5hmC Marks Research Articles

Mapping the epigenomic landscape of post-traumatic stress disorder in human cortical neurons.

The study conducted a comprehensive genome-wide analysis of differential 5mC and 5hmC modifications at both CpG and non-CpG sites in postmortem orbitofrontal neurons from 25 PTSD cases and 13 healthy controls. It was observed that PTSD patients exhibit a greater number of differential 5hmC sites compared to 5mC sites. Specifically, individuals with PTSD tend to show hyper-5mC/5hmC at CpG sites, particularly within CpG islands and promoter regions, and hypo-5mC/5hmC at non-CpG sites, especially within intragenic regions. Functional enrichment analysis indicated distinct yet interconnected roles for 5mC and 5hmC in PTSD. The 5mC marks primarily regulate cell-cell adhesion processes, whereas 5hmC marks are involved in embryonic morphogenesis and cell fate commitment. By integrating published PTSD findings from central and peripheral tissues through multi-omics approaches, several biological mechanisms were prioritized, including developmental processes, HPA axis regulation, and immune responses. Based on the consistent enrichment in developmental processes, we hypothesize that if epigenetic changes occur during early developmental stages, they may increase the risk of developing PTSD following trauma exposure. Conversely, if these epigenetic changes occur in adulthood, they may influence neuronal apoptosis and survival mechanisms.

Profiling neuronal methylome and hydroxymethylome of opioid use disorder in the human orbitofrontal cortex

Opioid use disorder (OUD) is influenced by genetic and environmental factors. While recent research suggests epigenetic disturbances in OUD, this is mostly limited to DNA methylation (5mC). DNA hydroxymethylation (5hmC) has been widely understudied. We conducted a multi-omics profiling of OUD in a male cohort, integrating neuronal-specific 5mC and 5hmC as well as gene expression profiles from human postmortem orbitofrontal cortex (OUD = 12; non-OUD = 26). Single locus methylomic analysis and co-methylation analysis showed a higher number of OUD-associated genes and gene networks for 5hmC compared to 5mC; these were enriched for GPCR, Wnt, neurogenesis, and opioid signaling. 5hmC marks also showed a higher correlation with gene expression patterns and enriched for GWAS of psychiatric traits. Drug interaction analysis revealed interactions with opioid-related drugs, some used as OUD treatments. Our multi-omics findings suggest an important role of 5hmC and reveal loci epigenetically dysregulated in OFC neurons of individuals with OUD.

Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer.

Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes. To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo. TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence. Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX. These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.

Effect of Hypoxia Preconditioning on the Regenerative Capacity of Adipose Tissue Derived Mesenchymal Stem Cells in a Model of Renal Artery Stenosis.

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

Analysis of archaic human haplotypes suggests that 5hmC acts as an epigenetic guide for NCO recombination

BackgroundNon-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates.MethodsNCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans.ResultsWe detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5′- and 3′-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and ‘poised’ bivalent regions.ConclusionOur results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci.

Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells.

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.

Ten-eleven translocation protein 1 modulates medulloblastoma progression

BackgroundMedulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes.ResultsWe investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes.ConclusionsThese results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Tfeb Links MYC Signaling to Epigenetic Control of Acute Myeloid Leukemia Cell Death and Differentiation

MYC gene amplification and somatic mutations are frequent in both adult and pediatric AML although how MYC drives and contributes to the development and maintenance of AML has not been resolved. Transcription factor EB (TFEB) is a master regulator of genes that control autophagy and lysosome biogenesis, a central catabolic recycling pathway that regulates cell survival. Given the oncogenic effects of MYC in AML and that the induction of autophagy compromises AML cell growth and survival, we tested if the oncogenic effect of MYC depends on its suppression of TFEB transcription programs in AML. In support of this hypothesis, inducible MYC expression in K562 and THP-1 leukemia cells was sufficient to suppress expression of TFEB and its target genes. Further and conversely, MYC knockdown in NB4 AML cells provoked increased expression of TFEB mRNA and protein, as well as increased expression of TFEB target genes. Notably, dose response studies demonstrated that expression of TFEBS211A, constitutively nuclear form of TFEB that is refractory to control by mTORC1 signaling, dramatically impairs proliferation of HL60, OCI-AML2 and OCI-AML3 AML cells. In addition, induction of TFEBS211A provoked rampant apoptosis. Of important, overexpression of TFEBS211A in HL-60 and OCI-AML3 cells was also sufficient to promote monocytic and granulocytic differentiation, as judged by morphological changes and the acquisition of mature monocytic and granulocytic markers including CD11b, Gr1, and CD15. To identify TFEB targets that might contribute to myeloid/granulocytic differentiation, we performed RNA-seq analysis of HL60 leukemia cells engineered to inducibly express the TFEBS211A transgene. Using a cut-off of fold change>4 with q<0.01, a total of 1152 genes were differentially regulated following the induction of TFEBS211A in HL60 cells. As expected, this included the robust induction of nearly all TFEB target genes associated with the autophagy-lysosome pathway, but also of STAT1, KLF4, KLF6, CEBPB, CSF1 and GATA2 genes that are necessary and/or sufficient to provoke terminal monocytic and granulocytic differentiation of AML cells. Surprisingly, among genes induced by TFEB in HL60 cells is IDH1, which catalyzes the production of α-ketoglutarate (α-KG), a required substrate of the TET family of dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In particular, TFEBS211A expression provoked increases in levels of α-KG and significant increases in global levels of 5hmC in genomic DNA of HL60 leukemia cells both ex vivo and in vivo. Furthermore, TFEB-mediated induction of IDH1/2 mRNA and protein, and of IDH1 promoter activity, was antagonized by inducible expression of MYC. To assess the global effects of TFEB on 5mC/5hmC landscapes, we performed paired reduced representation bisulfite (BS)- and oxidation bisulfite (oxBS)-sequencing. As predicted, TFEBS211A induced both loss and gains of 5mC, but there were more losses (n = 722) than gains (n = 459) across all 22 chromosomes. Quite remarkably, and consistent with TFEB-provoked increases of 5hmC signals, TFEBS211A exclusively induced 5hmC gains (in a total of 863 genes), and 37% and 36% of these 5hmC gains occurred in promoter regions and CpG islands, and across all 22 chromosomes. Comparison of BS- and oxBS-seq versus RNA-seq analyses of HL60 cells expressing TFEB revealed significant changes in mRNA levels and concomitant differential changes in 5mC and 5hmC marks in KLF4, KLF6, STAT3, TP73, andFOXO1 that have pivotal roles in controlling myeloid cell differentiation and death. Collectively, these findings demonstrate that MYC suppresses TFEB expression and function in AML cells, and that TFEB functions as a tumor suppressor that provokes AML cell differentiation and death. Strikingly, these responses rely on epigenetic control, where TFEB directly induces the transcription of IDH1 and IDH2 to provoke global hydroxylation of 5-methylcytosine and the expression of genes that drive terminal differentiation and apoptosis. Thus, a MYC-TFEB-IDH1/2-TET2 circuit controls AML cell fate. Disclosures Murphy: Merck: Research Funding; Puma Biotech: Research Funding. Ballabio:CASMA Therapeutics: Other: Co-Founder. Kaufmann:Takeda Pharmaceuticals: Research Funding.

Mapping 5-Hydroxymethylcytosine (5hmC) Modifications in Skeletal Tissues Using High-Throughput Sequencing.

Cytosine modifications can alter the epigenetic landscape of a cell, affecting the binding of transcription factors, chromatin organizing complexes, and ultimately affecting gene expression and cell fate. 5-Hydroxymethylcytosine (5hmC) modifications are generated by the Ten-eleven-translocation (TET) family of enzymes, TET 1, 2, and 3, through the oxidation of methylated cytosines (5mC). The TET family is capable of further oxidizing 5hmC to 5fC and 5caC, leading to eventual DNA demethylation. However, 5hmC marks can also exist stably in DNA. Stable 5hmC is enriched in the gene bodies of activated genes in multiple tissues, as well as associated with regulatory regions such as enhancers. Alterations to 5hmC patterns have now been found in multiple diseases including osteoarthritis. Here, we describe a method to map 5hmC modifications by next-generation sequencing using a technique based on the selective modification and enrichment of the 5hmC mark. We additionally provide a bioinformatic analysis pipeline to interpret the resulting data.

Global epigenetic alterations of mesenchymal stem cells in obesity: the role of vitamin C reprogramming

ABSTRACT Obesity promotes dysfunction and impairs the reparative capacity of mesenchymal stem/stromal cells (MSCs), and alters their transcription, protein content, and paracrine function. Whether these adverse effects are mediated by chromatin-modifying epigenetic changes remains unclear. We tested the hypothesis that obesity imposes global DNA hydroxymethylation and histone tri-methylation alterations in obese swine abdominal adipose tissue-derived MSCs compared to lean pig MSCs. MSCs from female lean (n = 7) and high-fat-diet fed obese (n = 7) domestic pigs were assessed using global epigenetic assays, before and after in-vitro co-incubation with the epigenetic modulator vitamin-C (VIT-C) (50 μg/ml). Dot blotting was used to measure across the whole genome 5-hydroxyemthycytosine (5hmC) residues, and Western blotting to quantify in genomic histone-3 protein tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. MSC migration and proliferation were studied in-vitro. Obese MSCs displayed reduced global 5hmC and H3K4m3 levels, but comparable H3K9me3 and H3K27me3, compared to lean MSCs. Global 5hmC, H3K4me3, and HK9me3 marks correlated with MSC migration and reduced proliferation, as well as clinical and metabolic characteristics of obesity. Co-incubation of obese MSCs with VIT-C enhanced 5hmC marks, and reduced their global levels of H3K9me3 and H3K27me3. Contrarily, VIT-C did not affect 5hmC, and decreased H3K4me3 in lean MSCs. Obesity induces global genomic epigenetic alterations in swine MSCs, involving primarily genomic transcriptional repression, which are associated with MSC function and clinical features of obesity. Some of these alterations might be reversible using the epigenetic modulator VIT-C, suggesting epigenetic modifications as therapeutic targets in obesity.

TET1 promotes growth of T-cell acute lymphoblastic leukemia and can be antagonized via PARP inhibition.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in T-ALL and is crucial for human T-ALL cell growth in vivo. Knockout of TET1 in mice and knockdown in human T cell did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was dependent on its catalytic property to maintain global 5-hydroxymethylcytosine (5hmC) marks, thereby regulate cell cycle, DNA repair genes, and T-ALL associated oncogenes. Furthermore, overexpression of the Tet1-catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of T-ALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in T-ALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in T-ALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks, and antagonized leukemic growth of T-ALL cells, opening a therapeutic avenue for this disease.

5-Hydroxymethylcytosine in cord blood and associations of DNA methylation with sex in newborns.

DNA methylation has been widely studied for associations with exposures and health outcomes. Both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are epigenetic marks that may function differently to impact gene expression; however, the most commonly used technology to assess methylation for population studies in blood use are the Illumina 450K and EPIC BeadChips, for which the traditional bisulfite conversion does not differentiate 5mC and 5hmC marks. We used a modified protocol originally developed by Stewart et al. to analyse oxidative bisulfite-converted and conventional bisulfite-converted DNA for the same subject in parallel by the EPIC chip, allowing us to isolate the two measures. We measured 5mC and 5hmC in cord blood of 41 newborn participants of the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort and investigated differential methylation of 5mC + 5hmC, isolated 5mC and isolated 5hmC with sex at birth as an example of a biological variable previously associated with DNA methylation. Results showed low levels of 5hmC throughout the epigenome in the cord blood samples in comparison to 5mC. The concordance of autosomal hits between 5mC + 5hmC and exclusive 5mC analyses were low (25%); however, overlap was larger with increased effect size difference. There were 43 autosomal cytosine nucleotide followed by a guanine nucleotide (CpG) sites where 5hmC was associated with sex, 21 of which were unique to 5hmC after adjustment for cell composition. 5hmC only accounts for a small portion of overall methylation in cord blood; however, it has the potential to impact interpretation of combined 5hmC + 5mC studies in cord blood, especially given that effect sizes of differential methylation analyses are often small. Several significant CpG sites were unique to 5hmC, suggesting some functions distinct from 5mC. More studies of genome-wide 5hmC in children are warranted.

Indoleamine 2,3-Dioxygenase-1 Expressing Dendritic Cell Populations Are Associated with Tumor-Induced Immune Tolerance & Aggressive Disease Biology in Chronic Myelomonocytic Leukemia

Introduction:Chronic myelomonocytic leukemia (CMML) is a chronic myeloid malignancy associated with monocytosis, autoimmunity (~30%) & an inherent risk for leukemic transformation. Bone marrow (BM) dendritic cell (DC) populations occur in ~30% of patients, with a poorly defined biological & prognostic role. The malignant immune microenvironment is regulated by indoleamine 2,3-dioxygenase-1 (IDO-1) expressing DCs, which modulate regulatory T (Treg) cells & block their conversion into proinflammatory T helper (Th17)-like cells. IDO-1 is a known immune checkpoint & functions by catabolizing tryptophan, an amino acid essential for T cell function. We hypothesized that distinct IDO-1 expressing DC populations in CMML modulate Tregs & contribute towards immune tolerance & aggressive disease biology.Methods:Primary diagnostic CMML peripheral blood mononuclear cells (PBMC) & BM biopsy specimens were obtained after Mayo Clinic IRB approval. A DC population was defined on H&E stained biopsy sections as focal collections (>10) of cells with characteristic elongated nuclei & cytoplasmic extensions. Transcriptomic & protein expression studies assessing IDO-1 expression were done by previously described methods. In addition, IHC expression of PD-1, PD-L1 & CTLA-4 was also done. IDO-1 promoter methylation studies with DIP-seq were performed. The impact on immune tolerance was assessed using mass cytometry (CyTOF).Results:Cohort: Twenty eight patients with CMML were included in the study, median age 70 (range: 51-80) years; 71% males. Eleven (39%) patients had coexisting autoimmune conditions. Of these, 8 (73%) had detectable DC populations either at diagnosis, or during the course of their disease. At a median follow-up of 46 (95% CI 27, 84) months, there were 14 (50%) deaths & 9 (32%) leukemic transformations.IHC results: Nine (32%) patients were identified to have a DC population at CMML diagnosis. CD123 & TCL1 staining was performed in 5 (56%) patients, with 3 being positive for both, & 2 positive for CD123 only (additional IHC studies ongoing). IDO1 expression by IHC was documented in all 9 (100%) cases (Fig 1A & 1B), while rare populations of PD-1, PD-L1 & CTLA-4 lymphocytes were also seen in all cases. Due to the low DC burdens (median cellularity ≤ 5%) & uniform staining intensity, IHC-based grading was not done. Samples at serial time-points, post-HMA therapy & at the time of blast transformation, were available in 5 & 3 patients respectively. Among the patients who did not have DC populations at diagnosis, 5 (42%) developed them post-HMA therapy, while 3 (50%) developed them at the time of LT. The development of DC populations was associated with loss of response to HMA (50%) & disease progression (50%).Transcriptomic analysis: RNA expression data was available on 7 (25%) patients, of whom only 1 (14%) had DC populations at diagnosis. The IDO-1 RPKM value in the former was higher than the mean pooled value in the latter group (330 versus 74, p=0.05).Methylation studies: DIP-seq was performed on 12 (43%) cases from the primary IHC cohort. Qualitative analysis of IDO-1 promoter hypomethylation was conducted & confirmed in all 9 (100%) cases with 5-mC & 5-hmC marks compared to input as displayed in figure 1C.Immune profiling: CyTOF was performed on 4 CMML samples (3 with IDO-1 expressing DC populations at diagnosis) from the primary IHC cohort & compared to a normal PBMC control. Results confirmed an increase in DC populations (fig 1D& 1E), & reduced % of Th17-like T cells in CMML samples compared to control (1.1 versus 5.07, p=0.05, fig 1F).Clinical correlates & survival analysis: With the exception that CMML patients with DC populations had a higher frequency of NRAS (P=0.007) mutations, the two groups were comparable for cytogenetic & molecular abnormalities. The median OS for the cohort was 45 (95% CI 29, 84) months. CMML patients with IDO-1 expressing DC populations at diagnosis had a shorter median OS, in comparison to those without (median OS 30 vs 45, p=0.03, Kaplan-Meier analysis in fig 1G).Conclusions:In conclusion, we demonstrate that DC populations are seen in ~30% of patients with CMML with a uniform expression of IDO-1 & limited expression of PD-1, PD-L1 & CTLA-4. CMML patients with BM DC populations have a higher frequency of NRAS mutations & DC IDO-1 expression is associated with tumor induced immune tolerance. Additional IHC, genomic & preclinical studies with IDO-1 inhibitors are ongoing. [Display omitted] DisclosuresAl-Kali:Novartis: Research Funding.

Longitudinal Effects of Developmental Bisphenol A Exposure on Epigenome-Wide DNA Hydroxymethylation at Imprinted Loci in Mouse Blood.

Background:Epigenetic machinery plays an important role in genomic imprinting, a developmental process that establishes parent-of-origin–specific monoallelic gene expression. Although a number of studies have investigated the role of 5-methylcytosine in imprinting control, the contribution of 5-hydroxymethylcytosine (5-hmC) to this epigenetic phenomenon remains unclear.Objectives:Using matched mouse blood samples (from mice at 2, 4, and 10 months of age), our objective was to examine the effects of perinatal bisphenol A (BPA) exposure ( diet) on longitudinal 5-hmC patterns at imprinted regions. We also aimed to test the hypothesis that 5-hmC would show defined patterns at imprinted genes that persist across the life course.Methods:Genome-wide 5-hmC levels were measured using hydroxymethylated DNA immunoprecipitation sequencing (HMeDIP-seq). Modeling of differential hydroxymethylation by BPA exposure was performed using a pipeline of bioinformatics tools, including the csaw R package.Results:Based on BPA exposure, we identified 5,950 differentially hydroxymethylated regions (DHMRs), including 12 DHMRs that were annotated to murine imprinted genes—Gnas, Grb10, Plagl1, Klf14, Pde10a, Snrpn, Airn, Cmah, Ppp1r9a, Kcnq1, Phactr2, and Pde4d. When visualized, these imprinted gene DHMRs showed clear, consistent patterns of differential 5-hmC by developmental BPA exposure that persisted throughout adulthood.Conclusions:These data show long-term establishment of 5-hmC marks at imprinted loci during development. Further, the effect of perinatal BPA exposure on 5-hmC at specific imprinted loci indicates that developmental exposure to environmental toxicants may alter long-term imprinted gene regulation via an epigenetic mechanism. https://doi.org/10.1289/EHP3441

Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory

Epigenetic mechanisms such as DNA methylation and histone methylation are critical regulators of gene transcription changes during memory consolidation. However, it is unknown how these epigenetic modifications coordinate control of gene expression following reactivation of a previously consolidated memory. Here, we found that retrieval of a recent contextual fear conditioned memory increased global levels of H3 lysine 4-trimethylation (H3K4me3) and DNA 5-hydroxymethylation (5hmC) in area CA1 of the dorsal hippocampus. Further experiments revealed increased levels of H3K4me3 and DNA 5hmC within a CpG-enriched coding region of the Npas4, but not c-fos, gene. Intriguingly, retrieval of a 30-day old memory increased H3K4me3 and DNA 5hmC levels at a CpG-enriched coding region of c-fos, but not Npas4, in the anterior cingulate cortex, suggesting that while these two epigenetic mechanisms co-occur following the retrieval of a recent or remote memory, their gene targets differ depending on the brain region. Additionally, we found that in vivo siRNA-mediated knockdown of the H3K4me3 methyltransferase Mll1 in CA1 abolished retrieval-induced increases in DNA 5hmC levels at the Npas4 gene, suggesting that H3K4me3 couples to DNA 5hmC mechanisms. Consistent with this, loss of Mll1 prevented retrieval-induced increases in Npas4 mRNA levels in CA1 and impaired fear memory. Collectively, these findings suggest an important link between histone methylation and DNA hydroxymethylation mechanisms in the epigenetic control of de novo gene transcription triggered by memory retrieval.

Epigenomic landscape of 5-hydroxymethylcytosine reveals its transcriptional regulation of lncRNAs in colorectal cancer

Background:DNA methylation at the 5 position of cytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) by the ten–eleven translocation family. The loss of global levels of 5hmC has been regarded as a hallmark in various cancers. 5-hydroxymethylcytosine is distributed at protein-coding gene bodies and promoters; however, the role and distribution of 5hmC at long non-coding RNAs (lncRNAs) is not clear. We investigated the distribution and regulatory roles of 5hmC for lncRNAs in colorectal cancer (CRC).Methods:We integrated genome-wide profiles of 5hmC, 5mC, transcriptome and histone marks in CRC patients and examined the 5hmC-based clinical outcomes in patients.Results:5-hydroxymethylcytosine was distributed at lncRNA loci and positively correlated with lncRNA transcription. Dysreulated CRC lncRNAs were regulated by 5hmC directly or through abnormal activities of typical and super-enhancers and promoters modified by 5hmC. In addition, 5hmC was involved in long-range chromatin interactions at lncRNA loci. Finally, lncRNAs regulated by differential 5hmC marks were correlated with different clinical outcomes and tumour status in patients.Conclusions:5-hydroxymethylcytosine is critical in regulating the transcription of lncRNA and serve as novel biomarkers for clinical prognosis in CRC.

Correlated 5-Hydroxymethylcytosine (5hmC) and Gene Expression Profiles Underpin Gene and Organ-Specific Epigenetic Regulation in Adult Mouse Brain and Liver.

BackgroundDNA methylation is an epigenetic mechanism essential for gene regulation and vital for mammalian development. 5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. Aside from being a key intermediate in cytosine demethylation, 5hmC may have potential regulatory functions with emerging importance in mammalian biology.MethodsHere, we investigate the global 5hmC enrichment in five brain structures, including cerebellum, cerebral cortex, hippocampus, hypothalamus and thalamus, as well as liver tissues from female and male adult mice by using chemical capture-based technique coupled with next-generation sequencing. At the same time, we carried out total RNA sequencing (RNA-seq) to analyze the transcriptomes of brain regions and liver tissues.ResultsOur results reveal preferential 5hmC enrichment in the gene bodies of expressed genes, and 5hmC levels of many protein-coding genes are positively correlated with RNA expression intensity. However, more than 75% of genes with low or no 5hmC enrichment are genes encode for mitochondrial proteins and ribosomal proteins despite being actively transcribed, implying different transcriptional regulation mechanisms of these housekeeping genes. Brain regions developed from the same embryonic structures have more similar 5hmC profiles. Also, the genic 5hmC enrichment pattern is highly tissue-specific, and 5hmC marks genes involving in tissue-specific biological processes. Sex chromosomes are mostly depleted of 5hmC, and the X inactive specific transcript (Xist) gene located on the X chromosome is the only gene to show sex-specific 5hmC enrichment.ConclusionsThis is the first report of the whole-genome 5hmC methylome of five major brain structures and liver tissues in mice of both sexes. This study offers a comprehensive resource for future work of mammalian cytosine methylation dynamics. Our findings offer additional evidence that suggests 5hmC is an active epigenetic mark stably maintained after the global reprogramming event during early embryonic development.

TET1 Promotes Leukemic Growth in T-ALL Via Maintenance of 5-Hydroxymethylation Marks and Can be Antagonized By the PARP Inhibitor Olaparib

T-cell acute lymphoid leukemia (T-ALL) is an aggressive hematological cancer predominantly occurring in children and adolescents. T-ALL is characterized by aberrant expression of genes involved in T-cell differentiation and self-renewal accompanied by aberrant DNA cytosine methylation patterns. The mechanisms underlying these aberrations are not well understood. The Ten-eleven translocation 1 (TET1) enzyme is an iron-dependent dioxygenase that catalyzes the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation and regulation of gene expression. Using quantitative real time (qRT)-PCR we now demonstrate that T-ALL is characterized by high and aberrant expression of TET1: TET1 was significantly and >20-fold higher expressed in human T-ALL cell lines compared to other B-lymphoid or myeloid leukemia cell lines (p<0.001, n=3). This was confirmed in primary patient samples, in which TET1 was more than 10-fold higher expressed in 95% of T-ALL cases (n=10) compared to other leukemia subtypes (B-ALL, n=29; AML, n=39; CLL, n=23, p<0.001). Furthermore, expression levels of TET1 in T-ALL patient samples were over 50-fold higher than in normal PB and BM derived healthy CD3+ T cells (n=6, p<0.001) in line with published cDNA microarray data from a large patient cohort of 174 T-ALL cases (Haferlach et al., JCO 2010). Knockdown (KD) of TET1 mRNA using two independent shRNAs in T-ALL cell lines adversely affected their cell growth (>70% reduction compared to scrambled, p<0.001, n=4) clonogenicity (>70% reduction compared to scrambled, p<0.001, n=4) and blocked cells in the G2/M phase of the cell cycle in vitro (n=3). Furthermore, TET1 KD significantly reduced the leukemic engraftment potential of T-ALL cell line Jurkat in xenografts (shRNA, n=16; scrambled, n=8). Global analysis of 5hmC levels using hMeDIP-seq performed on TET1 depleted Jurkat cells revealed lower global 5hmC levels as compared to cells transduced with scrambled control (n=2). Promoters (-5kb TSS) of 5,181 genes showed reduction of 5hmC marks (log2 fold diff.; p-value < 1e-10). Global 5mC analysis (MeDIP-seq) revealed 12,007 genes that gained 5mC marks at their promoters as a result of TET1 KD (log2 fold diff. > 1.5; p-value < 1-10). Of note, genes demonstrating a loss of 5hmC marks and corresponding a gain in 5mC marks on their promoters belonged to gene families involved innucleosome organization (HIST family), DNA damage response (DDR)(RAD, PARP), T-cell development (NOTCH3, RUNX1, LEF1) and homeobox genes (q-value < 1e-5). These changes were paralleled by changes in expression after TET1 depletion as measured by RNA-Seq in Jurkat cells: GO TERM analysis (log2 fold diff. > 1.5; p<0.05) revealed deregulation of genes belonging to nucleosome assembly, DNA damage, nucleosome organization and transcriptional deregulation in cancer. In line with these observations these pathways showed differential expression in T-ALL patients divided into the categories high vs. low TET1 expression based on previously published microarray data (Haferlach et al., JCO 2010) (median as cut-off; n=174)(log2 fold diff. >1.5; p<0.05).The aberrantly high TET1 expression in T-ALL cell lines shares a potential causal relationship with the hypomethylated status of the TET1 promoter (determined by LC/MS), and the enrichment of euchromatic marks such as H3K4me3 and acetylated H3 as determined by ChIP-qPCR. Since it was shown that PARP activity positively influences TET1 expression by regulating its promoter epigenetically, we tested the impact of the PARP inhibitor olaparib on TET1 activity in our model: of note, aberrant expression of TET1 could be reverted by the PARP inhibitor olaparib, which induced reduction of euchromatic marks on the TET1 promoter, in parallel with decreased TET1 expression and protein levels as well as 5hmC levels in Jurkat cells without affecting expression of other TET family members. Olaparib treatment caused a >75% reduction in cell growth and clonogenicity of T-ALL cell lines in vitro (n=3) and delayed leukemic engraftment in NSG mice (42%±8 in scrambled vs 9%±7 for the shRNAs, n=4 per arm; p<0.001). In conclusion, these data demonstrate for the first time that TET1 plays an important oncogenic role in T-ALL. We also identify PARPs as upstream regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologically in this poor prognosis leukemia. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

Neurofilament-labeled pyramidal neurons and astrocytes are deficient in DNA methylation marks in Alzheimer's disease

There is increasing evidence that epigenetic alterations may play a role in Alzheimer's disease (AD); yet, there is little information regarding epigenetic modifications in specific cell types. We assessed DNA methylation (5-methylcytosine [5mC]) and hydroxymethylation (5-hydroxymethylcytosine [5hmC]) marks specifically in neuronal and glial cell types in the inferior temporal gyrus of human AD cases and age-matched controls. Interestingly, neurofilament (NF)–labeled pyramidal neurons that are vulnerable to AD pathology are deficient in extranuclear 5mC in AD cases compared with controls. We also found that fewer astrocytes exhibited nuclear 5mC and 5hmC marks in AD cases compared with controls. However, there were no alterations in 5mC and 5hmC in disease-resistant calretinin interneurons or microglia in AD, and there was no alteration in the density of 5mC- or 5hmC-labeled nuclei in near-plaque versus plaque-free regions in late-AD cases. 5mC and 5hmC were present in a high proportion of neurofibrillary tangles, suggesting no loss of DNA methylation marks in tangle bearing neurons. We provide evidence that epigenetic dysregulation may be occurring in astrocytes and NF-positive pyramidal neurons in AD.

Abstract B1-32: Genome-wide integrative analysis correlating locus-specific DNA methylation and hydroxymethylation to gene expression in normal prostate cells

Abstract Aberrant epigenetic modification in the form of regional hypermethylation and global hypomethylation has been implicated in the dysregulation of gene expression in various cancers, including prostate cancer (PCa). Recently discovered 5-hydroxymethylated marks (5hmC), considered to be key intermediates in the process of DNA demethylation, have also been shown to exhibit significant global loss in solid tumors as compared to normal tissue. Similar to cytosine base methylation (5mC), 5hmC may also play a key role in the regulation of gene expression and, consequently, gene function in PCa. We hypothesize that dynamic interplay between 5mC and 5hmC enrichment in promoter, CpG island, and intragenic regions shows specific association patterns with gene expression. A systematic investigation of these methylation marks may provide novel insights into epigenomic architecture by elucidating novel pathways and candidate genes in prostate tumorigenesis. Using Next Generation Sequencing (NGS), we characterized genome-wide 5mC and 5hmC marks and RNA expression data in a normal prostate tissue-derived representative cell line (RWPE-1). We have completed NGS following methyl-binding protein capture (MBD-seq) and hydroxymethyl-selective chemical labeling (hMe-Seal), and have performed integrative analysis correlating enriched genes stratified by region (intergenic regions proximal to DNase I hypersensitivity sites, as well as promoter, CpG island, and intragenic regions) to expression data obtained from RNA-seq and also validated using publicly available microarray expression data (GEO Accession Number: GSM375783) We divided expression data into equal tiers representing genes with no expression, low expression, and high expression, and have performed pathway-based analysis on genes significantly enriched for either 5mC or 5hmC marks for each tier stratified by gene region. We found gene expression to exhibit a strong positive correlation with core promoter and CpG island hydroxymethylation, with a corresponding negative correlation to methylation of these regions. In contrast, gene expression was found to be positively correlated with both methylation and hydroxymethylation in both intragenic regions and intergenic regions proximal to ENCODE DNase I hypersensitivity sites (GEO Accession Number: GSM1008595), which are associated with open chromatin and may play a role in downstream gene regulation. Interestingly, pathway-based analysis of these stratified tiers, grouped via the Molecular Complex Detection algorithm (MCODE) to identify pathway clusters with high biological significance, showed 5hmC and 5mC enrichment of the same pathways in each expression tier, suggesting a possible co-operative role for intragenic co-expression of these marks in regulating biological pathways. Clustered pathways co-enriched in intragenic 5mC and 5hmC showed strong enrichment in Gene Ontology terms related to core cellular functions within each expression tier, such as ion channel and transport regulation, primary metabolic processes, and protein and RNA binding. Currently, we are investigating these pathways to identify key candidate genes regulated by 5hmC and 5mC marks in normal prostate. This is the first study to correlate locus-specific global 5hmC enrichment to expression in normal prostate cells. Our preliminary analysis has shown correlation between 5hmC and 5mC enrichment in genic, promoter, CpG island, and upstream DNase I hypersensitivity site-proximal regions and expression in RWPE-1 cells. Insights from this model will subsequently be explored via whole-genome differential analysis between normal prostate and PCa to determine the effect of epigenetic alterations in cancer on gene expression. Subsequently, our characterization of key cellular pathways exhibiting dynamic enrichment patterns for 5hmC or 5mC marks will potentially allow us to identify differentially epigenetically modified target genes implicated in prostate cancer tumorigenesis. Citation Format: Shivani N. Kamdar, Linh T. Ho, Ruth Isserlin, Ken J. Kron, Theodorus van der Kwast, Alexandre R. Zlotta, Neil E. Fleshner, Gary Bader, Bharati Bapat. Genome-wide integrative analysis correlating locus-specific DNA methylation and hydroxymethylation to gene expression in normal prostate cells. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-32.