male-specific.
Genes involved in X-chromosome inactivation were taken from previously published data . Briefly, surveyed genes were given a score from 0–9 based on the number of individual hybrid lines detected from the inactivated X chromosome. A score of 0 corresponds to complete inactivation, whereas a score of 9 corresponds to complete escape from X-inactivation. Intragenic 5hmC patterns on human X-linked genes were then compared between our female and male subjects.
Availability of supporting data
The data sets supporting the results of this article will be deposited in the GEO database.
Tet-assisted bisulfite sequencing
Whole genome bisulfite sequencing
Transcription start sites
Long interspersed elements
Short interspersed elements
Transcription end site
X-linked zinc finger protein
Y-linked zinc finger protein
Differentially hydroxymethylated regions
This study benefited from access to tissue from the Douglas-Bell Canada Brain Bank, which is supported by the Réseau québécois sur le suicide, les troubles de l'humeur et les troubles associés and Brain Canada. J.A.G. is supported by a CIHR Frederick Banting And Charles Best Doctoral fellowship. GT is supported by grants from the Canadian Institute of Health Research MOP93775, MOP11260, MOP119429, and MOP119430; from the National Institutes of Health 1R01DA033684-01; and by the Fonds de Recherche du Québec - Santé through a Chercheur National salary award and through the Quebec Network on Suicide, Mood Disorders, and Related Disorders.
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