5-hydroxymethylcytosines regulate gene expression as a passive DNA demethylation resisting epigenetic mark in proliferative somatic cells.

Enzymatic erasure of DNA methylation in mammals involves iterative 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (TET) family of DNA dioxygenase proteins. As the most abundant form of oxidized 5mC, the prevailing model considers 5-hydroxymethylcytosine (5hmC) as a key nexus in active DNA demethylation that can either indirectly facilitate replication-dependent depletion of 5mC by inhibiting maintenance DNA methylation machinery (UHRF1/DNMT1), or directly be iteratively oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) and restored to cytosine (C) through thymine DNA glycosylase (TDG)-mediated 5fC/5caC excision repair. In proliferative somatic cells, to what extent TET-dependent removal of 5mC entails indirect DNA demethylation via 5hmC-induced replication-dependent dilution or direct iterative conversion of 5hmC to 5fC/5caC is unclear. Here we leverage a catalytic processivity stalling variant of human TET1 (TET1.var: T1662E) to decouple the stepwise generation of 5hmC from subsequent 5fC/5caC generation, excision and repair. By using a CRISPR/dCas9-based epigenome-editing platform, we demonstrate that 5fC/5caC excision repair (by wild-type TET1, TET1.wt), but not 5hmC generation alone (by TET1.var), is requisite for robust restoration of unmodified cytosines and reversal of somatic silencing of the methylation-sensitive, germline-specific RHOXF2B gene promoter. Furthermore, integrated whole-genome multi-modal epigenetic sequencing reveals that hemi-hydroxymethylated CpG dyads predominantly resist replication-dependent depletion of 5mC on the opposing strand in TET1.var-expressing cells. Notably, TET1.var-mediated 5hmC generation is sufficient to induce similar levels of differential gene expression (compared to TET1.wt) without inducing major changes in unmodified cytosine profiles across the genome. Our study suggests 5hmC alone plays a limited role in driving replication-dependent DNA demethylation in the presence of functional DNMT1/UHRF1 mechanisms, but can regulate gene expression as a bona fide epigenetic mark in proliferative somatic cells.

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects.

Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multimodal single-cell data integration, enable accurate identification of neuronal subtypes and uncover context-specific regulatory effects on cell-type-specific genes by TET enzymes.

TET1 facilitates specification of early human lineages including germ cells.

Ten Eleven Translocation 1 (TET1) is a regulator of localized DNA demethylation through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). To examine DNA demethylation in human primordial germ cell-like cells (hPGCLCs) induced from human embryonic stem cells (hESCs), we performed bisulfite-assisted APOBEC coupled epigenetic sequencing (bACEseq) followed by integrated genomics analysis. Our data indicates that 5hmC enriches at hPGCLC-specific NANOG, SOX17 or TFAP2C binding sites on hPGCLC induction, and this is accompanied by localized DNA demethylation. Using CRISPR-Cas9, we show that deleting the catalytic domain of TET1 reduces hPGCLC competency when starting with hESC cultured on mouse embryonic fibroblasts, and this phenotype can be rescued after transitioning hESCs to defined media and a recombinant substrate. Taken together, our study demonstrates the importance of 5hmC in facilitating hPGCLC competency, and the role of hESC culture conditions in modulating this effect.

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

An evaluation of the discriminating power of an Integrated Ballistics Identification System® Heritage™system with the NIST standard cartridge case (Standard Reference Material 2461).

Through analysis and comparison of firing pin, breech face, and ejector impressions, where appropriate, firearm examiners may connect a cartridge case to a suspect firearm with a certain likelihood in a criminal investigation. When a firearm is not present, an examiner may use the Integrated Ballistics Identification System (IBIS®), an automated search and retrieval system coupled with the National Integrated Ballistics Information Network (NIBIN), a database of images showing the markings on fired cartridge cases and bullets from crime scenes along with test fired firearms. For the purpose of measurement quality control of these IBIS® systems the National Institute of Standards and Technology (NIST) initiated the Standard Reference Material (SRM) 2460/2461 standard bullets and cartridge cases project. The aim of this study was to evaluate the overall performance of the IBIS® system by using NIST standard cartridge cases. By evaluating the resulting correlation scores, error rates, and percent recovery, both the variability between and within examiners when using IBIS®, in addition to any inter- and intra-variability between SRM cartridge cases was observed.

In vitro cytotoxicity assessment of a West Virginia chemical spill mixture involving 4-methylcyclohexanemethanol and propylene glycol phenyl ether.

Thousands of gallons of industrial chemicals, crude 4-methylcyclohexanemethanol (MCHM) and propylene glycol phenyl ether (PPh), leaked from industrial tanks into the Elk River in Charleston, West Virginia, USA, on January 9, 2014. A considerable number of people were reported to exhibit symptoms of chemical exposure and an estimated 300,000 residents were advised not to use or drink tap water. At the time of the spill, the existing toxicological data of the chemicals were limited for a full evaluation of the health risks, resulting in concern among those in the impacted regions. In this preliminary study, we assessed cell viability and plasma membrane degradation following a 24-h exposure to varying concentrations (0-1000 µM) of the two compounds, alone and in combination. Evaluation of different cell lines, HEK-293 (kidney), HepG2 (liver), H9c2 (heart), and GT1-7 (brain), provided insight regarding altered cellular responses in varying organ systems. Single exposure to MCHM or PPh did not affect cell viability, except at doses much higher than the estimated exposure levels. Certain co-exposures significantly reduced metabolic activity and increased plasma membrane degradation in GT1-7, HepG2, and H9c2 cells. These findings highlight the importance of examining co-exposures to fully understand the potential toxic effects.

Spatiotemporal phosphoprotein distribution and associated cytokine response of a traumatic injury.

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/ß, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options.