Substantial DNA methylation differences between two major neuronal subtypes in human brain

Abstract The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons—GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.

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Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1411269112 ◽

2015 ◽

Vol 112 (22) ◽

pp. 6800-6806 ◽

Cited By ~ 129

Author(s):

Benyam Kinde ◽

Harrison W. Gabel ◽

Caitlin S. Gilbert ◽

Eric C. Griffith ◽

Michael E. Greenberg

Keyword(s):

Dna Methylation ◽

Binding Protein ◽

Cell Types ◽

Cpg Methylation ◽

Early Postnatal Development ◽

Cpg Dinucleotides ◽

Epigenetic Regulator ◽

And Function ◽

Syndrome Protein ◽

The Brain

DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.

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Analysis of DNA Methylation and Transcription During Granulopoiesis Reveals Timed Methylation Changes in Low CpG Areas That Correlate with Changed Transcriptional Activity.

Blood ◽

10.1182/blood.v120.21.2334.2334 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2334-2334

Author(s):

Rönnerblad Michelle ◽

Olofsson Tor ◽

Iyadh Douagi ◽

Sören Lehmann ◽

Karl Ekwall ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Conflicts Of Interest ◽

Cell Types ◽

Cpg Methylation ◽

Epigenetic Mechanism ◽

450K Array ◽

Chi Square ◽

Heterochromatin Formation ◽

Chi Square Test

Abstract Abstract 2334 Accumulating evidence demonstrates that epigenetic changes, including DNA methylation play a central role in differentiation, providing cellular memory and stabilizing lineage choice in hematopoiesis1–3. DNA methylation is an important epigenetic mechanism involved in transcriptional regulation, heterochromatin formation and the normal development of many organisms. In this study we investigated the DNA methylome and transcriptome of human cells in four separate differentiation stages in granulopoiesis, ranging from the multipotent Common Myeloid progenitor (CMP) to terminally differentiated bone marrow neutrophils (PMN). To this end we employed HumanMethylation 450 BeadChip (450K array) from Illumina with extensive genomic coverage and mRNA expression arrays (Illumina). Temporally distinct methylation changes during granulopoiesis Differential methylation between two cell stages was defined as an average difference in β value of at least 0.17 (p ≤ 0.05). We detected 12132 DMSs during granulopoiesis. Of these the majority showed decreased methylation during granulopoeisis (10771 CpGs) and a smaller set gained methylation (1658 CpGs). Strikingly, increases in methylation predominantly occur between CMP and GMP, the two least mature cell types. Some CpGs also show increased methylation in the GMP-PMC transition, while very few CpG sites increase at the final stage of differentiation from PMC to PMN. Although reduction of methylation occurs at all stages of granulopoiesis, the greatest change is between GMP and PMC. It is striking that the DNA methylation patterns preferentially change at points of lineage restriction, and that the greatest change occurs upon loss of oligopotency between GMP and PMC. DMSs within CGIs were greatly underrepresented (p<0.001 with chi-square test), while DMSs were overrepresented in shelves (p<0.001) and open sea (p<0.001). Thus, methylation appears to be more dynamic outside of CGIs during granulocytic development. For all regions the variation within enhancers was greater than outside of enhancers indicating greater methylation changes in enhancers compared to non-enhancers. In addition, CpGs in enhancer regions are significantly enriched in the list of DMSs (p<0.001, chi-square test) further supporting the observation that enhancer regions display dynamic DNA methylation changes during granulopoiesis. Changes in gene expression correlate with DNA methylation changes There was a significant overlap between genes showing decreased methylation and genes with increased expression as well as for the reverse comparison between genes with increased methylation and decreased expression. Thus, support a general anticorrelation between DNA methylation and gene expression. Azurophilic granule proteins showed increased expression peaking in PMC and a rapid decrease toward PMN. CpG methylation levels for those genes decreased concomitantly with the peak in expression. We report cell population specific changes of DNA methylation levels. The main reduction of CpG methylation coincides with the loss of oligopotency at the transition from GMP-PMC. This suggests a role of DNA methylation in regulating cell plasticity and lineage choice. Disclosures: No relevant conflicts of interest to declare.

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Correlation Patterns Between DNA Methylation and Gene Expression in The Cancer Genome Atlas

Cancer Informatics ◽

10.1177/1176935119828776 ◽

2019 ◽

Vol 18 ◽

pp. 117693511982877 ◽

Cited By ~ 28

Author(s):

John CG Spainhour ◽

Hong Seo Lim ◽

Soojin V Yi ◽

Peng Qiu

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Region ◽

The Cancer Genome Atlas ◽

Gene Body ◽

Cpg Sites ◽

Cpg Site ◽

Cancer Genome Atlas ◽

Genome Atlas

Background: DNA methylation is a form of epigenetic modification that has been shown to play a significant role in gene regulation. In cancer, DNA methylation plays an important role by regulating the expression of oncogenes. The role of DNA methylation in the onset and progression of various cancer types is now being elucidated as more large-scale data become available. The Cancer Genome Atlas (TCGA) provides a wealth of information for the analysis of various molecular aspects of cancer genetics. Gene expression data and DNA methylation data from TCGA have been used for a variety of studies. A traditional understanding of the effects of DNA methylation on gene expression has linked methylation of CpG sites in the gene promoter region with the decrease in gene expression. Recent studies have begun to expand this traditional role of DNA methylation. Results: Here we present a pan-cancer analysis of correlation patterns between CpG methylation and gene expression. Using matching patient data from TCGA, 33 cancer-specific correlations were calculated for each CpG site and the expression level of its corresponding gene. These correlations were used to identify patterns on a per-site basis as well as patterns of methylation across the gene body. Using these identified patterns, we found genes that contain conflicting methylation signals beyond the commonly accepted association between the promoter region methylation and silencing of gene expression. Beyond gene body methylation in whole, we examined individual CpG sites and show that, even in the same gene body, some sites can have a contradictory effect on gene expression in cancers. Conclusions: We observed that within promoter regions there was a substantial amount of positive correlation between methylation and gene expression, which contradicts the commonly accepted association. We observed that the correlation between CpG methylation and gene expression does not exhibit in a tissue-specific manner, suggesting that the effects of methylation on gene expression are largely tissue independent. The analysis of correlation associated with the location of the CpG site in the gene body has led to the identification of several different methylation patterns that affect gene expression, and several examples of methylation activating gene expression were observed. Distinctly opposing or conflicting effects were seen in close proximity on the gene body, where negative and positive correlations were seen at the neighboring CpG sites.

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The Role of DNA Methylation in Regulating Tissue-Specific Gene Expression During Gametogenesis.John R. McCarrey, Ph.D.

Biology of Reproduction ◽

10.1093/biolreprod/81.s1.62 ◽

2009 ◽

Vol 81 (Suppl_1) ◽

pp. 62-62 ◽

Cited By ~ 1

Author(s):

John R. McCarrey

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Specific Gene ◽

Tissue Specific ◽

Tissue Specific Gene ◽

Specific Gene Expression

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Epigenetic Conservation Is a Beacon of Function: An Analysis Using Methcon5 Software for Studying Gene Methylation

JCO Clinical Cancer Informatics ◽

10.1200/cci.19.00109 ◽

2020 ◽

pp. 100-107 ◽

Cited By ~ 1

Author(s):

Emil Hvitfeldt ◽

Chao Xia ◽

Kimberly D. Siegmund ◽

Darryl Shibata ◽

Paul Marjoram

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Software Package ◽

Human Colon ◽

Cell Types ◽

Gene Promoters ◽

Cpg Sites ◽

Conserved Genes ◽

Multiple Cell ◽

Promoter Dna

PURPOSE Different epigenetic configurations allow one genome to develop into multiple cell types. Although the rules governing what epigenetic features confer gene expression are increasingly being understood, much remains uncertain. Here, we used a novel software package, Methcon5, to explore whether the principle of biologic conservation can be used to identify expressed genes. The hypothesis is that epigenetic configurations of important expressed genes will be conserved within a tissue. MATERIALS AND METHODS We compared the DNA methylation of approximately 850,000 CpG sites between multiple clonal crypts or glands of human colon, small intestine, and endometrium. We performed this analysis using the new software package, Methcon5, which enables detection of regions of high (or low) conservation. RESULTS We showed that DNA methylation is preferentially conserved at gene-associated CpG sites, particularly in gene promoters (eg, near the transcription start site) or the first exon. Furthermore, higher conservation correlated well with gene expression levels and performed better than promoter DNA methylation levels. Most conserved genes are in canonical housekeeping pathways. CONCLUSION This study introduces the new software package, Methcon5. In this example application, we showed that epigenetic conservation provides an alternative method for identifying functional genomic regions in human tissues.

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CpG traffic lights are markers of regulatory regions in humans

10.1101/095968 ◽

2016 ◽

Author(s):

Abdullah M. Khamis ◽

Anna V. Lioznova ◽

Artem V. Artemov ◽

Vasily Ramensky ◽

Vladimir B. Bajic ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Regulation Of Gene Expression ◽

Cell Types ◽

Regulatory Elements ◽

Cpg Methylation ◽

Population Heterogeneity ◽

Gene Body Methylation ◽

Enhancer Activity ◽

Traffic Lights

AbstractDNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

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Mapping eQTL by leveraging multiple tissues and DNA methylation

10.1101/069534 ◽

2016 ◽

Author(s):

Chaitanya R. Acharya ◽

Kouros Owzar ◽

Andrew S. Allen

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Specific Gene ◽

Model Parameters ◽

Eqtl Mapping ◽

Joint Tissue ◽

Tissue Specific ◽

Cpg Sites ◽

Micro Rnas ◽

Mapping Techniques

AbstractBackgroundDNA methylation is an important tissue-specific epigenetic event that influences transcriptional regulation of gene expression. Differentially methylated CpG sites may act as mediators between genetic variation and gene expression, and this relationship can be exploited while mapping multi-tissue expression quantitative trait loci (eQTL). Current multi-tissue eQTL mapping techniques are limited to only exploiting gene expression patterns across multiple tissues either in a joint tissue or tissue-by-tissue frameworks. We present a new statistical approach that enables us to model the effect of germ-line variation on tissue-specific gene expression in the presence of effects due to DNA methylation.ResultsOur method efficiently models genetic and epigenetic variation to identify genomic regions of interest containing combinations of mRNA transcripts, CpG sites, and SNPs by jointly testing for genotypic effect and higher order interaction effects between genotype, methylation and tissues. We demonstrate using Monte Carlo simulations that our approach, in the presence of both genetic and DNA methylation effects, gives an improved performance (in terms of statistical power) to detect eQTLs over the current eQTL mapping approaches. When applied to an array-based dataset from 150 neuropathologically normal adult human brains, our method identifies eQTLs that were undetected using standard tissue-by-tissue or joint tissue eQTL mapping techniques. As an example, our method identifies eQTLs in a BAX inhibiting gene (TMBIM1), which may have a role in the pathogenesis of Alzheimer disease.ConclusionsOur score test-based approach does not need parameter estimation under the alternative hypothesis. As a result, our model parameters are estimated only once for each mRNA - CpG pair. Our model specifically studies the effects of non-coding regions of DNA (in this case, CpG sites) on mapping eQTLs. However, we can easily model micro-RNAs instead of CpG sites to study the effects of post-transcriptional events in mapping eQTL. Our model’s flexible framework also allows us to investigate other genomic events such as alternative gene splicing by extending our model to include gene isoform-specific data.

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Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells

Nucleic Acids Research ◽

10.1093/nar/gkt1306 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3009-3016 ◽

Cited By ~ 32

Author(s):

Weilong Guo ◽

Wen-Yu Chung ◽

Minping Qian ◽

Matteo Pellegrini ◽

Michael Q. Zhang

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem ◽

Cell Types ◽

Cpg Methylation ◽

Antisense Strand ◽

Long Terminal Repeats ◽

Cpg Sites ◽

Specific Distribution ◽

Highly Expressed Genes

Abstract DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.

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Genetic, epigenetic, and environmental factors controlling oxytocin receptor gene expression

Clinical Epigenetics ◽

10.1186/s13148-021-01017-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Joshua S. Danoff ◽

Kelly L. Wroblewski ◽

Andrew J. Graves ◽

Graham C. Quinn ◽

Allison M. Perkeybile ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Receptor Gene ◽

Oxytocin Receptor ◽

Prairie Vole ◽

Alternative Transcript ◽

Prairie Voles ◽

Oxytocin Receptor Gene ◽

Cpg Sites ◽

The Brain

Abstract Background The neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown. Results Here, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3′ portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2. Conclusions These results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3′ portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3′ portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.

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