Differentially Hydroxymethylated Regions Research Articles

Circadian clock regulator Bmal1 gates axon regeneration via Tet3 epigenetics in mouse sensory neurons

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.

Role of TET1-mediated epigenetic modulation in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder influenced by a complex interplay of environmental, epigenetic, and genetic factors. DNA methylation (5mC) and hydroxymethylation (5hmC) are DNA modifications that serve as tissue-specific and temporal regulators of gene expression. TET family enzymes dynamically regulate these epigenetic modifications in response to environmental conditions, connecting environmental factors with gene expression. Previous epigenetic studies have identified 5mC and 5hmC changes associated with AD. In this study, we performed targeted resequencing of TET1 on a cohort of early-onset AD (EOAD) and control samples. Through gene-wise burden analysis, we observed significant enrichment of rare TET1 variants associated with AD (p = 0.04). We also profiled 5hmC in human postmortem brain tissues from AD and control groups. Our analysis identified differentially hydroxymethylated regions (DhMRs) in key genes responsible for regulating the methylome: TET3, DNMT3L, DNMT3A, and MECP2. To further investigate the role of Tet1 in AD pathogenesis, we used the 5xFAD mouse model with a Tet1 KO allele to examine how Tet1 loss influences AD pathogenesis. We observed significant changes in neuropathology, 5hmC, and RNA expression associated with Tet1 loss, while the behavioral alterations were not significant. The loss of Tet1 significantly increased amyloid plaque burden in the 5xFAD mouse (p = 0.044) and lead to a non-significant trend towards exacerbated AD-associated stress response in 5xFAD mice. At the molecular level, we found significant DhMRs enriched in genes involved in pathways responsible for neuronal projection organization, dendritic spine development and organization, and myelin assembly. RNA-Seq analysis revealed a significant increase in the expression of AD-associated genes such as Mpeg1, Ctsd, and Trem2. In conclusion, our results suggest that TET enzymes, particularly TET1, which regulate the methylome, may contribute to AD pathogenesis, as the loss of TET function increases AD-associated pathology.

Genome-wide 5-hydroxymethylcytosine (5hmC) reassigned in Pten-depleted mESCs along neural differentiation.

DNA methylation and hydroxymethylation have been implicated in the regulatory dynamics of gene expression in normal development and differentiation. 5-Hydroxymethylcytosine (5hmC), created by the ten-eleven translocation (TET) protein-catalyzed oxidation of 5-methylcytosine (5mC), is abundant in the brain, but the genome-wide distribution and impact of 5hmC during diverse neuronal differentiation remain unknown. Here, we used an in vitro model to differentiate mouse embryonic stem cells (mESCs) into ventral midbrain and hindbrain neural progenitors, followed by characterizing global 5hmC distribution using a nano-5hmC-seal approach. The 5hmC pattern was dynamic in promoter, exon, and enhancer regions, associated with gene activation and repression. For example, ventral midbrain markers (Lmx1a, Otx2, and Th) and hindbrain markers (Hoxa1, Zic1, and Tph1) acquire 5hmC and are upregulated during differentiation. Among the differentially expressed genes involved in both midbrain and hindbrain lineage commitment, phosphatase and tensin homolog (Pten) was identified as a key regulator for neuronal development. We confirmed that Pten knockout disrupted the normal differentiation of midbrain/hindbrain neural progenitors, resulting in immature neurons. In addition, 5421 and 4624 differentially hydroxymethylated regions (DhMRs) were identified in the differentiation of Pten-/- mESC into ventral midbrain and hindbrain progenitors, respectively. Gene ontology analysis showed that the majority of these DhMRs were associated with neurogenesis, ectoderm development, and signal transduction. Moreover, further combinational analysis of the 5hmC pattern and transcriptomic profile in the midbrain progenitor cells demonstrated Pten as a toggle to modulate mitochondrial associated pathways. Therefore, our findings elucidated the molecular mechanisms underlying lineage-specific differentiation of pluripotent stem cells to the midbrain/hindbrain progenitors, where Pten participates as one key regulator.

Dynamic 5-Hydroxymethylcytosine Change: Implication for Aging of Non-Human Primate Brain.

Profiling of 5-hydroxymethylcytosine (5hmC) in the brain regions of rhesus monkey at different ages reveals accumulation and tissue-specific patterns of 5hmC with aging. Region-specific differentially hydroxymethylated regions (DhMRs) are involved in neuronal functions and signal transduction. These data suggest that 5hmC may be a key regulator of gene transcription in neurodevelopment and thus a potential candidate for the epigenetic clock. Importantly, non-human primates are the ideal animal models for investigation of human aging and diseases not only because they are more genetically similar to humans but also epigenetically.

Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis.

HighlightsThe conversion of 5-methylcytosine to 5-hydroxymethylcytosine relaxes the repressive marks of gene expression, and their imbalance leads to cancer development.Global loss of 5-hmC is found to be associated with the downregulation of TET1 and TET3 genes in breast cancer.Genome-wide analysis revealed 4809 differentially methylated regions and 4841 differentially hydroxymethylated regions in breast cancer.The abundance of 5-mC was observed at gene promoter regions while the 5-hmC was profusely distributed at the distal regulatory regions of the breast cancer genome. The accumulation of 5-hmC at distal regulatory sites can potentially enhance gene transcription.Alteration in the 5-hmC levels was positively associated with the respective gene expression. Novel 5-hmC candidates such as TXNL1, CNIH3, BNIPL, and CHODL were found to be promising diagnostic and therapeutic markers for breast cancer.An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>−5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

Brain Region- and Age-Dependent 5-Hydroxymethylcytosine Activity in the Non-Human Primate

Because of the difficulty in collecting fresh brains of humans at different ages, it remains unknown how epigenetic regulation occurs in the primate brains during aging. In the present study, we examined the genomic distribution of 5hmC, an indicator of DNA methylation, in the brain regions of non-human primates (rhesus monkey) at the ages of 2 (juvenile), 8 (young adult), and 17 (old) years. We found that genomic 5hmC distribution was accumulated in the monkey brain as age increased and displayed unique patterns in the cerebellum and striatum in an age-dependent manner. We also observed a correlation between differentially hydroxymethylated regions (DhMRs) and genes that contribute to brain region-related functions and diseases. Our studies revealed, for the first time, the brain-region and age-dependent 5hmC modifications in the non-human primate and the association of these 5hmC modifications with brain region-specific function and potentially aging-related brain diseases.

Altered hydroxymethylome in the substantia nigra of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and aging and genetic and environmental exposure can contribute to its pathogenesis. DNA methylation has been suggested to play a pivotal role in neurodevelopment and neurodegenerative diseases. 5-hydroxymethylcytosine (5hmC) is generated through 5-methylcytosine (5mC) oxidization by ten-eleven translocation proteins and is particularly enriched in the brain. Although 5hmC has been linked to multiple neurological disorders, little is known about 5hmC alterations in the substantia nigra of patients with PD. To determine the specific alterations in DNA methylation and hydroxymethylation in PD brain samples, we examined the genome-wide profiles of 5mC and 5hmC in the substantia nigra of patients with PD and Alzheimer's disease (ad). We identified 4119 differentially hydroxymethylated regions (DhMRs) and no differentially methylated regions (DMRs) in the postmortem brains of patients with PD compared with those of controls. These DhMRs were PD-specific when compared with the results of AD. Gene ontology analysis revealed that several signaling pathways, such as neurogenesis and neuronal differentiation, were significantly enriched in PD DhMRs. KEGG enrichment analysis revealed substantial alterations in multiple signaling pathways, including phospholipase D (PLD), cAMP and Rap1. In addition, using a PD Drosophila model, we found that one of the 5hmC-modulated genes, PLD1, modulated α-synuclein toxicity. Our analysis suggested that 5hmC may act as an independent epigenetic marker and contribute to the pathogenesis of PD.

Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine Reprogramming During Early Human Development

After implantation, complex and highly specialized molecular events render functionally distinct organ formation, whereas how the epigenome shapes organ-specific development remains to be fully elucidated. Here, nano-hmC-Seal, RNA bisulfite sequencing (RNA-BisSeq), and RNA sequencing (RNA-Seq) were performed, and the first multilayer landscapes of DNA 5-hydroxymethylcytosine (5hmC) and RNA 5-methylcytosine (m5C) epigenomes were obtained in the heart, kidney, liver, and lung of the human foetuses at 13–28 weeks with 123 samples in total. We identified 70,091 and 503 organ- and stage-specific differentially hydroxymethylated regions (DhMRs) and m5C-modified mRNAs, respectively. The key transcription factors (TFs), T-box transcription factor 20 (TBX20), paired box 8 (PAX8), krueppel-like factor 1 (KLF1), transcription factor 21 (TCF21), and CCAAT enhancer binding protein beta (CEBPB), specifically contribute to the formation of distinct organs at different stages. Additionally, 5hmC-enriched Alu elements may participate in the regulation of expression of TF-targeted genes. Our integrated studies reveal a putative essential link between DNA modification and RNA methylation, and illustrate the epigenetic maps during human foetal organogenesis, which provide a foundation for an in-depth understanding of the epigenetic mechanisms underlying early development and birth defects.

5-Hydroxymethylcytosine Signatures in Circulating Cell-Free DNA as Diagnostic Biomarkers for Late-Onset Alzheimer's Disease.

5-Hydroxymethylcytosine (5hmC) is an epigenetic DNA modification that is highly abundant in central nervous system. It has been reported that DNA 5hmC dysregulation play a critical role in Alzheimer's disease (AD) pathology. Changes in 5hmC signatures can be detected in circulating cell-free DNA (cfDNA), which has shown potential as a non-invasive liquid biopsy material. However, the genome-wide profiling of 5hmC in cfDNA and its potential for the diagnosis of AD has not been reported to date. We carried out a case-control study and used a genome-wide chemical capture followed by high-throughput sequencing to detect the genome-wide profiles of 5hmC in human cfDNA and identified differentially hydroxymethylated regions (DhMRs) in late-onset AD patients and the control. We discovered significant differences of 5hmC enrichment in gene bodies which were linked to multiple AD pathogenesis-associated signaling pathways in AD patients compared with cognitively normal controls, indicating they can be well distinguished from normal controls by DhMRs in cfDNA. Specially, we identified 7 distinct genes (RABEP1, CPNE4, DNAJC15, REEP3, ROR1, CAMK1D, and RBFOX1) with predicting diagnostic potential based on their significant correlations with MMSE and MoCA scores of subjects. The present results suggest that 5hmC markers derived from plasma cfDNA can served as an effective, minimally invasive biomarkers for clinical auxiliary diagnosis of late-onset AD.

Tet1-mediated modulation of Alzheimer's disease.

Human sequencing studies have identified roughly 30 risk loci associated Alzheimer's disease (AD). However, these loci only explain a portion of disease pathogenicity. Recent studies support a link between gene-environment interactions and AD, and published work from our lab indicates Tet1 mediates gene-environment interactions and Tet1 KO reduces stress response in mice [1]. Furthermore, DNA deep sequencing studies identified an enrichment of rare Tet2 variants associated with AD [2]. This study examines how knockout of the Tet1, a DNA 5-hydroxymethylcytosine (5hmC) regulator, influences AD-associated transcription and pathological outcomes in the 5xFAD AD mouse model. We measured cognitive/behavioral, 5hmC, and expression profiles of a Tet1 heterozygous knockout in the 5xFAD mouse model to examine the influences of Tet1 KO on AD pathogenicity. We show significant differences in behavior, cognition, 5hmC methylation and expression profiles of FAD/Tet1+/- mice relative to WT and FAD. In relation to FAD, FAD/Tet1+/- improved stress response in the Tail-Suspension Assay (p = 0.0438), and relative to WT mice, FAD/Tet1+/- mice display a reduction in their latency to mount the platform in the Morris water maze (p = 0.01). Analysis of 5hmC capture data revealed 2536 differentially hydroxymethylated regions (DhMRs) upregulated in FADTet1 and 410 DhMRs downregulated relative FAD (FDR > 0.05). Analysis of the expression data highlighted 132 differentially expressed genes (DEGs) in FAD/Tet1+/- relative to FAD (FDR > 0.05). Among the genes exclusive to the FAD/Tet1 mice were Nr4A1, Arc, and APLP1. Notably, Nr4A1 appeared in both the 5hmC and RNA-Seq data with increased levels of 5hmC and gene expression relative to FAD mice. Gene ontology analysis of RNA expression data identified significant changes in pathways involved in regulation of ion transmembrane transport, behavioral fear response, regulation of trans-synaptic signaling, and learning or memory. Taken together, these results suggest Tet1 modulates AD-associated gene regulation, expression and pathology.

Tet1 Regulates Astrocyte Development and Cognition of Mice Through Modulating GluA1.

Tet (Ten eleven translocation) family proteins-mediated 5-hydroxymethylcytosine (5hmC) is highly enriched in the neuronal system, and is involved in diverse biological processes and diseases. However, the function of 5hmC in astrocyte remains completely unknown. In the present study, we show that Tet1 deficiency alters astrocyte morphology and impairs neuronal function. Specific deletion of Tet1 in astrocyte impairs learning and memory ability of mice. Using 5hmC high-throughput DNA sequencing and RNA sequencing, we present the distribution of 5hmC among genomic features in astrocyte and show that Tet1 deficiency induces differentially hydroxymethylated regions (DhMRs) and alters gene expression. Mechanistically, we found that Tet1 deficiency leads to the abnormal Ca2+ signaling by regulating the expression of GluA1, which can be rescued by ectopic GluA1. Collectively, our findings suggest that Tet1 plays important function in astrocyte physiology by regulating Ca2+ signaling.

Genome-Wide DNA Methylation and Hydroxymethylation Changes Revealed Epigenetic Regulation of Neuromodulation and Myelination in Yak Hypothalamus.

Both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are important epigenetic modifications in neurodevelopment. However, there is little research examining the genome-wide patterns of 5mC and 5hmC in brain regions of animals under natural high-altitude conditions. We used oxidative reduced representation bisulfite sequencing (oxRRBS) to determine the 5mC and 5hmC sites in the brain, brainstem, cerebellum, and hypothalamus of yak and cattle. We reported the first map of genome-wide DNA methylation and hydroxymethylation in the brain, brainstem, cerebellum, and hypothalamus of yak (living at high altitudes) and cattle. Overall, we found striking differences in 5mC and 5hmC between the hypothalamus and other brain regions in both yak and cattle. Genome-wide profiling revealed that 5mC level decreased and 5hmC level increased in the hypothalamus than in other regions. Furthermore, we identified differentially methylated regions (DMRs) and differentially hydroxymethylated regions (DhMRs), most of which overlapped with each other. Interestingly, transcriptome results for these brain regions also showed distinctive gene levels in the hypothalamus. Finally, differentially expressed genes (DEGs) regulated by DMRs and DhMRs may play important roles in neuromodulation and myelination. Overall, our results suggested that mediation of 5mC and 5hmC on epigenetic regulation may broadly impact the development of hypothalamus and its biological functions.

Whole-Genome Differentially Hydroxymethylated DNA Regions among Twins Discordant for Cardiovascular Death.

Epigenetics is a mechanism underlying cardiovascular disease. It is unknown whether DNA hydroxymethylation is prospectively associated with the risk for cardiovascular death independent of germline and common environment. Male twin pairs middle-aged in 1969–1973 and discordant for cardiovascular death through December 31, 2014, were included. Hydroxymethylation was quantified in buffy coat DNA collected in 1986–1987. The 1893 differentially hydroxymethylated regions (DhMRs) were identified after controlling for blood leukocyte subtypes and age among 12 monozygotic (MZ) pairs (Benjamini–Hochberg False Discovery Rate < 0.01), of which the 102 DhMRs were confirmed with directionally consistent log2-fold changes and p < 0.01 among additional 7 MZ pairs. These signature 102 DhMRs, independent of the germline, were located on all chromosomes except for chromosome 21 and the Y chromosome, mainly within/overlapped with intergenic regions and introns, and predominantly hyper-hydroxymethylated. A binary linear classifier predicting cardiovascular death among 19 dizygotic pairs was identified and equivalent to that generated from MZ via the 2D transformation. Computational bioinformatics discovered pathways, phenotypes, and DNA motifs for these DhMRs or their subtypes, suggesting that hydroxymethylation was a pathophysiological mechanism underlying cardiovascular death that might be influenced by genetic factors and warranted further investigations of mechanisms of these signature regions in vivo and in vitro.

The Role of Tet2-mediated Hydroxymethylation in Poststroke Depression

Poststroke depression (PSD) is a common complication of stroke and has long been a serious threat to human health. PSD greatly affects neurological recovery, quality of life and mortality. Recent studies have shown that 5-hydroxymethylcytosine (5hmC), an important epigenetic modification, is enriched in the brain and associated with many neurological diseases. However, its role in PSD is still unclear. In this study, middle cerebral artery occlusion (MCAO) and spatial restraint stress were used to successfully induce a PSD mouse model and resulted in reduced 5hmC levels, which were caused by Tet2. Furthermore, genome-wide analysis of 5hmC revealed that differentially hydroxymethylated regions (DhMRs) were associated with PSD. DhMRs were enriched among genes involved in the Wnt signaling pathway, neuron development and learning or memory. In particular,DhMRs were strongly enriched in genes with lymphoid enhancer factor 1 (LEF1) binding motifs. Finally, we demonstrated that decreases in TET2 expression in the brain caused PSD by decreasing Wnt/β-catenin/LEF1 pathway signaling to promote inflammatory factor IL-18 expression. In conclusion, our data highlight the potential for 5hmC modification as a therapeutic target for PSD.

Abstract P775: Ascorbate Prevents Secondary Brain Damage Following Stroke Through Epigenetic Gene Regulation

The antioxidant ascorbate is an enzyme cofactor with established roles in maintaining brain function and providing neuroprotection in response to oxidative stress. Recently ascorbate has been identified as an epigenetic regulator through its ability to induce the activity of the ten-eleven translocase (TET) enzymes that produce 5-hydroxymethylcytosine (5-hmC), a CNS-enriched epigenetic modification that is associated with transcriptional activation and neuroprotection. In the current study, we evaluated the role of ascorbate on 5-hmC and its therapeutic potential against ischemic brain injury in comorbid conditions associated with stroke in mice. Adult mice subjected to transient middle cerebral artery occlusion (MCAO) were injected intraperitoneally (i.p.) with ascorbate at 30 min of reperfusion. Young adult male and female mice showed robust induction of 5hmC in the peri-infarct region of the cortex, reduced infarct size and improved motor function. Knockdown of TET3 by intracerebral injection using siRNA blocked the ascorbate-induced increases in 5hmC and led to increased brain degeneration and motor function deficits. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that ascorbate increased 5hmC at the promoters of genes associated with protection against ischemia pathophysiology. Furthermore, ascorbate treatment reduced infarct and improved functional recovery in aged, obese diabetic and hypertensive male and female mice. Delayed ascorbate treatment at 6h of reperfusion was also protective against secondary brain damage and motor deficits following experimental stroke. Collectively, these results indicate that ascorbate regulates the 5hmC epigenetic modification in a neuroprotective manner and may represent a promising therapeutic target for stroke treatment.

Multi-Omics Characterization of Inflammatory Bowel Disease-Induced Hyperplasia/Dysplasia in the Rag2−/−/Il10−/− Mouse Model

Epigenetic dysregulation is hypothesized to play a role in the observed association between inflammatory bowel disease (IBD) and colon tumor development. In the present work, DNA methylome, hydroxymethylome, and transcriptome analyses were conducted in proximal colon tissues harvested from the Helicobacter hepaticus (H. hepaticus)-infected murine model of IBD. Reduced representation bisulfite sequencing (RRBS) and oxidative RRBS (oxRRBS) analyses identified 1606 differentially methylated regions (DMR) and 3011 differentially hydroxymethylated regions (DhMR). These DMR/DhMR overlapped with genes that are associated with gastrointestinal disease, inflammatory disease, and cancer. RNA-seq revealed pronounced expression changes of a number of genes associated with inflammation and cancer. Several genes including Duox2, Tgm2, Cdhr5, and Hk2 exhibited changes in both DNA methylation/hydroxymethylation and gene expression levels. Overall, our results suggest that chronic inflammation triggers changes in methylation and hydroxymethylation patterns in the genome, altering the expression of key tumorigenesis genes and potentially contributing to the initiation of colorectal cancer.

Abstract PO-05: TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center (GC) B cells. Despite progress in the treatment of DLBCL, approximately 40% of patients relapse or are refractory to the treatment, which usually leads to fatality. DLBCLs are characterized by aberrant DNA methylation and this feature correlates with poor clinical outcome. We and others have shown that deregulated epigenetic mechanisms contribute to lymphoma formation. Particularly, we have demonstrated that TET2, an enzyme that converts methylcytosine (mC) into hydroxymethylcytosine (hmC) and is mutated in ~10% of DLBCLs, is a B-cell tumor suppressor. GC-specific deletion of TET2 (Cg1Cre/Tet2-/-) resulted in accelerated lymphomagenesis in DLBCL mouse models driven by BCL6 overexpression, with 100% Cg1Cre/Tet2-/-;ImBcl6 mice developing lymphoma at 7 months compared to only 50% in ImBcl6 control mice. In addition, TET2 deletion in hematopoietic stem cells (VavCre/Tet2-/-) induced GC B-cell hyperplasia (B220+GL7+CD95+ cells; 10% VavCre/Tet2-/- vs 5% VavCre/Tet2+/+), promoting malignant transformation. Further analysis of the GC reaction revealed that TET2-deficient GC B cells displayed anomalous patterns of DNA methylation. GC B cells from VavCre/Tet2-/- mice presented 1) focal loss of hmC—using hMeDIPseq—with 25,000 differentially hydroxymethylated regions (DHMR) lost compared to VavCre/Tet2+/+ GC B cells and 2) increased mC—using RRBS—with almost 11,000 differentially methylated cytosines (DMCs), 84% hypermethylated, compared to VavCre/Tet2+/+ GC B cells. TET2-mediated reduction of hmC and hypermethylation affected enhancers and promoters, respectively, of genes mediating GC exit and terminal differentiation of GC B cells, especially those regulated at enhancers by the opposing functions of CREBBP and HDAC3. We are currently investigating the potential cooperative role between TET2-mediated hmC and CREBBP-mediated H3K27Ac, supported by reduced H3K27Ac at enhancers activated by CREBBP in VavCre/Tet2-/- GC B cells and mutual exclusion between TET2-mutant and CREBBP-mutant primary DLBCL. RNA sequencing analysis revealed that the genes epigenetically regulated by TET2 were aberrantly repressed in VavCre/Tet2+/+ GC B cells, explaining the observed GC hyperplasia in TET2-deficient GC B cells since these genes control the differentiation of GC B cells into plasma cells. Importantly, TET2-mutant DLBCL primary samples display a similar repressive transcriptional signature associated with GC B-cell terminal differentiation. Our data show how TET2-induced epigenetic changes contribute to lymphoma development and highlight the multilayered nature of the epigenome, which can be therapeutically exploited. We are evaluating the therapeutic potential in TET2-mutant DLBCL of a combinatorial therapy consisting of DNA methylation inhibitors (DNMTi), to revert hypermethylation at promoters, plus specific HDAC3 inhibitors, to compensate for the loss of hmC at enhancers. Citation Format: Pilar M. Dominguez, Wojciech Rosikiewicz, Xiaowen Chen, Hussein Ghamlouch, Said Aoufouchi, Olivier A. Bernard, Ari M. Melnick, Sheng Li, Ricky W. Johnstone. TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-05.

DNA methylation and hydroxymethylation have distinct genome-wide profiles related to axonal regeneration

ABSTRACT Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

TET3 regulates DNA hydroxymethylation of neuroprotective genes following focal ischemia

The 5-hydroxymethylcytosine (5hmC) epigenetic modification is highly enriched in the CNS and a critical modulator of neuronal function and development. We found that cortical 5hmC was enhanced from 5 min to three days of reperfusion following focal ischemia in adult mice. Blockade of the 5hmC-producing enzyme ten-eleven translocase 3 (TET3) increased edema, infarct volume, and motor function impairments. To determine the mechanism by which TET3 provides ischemic neuroprotection, we assessed the genomic regions where TET3 modulates 5hmC. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that focal ischemia robustly increased 5hmC at the promoters of thousands of genes in a TET3-dependent manner. TET3 inhibition reduced 5hmC at the promoters of neuroprotective genes involved in cell survival, angiogenesis, neurogenesis, antioxidant defense, DNA repair, and metabolism demonstrating a role for TET3 in endogenous protection against stroke. The mRNA expression of several genes with known involvement in ischemic neuroprotection were also reduced with TET3 knockdown in both male and female mice, establishing a correlation between decreased promoter 5hmC levels and decreased gene expression. Collectively, our results indicate that TET3 globally increases 5hmC at regulatory regions and overwhelmingly modulates 5hmC in several neuroprotective pathways that may improve outcome after ischemic injury.

Ethnicity-specific and overlapping alterations of brain hydroxymethylome in Alzheimer's disease.

5-Methylcytosine (5mC), generated through the covalent addition of a methyl group to the fifth carbon of cytosine, is the most prevalent DNA modification in humans and functions as a critical player in the regulation of tissue and cell-specific gene expression. 5mC can be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) enzymes, which is enriched in brain. Alzheimer's disease (AD) is the most common neurodegenerative disorder, and several studies using the samples collected from Caucasian cohorts have found that epigenetics, particularly cytosine methylation, could play a role in the etiological process of AD. However, little research has been conducted using the samples of other ethnic groups. Here we generated genome-wide profiles of both 5mC and 5hmC in human frontal cortex tissues from late-onset Chinese AD patients and cognitively normal controls. We identified both Chinese-specific and overlapping differentially hydroxymethylated regions (DhMRs) with Caucasian cohorts. Pathway analyses revealed specific pathways enriched among Chinese-specific DhMRs, as well as the shared DhMRs with Caucasian cohorts. Furthermore, two important transcription factor-binding motifs, hypoxia-inducible factor 2α (HIF2α) and hypoxia-inducible factor 1α (HIF1α), were enriched in the DhMRs. Our analyses provide the first genome-wide profiling of DNA hydroxymethylation of the frontal cortex of AD patients from China, emphasizing an important role of 5hmC in AD pathogenesis and highlighting both ethnicity-specific and overlapping changes of brain hydroxymethylome in AD.