Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine.

BACKGROUND: Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing. RESULTS: Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods. CONCLUSIONS: This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.

Molecular convergence of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are caused by mutations in diverse genes involved in different cellular functions, although there can be crosstalk, or convergence, between molecular pathways affected by different NDDs. To assess molecular convergence, we generated human neural progenitor cell models of 9q34 deletion syndrome, caused by haploinsufficiency of EHMT1, and 18q21 deletion syndrome, caused by haploinsufficiency of TCF4. Using next-generation RNA sequencing, methylation sequencing, chromatin immunoprecipitation sequencing, and whole-genome miRNA analysis, we identified several levels of convergence. We found mRNA and miRNA expression patterns that were more characteristic of differentiating cells than of proliferating cells, and we identified CpG clusters that had similar methylation states in both models of reduced gene dosage. There was significant overlap of gene targets of TCF4 and EHMT1, whereby 8.3% of TCF4 gene targets and 4.2% of EHMT1 gene targets were identical. These data suggest that 18q21 and 9q34 deletion syndromes show significant molecular convergence but distinct expression and methylation profiles. Common intersection points might highlight the most salient features of disease and provide avenues for similar treatments for NDDs caused by different genetic mutations.

BisQC: an operational pipeline for multiplexed bisulfite sequencing.

BACKGROUND: Bisulfite sequencing is the most efficient single nucleotide resolution method for analysis of methylation status at whole genome scale, but improved quality control metrics are needed to better standardize experiments. RESULTS: We describe BisQC, a step-by-step method for multiplexed bisulfite-converted DNA library construction, pooling, spike-in content, and bioinformatics. We demonstrate technical improvements for library preparation and bioinformatic analyses that can be done in standard laboratories. We find that decoupling amplification of bisulfite converted (bis) DNA from the indexing reaction is an advantage, specifically in reducing total PCR cycle number and pre-selecting high quality bis-libraries. We also introduce a progressive PCR method for optimal library amplification and size-selection. At the sequencing stage, we thoroughly test the benefits of pooling non-bis DNA library with bis-libraries and find that BisSeq libraries can be pooled with a high proportion of non-bis DNA libraries with minimal impact on BisSeq output. For informatics analysis, we propose a series of optimization steps including the utilization of the mitochondrial genome as a QC standard, and we assess the validity of using duplicate reads for coverage statistics. CONCLUSION: We demonstrate several quality control checkpoints at the library preparation, pre-sequencing, post-sequencing, and post-alignment stages, which should prove useful in determining sample and processing quality. We also determine that including a significant portion of non-bisulfite converted DNA with bisulfite converted DNA has a minimal impact on usable bisulfite read output.

Estrogen receptor 1 promoter polymorphism and digit ratio in men.

OBJECTIVES: The 2D:4D digit ratio, the relative lengths of the index and ring fingers in humans, is a widely used proxy measure for prenatal testosterone exposure. Varying distributions of androgen and estrogen receptors on the second and fourth digits, both of which regulate digit development, appears to be the basis for this effect. Polymorphism in a tandem repeat in the gene coding for the estrogen receptor α (ESR1) in zebra finches (Taeniopygia guttata) not only explains a significant amount of variation in digit ratio but also seems to explain the significant correlation between digit ratio and sexual behavior in these birds. Here, we investigate the effect of TA polymorphism in ESR1 on 2D:4D and aggressive behavior in men. METHODS: We genotyped ESR1 polymorphism in samples collected for a previous study in which we had demonstrated an association between androgen receptor polymorphism and aggression, but not 2D:4D. RESULTS: We found a significant effect of ESR1 TA repeat number on left hand 2D:4D ratio. More TA repeats were associated with higher, more feminized, digit ratios. We found no effect on right hand 2D:4D. We also found an effect of ESR1 polymorphism on aggressive behavior. Greater heterozygosity in TA(n) was associated with lower physical aggression. CONCLUSIONS: Our results suggest that a significant amount of left hand 2D:4D variation and aggressive behavior is due to this variation in ESR1, and that some of the correlation between digit ratio and social behavior is due to pleiotropic effects of ESR1 variation on the two traits.

Aggression, digit ratio and variation in androgen receptor and monoamine oxidase a genes in men.

Variation in prenatal exposure to androgens is thought to be responsible for some of the individual differences in aggressive behavior among adults. A putative indicator of prenatal testosterone exposure, 2D:4D (the index to ring finger length) ratios have shown a weak correlation with aggression. Variation in sensitivity of the androgen receptor, resulting from polymorphism in the AR gene, is also thought to influence the relative expression of sexually dimorphic traits within each sex, including aggressive behavior and 2D:4D. Here we examine variation in aggression, 2D:4D, and polymorphism in the AR and MAO-A genes in a sample of 188 men. We find no evidence of AR gene influence on right hand 2D:4D, and a weak trend towards more feminine-typical left hand 2D:4D in men with more sensitive androgen receptors. Men with more sensitive androgen receptors tended to score lower on many of the subscales of the Aggression Questionnaire and Indirect Aggression Questionnaire. We found no influence of MAO-A allele on either digit ratio or aggressive behavior. We conclude that more masculine-typical 2D:4D does not reflect greater sensitivity to testosterone through variation in this locus on the AR gene, and that AR alleles conferring greater sensitivity to testosterone are associated with lower, not higher propensity to aggression.

Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum.

Cocaine dependence is a chronic, relapsing disorder caused by lasting changes in the brain. Animal studies have identified cocaine-related alterations in striatal DNA methylation; however, it is unclear how methylation is related to cocaine dependence in humans. We generated methylomic profiles of the nucleus accumbens using human postmortem brains from a cohort of individuals with cocaine dependence and healthy controls (n = 25 per group). We found hypermethylation in a cluster of CpGs within the gene body of tyrosine hydroxylase (TH), containing a putative binding site for the early growth response 1 (EGR1) transcription factor, which is hypermethylated in the caudate nucleus of cocaine-dependent individuals. We replicated this finding and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity which is attenuated by methylation and enhanced by EGR1 overexpression. These results suggest that cocaine dependence alters the epigenetic regulation of dopaminergic signaling genes.

DNA Methylation Dynamics and Cocaine in the Brain: Progress and Prospects.

Cytosine modifications, including DNA methylation, are stable epigenetic marks that may translate environmental change into transcriptional regulation. Research has begun to investigate DNA methylation dynamics in relation to cocaine use disorders. Specifically, DNA methylation machinery, including methyltransferases and binding proteins, are dysregulated in brain reward pathways after chronic cocaine exposure. In addition, numerous methylome-wide and candidate promoter studies have identified differential methylation, at the nucleotide level, in rodent models of cocaine abuse and drug seeking behavior. This review highlights the current progress in the field of cocaine-related methylation, and offers considerations for future research.

Association of a History of Child Abuse With Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence.

OBJECTIVE: Child abuse has devastating and long-lasting consequences, considerably increasing the lifetime risk of negative mental health outcomes such as depression and suicide. Yet the neurobiological processes underlying this heightened vulnerability remain poorly understood. The authors investigated the hypothesis that epigenetic, transcriptomic, and cellular adaptations may occur in the anterior cingulate cortex as a function of child abuse. METHOD: Postmortem brain samples from human subjects (N=78) and from a rodent model of the impact of early-life environment (N=24) were analyzed. The human samples were from depressed individuals who died by suicide, with (N=27) or without (N=25) a history of severe child abuse, as well as from psychiatrically healthy control subjects (N=26). Genome-wide DNA methylation and gene expression were investigated using reduced representation bisulfite sequencing and RNA sequencing, respectively. Cell type-specific validation of differentially methylated loci was performed after fluorescence-activated cell sorting of oligodendrocyte and neuronal nuclei. Differential gene expression was validated using NanoString technology. Finally, oligodendrocytes and myelinated axons were analyzed using stereology and coherent anti-Stokes Raman scattering microscopy. RESULTS: A history of child abuse was associated with cell type-specific changes in DNA methylation of oligodendrocyte genes and a global impairment of the myelin-related transcriptional program. These effects were absent in the depressed suicide completers with no history of child abuse, and they were strongly correlated with myelin gene expression changes observed in the animal model. Furthermore, a selective and significant reduction in the thickness of myelin sheaths around small-diameter axons was observed in individuals with history of child abuse. CONCLUSIONS: The results suggest that child abuse, in part through epigenetic reprogramming of oligodendrocytes, may lastingly disrupt cortical myelination, a fundamental feature of cerebral connectivity.

Effects of postmortem interval on biomolecule integrity in the brain.

Postmortem brain research is invaluable to the study of neurologic and neuropsychiatric disorders, including Alzheimer disease, schizophrenia, and major depression. A major confounder in molecular studies using human brain tissue is postmortem interval (i.e. the amount of time between a subject's death and processing of tissue). We examined the integrity of biomolecules that were of interest to molecular studies of neurologic disorders, including RNA, microRNA, histone modifications, and proteins, at various postmortem intervals in an animal model to assess their robustness and suitability for experimentation. Sprague-Dawley rats were selected as model and subjected to 2 conditions: a variable postmortem interval at room temperature and a fixed time of 24 hours at 4°C, which simulates the period commonly spent in the morgue before brain collection. Eight time points were investigated. MicroRNA was impressively resistant to postmortem intervals; methylated histone modifications showed a threshold between 72 and 96 hours, mirroring results from histone proteins at 72 hours. RNA degradation was transcript-specific, with housekeeping genes being more robust than genes with lower expression. Our results suggest that molecules commonly investigated in genetic and epigenetic studies were highly stable through the postmortem intervals investigated. These results support the continued use of postmortem tissue for neuropsychiatric research.