Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC.

Attention-Deficit Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders and manifests inattention, hyperactivity, and impulsivity symptoms in childhood that can last throughout life. Genetic and environmental studies implicate the dopamine system in ADHD pathogenesis. Work from our group and that of others indicates that deltamethrin insecticide and stress exposure during neurodevelopment leads to alterations in dopamine function, and we hypothesized that exposure to both of these factors together would lead to synergistic effects on DNA methylation of key genes within the midbrain, a highly dopaminergic region, that could contribute to these findings. Through targeted next-generation sequencing of a panel of cortisol and dopamine pathway genes, we observed hypermethylation of the glucocorticoid receptor gene, Nr3c1, in the midbrain of C57/BL6N males in response to dual deltamethrin and corticosterone exposures during development. This is the first description of DNA methylation studies of Nr3c1 and key dopaminergic genes within the midbrain in response to a pyrethroid insecticide, corticosterone, and these two exposures together. Our results provide possible connections between environmental exposures that impact the dopamine system and the hypothalamic-pituitary-adrenal axis via changes in DNA methylation and provides new information about the presence of epigenetic effects in adulthood after exposure during neurodevelopment.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.

Poor placental function is a common cause of intrauterine growth restriction, which in turn is associated with increased risks of adverse health outcomes. Our prior work suggests that birthweight and childhood obesity-associated genetic variants functionally impact placental function and that placental microRNA are associated with birthweight. To address the influence of the placenta beyond birth, we assessed the relationship between placental microRNAs and early childhood growth.

Topologically associating domains (TADs), CTCF loop domains, and A/B compartments have been identified as important structural and functional components of 3D chromatin organization, yet the relationship between these features is not well understood. Using high-resolution Hi-C and HiChIP, we show that Drosophila chromatin is organized into domains we term compartmental domains that correspond precisely with A/B compartments at high resolution. We find that transcriptional state is a major predictor of Hi-C contact maps in several eukaryotes tested, including C. elegans and A. thaliana. Architectural proteins insulate compartmental domains by reducing interaction frequencies between neighboring regions in Drosophila, but CTCF loops do not play a distinct role in this organism. In mammals, compartmental domains exist alongside CTCF loop domains to form topological domains. The results suggest that compartmental domains are responsible for domain structure in all eukaryotes, with CTCF playing an important role in domain formation in mammals.

Prenatal cadmium (Cd) exposure has been implicated in both placental toxicity and adverse neurobehavioral outcomes. Placental microRNAs (miRNAs) may function to developmentally program adverse pregnancy and newborn health outcomes in response to gestational Cd exposure.

Neonatal molecular biomarkers of neurobehavioral responses (measures of brain-behavior relationships), when combined with neurobehavioral performance measures, could lead to better predictions of long-term developmental outcomes. To this end, we examined whether variability in buccal cell DNA methylation (DNAm) associated with neurobehavioral profiles in a cohort of infants born less than 30 weeks postmenstrual age (PMA) and participating in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study (N = 536). We tested whether epigenetic age, age acceleration, or DNAm levels at individual loci differed between infants based on their NICU Network Neurobehavioral Scale (NNNS) profile classifications. We adjusted for recruitment site, infant sex, PMA, and tissue heterogeneity. Infants with an optimally well-regulated NNNS profile had older epigenetic age compared to other NOVI infants (β1 = 0.201, p-value = 0.026), but no significant difference in age acceleration. In contrast, infants with an atypical NNNS profile had differential methylation at 29 CpG sites (FDR < 10%). Some of the genes annotated to these CpGs included PLA2G4E, TRIM9, GRIK3, and MACROD2, which have previously been associated with neurological structure and function, or with neurobehavioral disorders. These findings contribute to the existing evidence that neonatal epigenetic variations may be informative for infant neurobehavior.