Maintaining DNA Methylation Research Articles

1,25-Dihydroxyvitamin D3 increases the methionine cycle, CD4+ T cell DNA methylation and Helios+Foxp3+ T regulatory cells to reverse autoimmune neurodegenerative disease

We investigated how one calcitriol dose plus vitamin D3 reverses experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. This protocol rapidly increased CD4+ T cell Ikzf2 transcripts, Helios protein, and CD4+Helios+FoxP3+ T regulatory cells. It also rapidly increased CD4+ T cell Bhmt1 transcripts, betaine:homocysteine methyltransferase-1 (BHMT1) enzyme activity, and global DNA methylation. BHMT1 transmethylates homocysteine to replenish methionine. Targeting the Vdr gene in T cells decreased Ikzf2 and Bhmt1 gene expression, reduced DNA methylation, and elevated systemic homocysteine in mice with EAE. We hypothesize that calcitriol drives a transition from encephalitogenic CD4+ T cell to Treg cell dominance by upregulating Ikzf2 and Bhmt1, recycling homocysteine to methionine, reducing homocysteine toxicity, maintaining DNA methylation, and stabilizing CD4+Helios+FoxP3+Tregulatory cells. Conserved vitamin D-responsive element (VDRE)-type sequences in the Bhmt1 and Ikzf2 promoters, the universal need for methionine in epigenetic regulation, and betaine's protective effects in MTHFR-deficiency suggest similar regulatory mechanisms exist in humans.

Natural variation of DNA methylation and gene expression may determine local adaptations of Scots pine populations.

Long-lived conifers are vulnerable to climate change because classical evolutionary processes are slow in developing adaptive responses. Therefore, the capacity of a genotype to adopt different phenotypes is important. Gene expression is the primary mechanism that converts genome-encoded information into phenotypes, and DNA methylation is employed in the epigenetic regulation of gene expression. We investigated variations in global DNA methylation and gene expression between three Scots pine (Pinus sylvestris L.) populations located in northern and southern Finland using mature seeds. Gene expression levels were studied in six DNA methyltransferase (DNMT) genes, which were characterized in this study, and in 19 circadian clock genes regulating adaptive traits. In embryos, expression diversity was found for three DNMT genes, which maintain DNA methylation. The expression of two DNMT genes was strongly correlated with climate variables, which suggests a role for DNA methylation in local adaptation. For adaptation-related genes, expression levels showed between-population variation in 11 genes in megagametophytes and in eight genes in embryos, and many of these genes were linked to climate factors. Altogether, our results suggest that differential DNA methylation and gene expression contribute to local adaptation in Scots pine populations and may enhance the fitness of trees under rapidly changing climatic conditions.

A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1–Dio3 domain during embryonic development

Genomic imprinting is a phenomenon that causes parent-origin-specific monoallelic expression of a small subset of genes, known as imprinted genes, by parentally inherited epigenetic marks. Imprinted genes at the delta-like homolog 1 gene (Dlk1)-type III iodothyronine deiodinase gene (Dio3) imprinted domain, regulated by intergenic differentially methylated region (IG-DMR), are essential for normal development of late embryonic stages. Although the functions of IG-DMR have been reported by generating knockout mice, molecular details of the regulatory mechanisms are not fully understood as the specific sequence(s) of IG-DMR have not been identified. Here, we generated mutant mice by deleting a 216 bp tandem repeated sequence in IG-DMR, which comprised seven repeats of 24 bp motifs, by genome editing technologies. The mutant mice showed that paternal transmission of the deletion allele, but not maternal transmission, induces severe growth retardation and perinatal lethality, possibly due to placental defects. Embryos with a paternally transmitted deletion allele showed biallelic expression of maternally expressed genes and repression of paternally expressed genes. DNA methylation status also showed loss of methylation at IG-DMR and Gtl2-DMR, indicating that the tandem repeat sequence of IG-DMR is one of the functional sequences of IG-DMR, which is required for maintaining DNA methylation imprints of paternal allele at IG-DMR.

Subtle Perturbations of the Maize Methylome Reveal Genes and Transposons Silenced by Chromomethylase or RNA-Directed DNA Methylation Pathways.

DNA methylation is a chromatin modification that can provide epigenetic regulation of gene and transposon expression. Plants utilize several pathways to establish and maintain DNA methylation in specific sequence contexts. The chromomethylase (CMT) genes maintain CHG (where H = A, C or T) methylation. The RNA-directed DNA methylation (RdDM) pathway is important for CHH methylation. Transcriptome analysis was performed in a collection of Zea mays lines carrying mutant alleles for CMT or RdDM-associated genes. While the majority of the transcriptome was not affected, we identified sets of genes and transposon families sensitive to context-specific decreases in DNA methylation in mutant lines. Many of the genes that are up-regulated in CMT mutant lines have high levels of CHG methylation, while genes that are differentially expressed in RdDM mutants are enriched for having nearby mCHH islands, implicating context-specific DNA methylation in the regulation of expression for a small number of genes. Many genes regulated by CMTs exhibit natural variation for DNA methylation and transcript abundance in a panel of diverse inbred lines. Transposon families with differential expression in the mutant genotypes show few defining features, though several families up-regulated in RdDM mutants show enriched expression in endosperm tissue, highlighting the potential importance for this pathway during reproduction. Taken together, our findings suggest that while the number of genes and transposon families whose expression is reproducibly affected by mild perturbations in context-specific methylation is small, there are distinct patterns for loci impacted by RdDM and CMT mutants.

Np95/Uhrf1 regulates tumor suppressor gene expression of neural stem/precursor cells, contributing to neurogenesis in the adult mouse brain

Adult neurogenesis is a process of generating new neurons from neural stem/precursor cells (NS/PCs) in restricted adult brain regions throughout life. It is now generally known that adult neurogenesis in the hippocampal dentate gyrus (DG) and subventricular zone participates in various higher brain functions, such as learning and memory formation, olfactory discrimination and repair after brain injury. However, the mechanisms underlying adult neurogenesis remain to be fully understood. Here, we show that Nuclear protein 95 KDa (Np95, also known as UHRF1 or ICBP90), which is an essential protein for maintaining DNA methylation during cell division, is involved in multiple processes of adult neurogenesis. Specific ablation of Np95 in adult NS/PCs (aNS/PCs) led to a decrease in their proliferation and an impairment of neuronal differentiation and to suppression of neuronal maturation associated with the impairment of dendritic formation in the hippocampal DG. We also found that deficiency of Np95 in NS/PCs increased the expression of tumor suppressor genes p16 and p53, and confirmed that expression of these genes in NS/PCs recapitulates the phenotype of Np95-deficient NS/PCs. Taken together, our findings suggest that Np95 plays an essential role in proliferation and differentiation of aNS/PCs through the regulation of tumor suppressor gene expression in adult neurogenesis.

Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation.

UHRF1 plays multiple roles in regulating DNMT1-mediated DNA methylation maintenance during DNA replication. The UHRF1 C-terminal RING finger functions as an ubiquitin E3 ligase to establish histone H3 ubiquitination at Lys18 and/or Lys23, which is subsequently recognized by DNMT1 to promote its localization onto replication foci. Here, we present the crystal structure of DNMT1 RFTS domain in complex with ubiquitin and highlight a unique ubiquitin binding mode for the RFTS domain. We provide evidence that UHRF1 N-terminal ubiquitin-like domain (UBL) also binds directly to DNMT1. Despite sharing a high degree of structural similarity, UHRF1 UBL and ubiquitin bind to DNMT1 in a very distinct fashion and exert different impacts on DNMT1 enzymatic activity. We further show that the UHRF1 UBL-mediated interaction between UHRF1 and DNMT1, and the binding of DNMT1 to ubiquitinated histone H3 that is catalyzed by UHRF1 RING domain are critical for the proper subnuclear localization of DNMT1 and maintenance of DNA methylation. Collectively, our study adds another layer of complexity to the regulatory mechanism of DNMT1 activation by UHRF1 and supports that individual domains of UHRF1 participate and act in concert to maintain DNA methylation patterns.

DDM1 guards against telomere truncation in Arabidopsis.

Prolonged hypomethylation of DNA leads to telomere truncation correlated with increased telomere recombination, transposon mobilization and stem cell death. Epigenetic pathways, including DNA methylation, are crucial for telomere maintenance. Deficient in DNA Methylation 1 (DDM1) encodes a nucleosome remodeling protein, required to maintain DNA methylation in Arabidopsis thaliana. Plants lacking DDM1 can be self-propagated, but in the sixth generation (G6) hypomethylation leads to rampant transposon activation and infertility. Here we examine the role of DDM1 in telomere length homeostasis through a longitudinal study of successive generations of ddm1-2 mutants. We report that bulk telomere length remains within the wild-type range for the first five generations (G1-G5), and then precipitously drops in G6. While telomerase activity becomes more variable in later generation ddm1-2 mutants, there is no correlation between enzyme activity and telomere length. Plants lacking DDM1 also exhibit no dysregulation of several known telomere-associated transcripts, including TERRA. Instead, telomere shortening coincides with increased G-overhangs and extra-chromosomal circles, consistent with deletional recombination. Telomere shortening also correlates with transcriptional activation of retrotransposons, and a hypersensitive DNA damage response in root apical meristems. Since abiotic stresses, including DNA damage, stimulate homologous recombination, we hypothesize that telomere deletion in G6 ddm1-2 mutants is a by-product of elevated genome-wide recombination in response to transposon mobilization. Further, we speculate that telomere truncation may be beneficial in adverse environmental conditions by accelerating the elimination of stem cells with aberrant genomes.

The DNA methyltransferase family: a versatile toolkit for epigenetic regulation.

The DNA methyltransferase (DNMT) family comprises a conserved set of DNA-modifying enzymes that have a central role in epigenetic gene regulation. Recent studies have shown that the functions of the canonical DNMT enzymes - DNMT1, DNMT3A and DNMT3B - go beyond their traditional roles of establishing and maintaining DNA methylation patterns. This Review analyses how molecular interactions and changes in gene copy numbers modulate the activity of DNMTs in diverse gene regulatory functions, including transcriptional silencing, transcriptional activation and post-transcriptional regulation by DNMT2-dependent tRNA methylation. This mechanistic diversity enables the DNMT family to function as a versatile toolkit for epigenetic regulation.

DNMT1 overexpression predicting gastric carcinogenesis, subsequent progression and prognosis: a meta and bioinformatic analysis.

DNMT1 is important in maintaining DNA methylation, and participates in the oncogenesis via up- or down-regulation leading to hyper-methylation or hypo-methylation. In the meta and bioinformatic analysis, we found that DNMT1 expression was higher in gastric cancer, compared with normal (p < 0.00001), para-cancerous (p = 0.0004) and dysplasia (p < 0.00001) tissues. DNMT1 up-regulation was associated with gender (OR = 2.27, p = 0.006), differentiation (OR = 0.21, p = 0.01) and TNM stage (OR = 0.31, p = 0.0005). Through TCGA database, DNMT1 overexpression increased gastric cancer risk, but unrelated with clinicopathological parameters and prognosis. Kaplan-Meier plotter showed, an increasing expression of DNMT1 was positive for overall survival rates of patients with stage III and IV (P = 0.044; P = 0.047), N2 and N1-3 phases of lymph node metastasis (P = 0.023; P = 0.032), as well as those with or without distant metastasis (P = 0.0052; P = 0.021). For DNMT1 negative patients, the progression-free survival rates was better in patients with Her2+ or Her2- than positive ones (P = 0.00015; P = 0.031). Besides, surgery alone was effective for the overall survival rates in patients with DNMT1 high expression (P = 0.035), while 5-Fu was useful for those with low expression (P < 0.05). In conclusion, these findings provided evidence that DNMT1 expression might be employed as a potential marker to indicate gastric carcinogenesis and subsequent progression, even prognosis.

5-Aza-2′-deoxycytidine, a DNA methylation inhibitor, induces cytotoxicity, cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells

ABSTRACTEpigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 μmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.

Potential Roles of Intrinsic Disorder in Maternal-Effect Proteins Involved in the Maintenance of DNA Methylation.

DNA methylation is an important epigenetic modification that needs to be carefully controlled as a prerequisite for normal early embryogenesis. Compelling evidence now suggests that four maternal-effect proteins, primordial germ cell 7 (PGC7), zinc finger protein 57 (ZFP57), tripartite motif-containing 28 (TRIM28) and DNA methyltransferase (cytosine-5) 1 (DNMT1) are involved in the maintenance of DNA methylation. However, it is still not fully understood how these maternal-effect proteins maintain the DNA methylation imprint. We noticed that a feature common to these proteins is the presence of significant levels of intrinsic disorder so in this study we started from an intrinsic disorder perspective to try to understand these maternal-effect proteins. To do this, we firstly analysed the intrinsic disorder predispositions of PGC7, ZFP57, TRIM28 and DNMT1 by using a set of currently available computational tools and secondly conducted an intensive literature search to collect information on their interacting partners and structural characterization. Finally, we discuss the potential effect of intrinsic disorder on the function of these proteins in maintaining DNA methylation.

Abstract LB-091: CpG island DNA methylation regulates alternative cleavage and polyadenylation

Abstract Abnormal DNA methylation pattern is a hallmark of human cancer, and our work in genomic profiling of DNA methylation patterns in prostate and colon cancers have identified the majority of unique DNA hypermethylation to occur outside of gene promoters. While promoter hypermethylation causes transcriptional silencing, the functions of non-promoter DNA methylation are poorly defined. Thus, we began investigating the impact of non-promoter DNA methylation on the transcriptome and specifically focused on studying the functions of DNA methylation near gene 3' ends. Using a pair of isogenic cancer cells (HCT116 and DKO cells) that differ specifically in their ability to maintain DNA methylation, we discovered an intriguing association between gene 3' end differential DNA methylation and alternative cleavage and polyadenylation (APA) events. Briefly, pre-mRNAs undergo cleavage and polyadenylation as part of normal mRNA 3' end formation, and alternative sites of cleavage and polyadenylation can be utilized to produce transcripts with varying regulatory sequences in the 3' untranslated regions (3' UTRs) or protein isoforms via APA within coding sequences. Previous studies have demonstrated that cancer cells can hijack the APA pathway to skew expression of short 3' UTRs in oncogenes to evade negative regulation, highlighting APA as an important process involved in cancer initiation and progression. By using pharmacologic inhibition of DNA methylation and reporter constructs, we demonstrated that CpG island DNA methylation near polyadenylation sites is necessary and sufficient for determining relative usage, most likely by modulating RNA polymerase II elongation rate. By leveraging the Cancer Genome Atlas (TCGA) and the Roadmap Epigenomics data, we could observe the correlation between gene 3' DNA methylation and APA at select loci in vivo. The results of our study will improve overall functional understanding of non-promoter DNA methylation, provide a novel mechanism for APA regulation, and ultimately accelerate discovery of novel targets for cancer management. Note: This abstract was not presented at the meeting. Citation Format: Thomas Sweet, Lihua Jin, Josephine Dermawan, Angela H. Ting. CpG island DNA methylation regulates alternative cleavage and polyadenylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-091. doi:10.1158/1538-7445.AM2017-LB-091

P53 is essential for DNA methylation homeostasis in naïve embryonic stem cells, and its loss promotes clonal heterogeneity.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53-/-) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53-/- mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53-/- mESCs display increased cellular heterogeneity both in the "naïve" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.

The epigenetic integrator UHRF1: on the road to become a universal biomarker for cancer.

Cancer is one of the deadliest diseases in the world causing record number of mortalities in both developed and undeveloped countries. Despite a lot of advances and breakthroughs in the field of oncology still, it is very hard to diagnose and treat the cancers at early stages. Here in this review we analyze the potential of Ubiquitin-like containing PHD and Ring Finger domain 1 (UHRF1) as a universal biomarker for cancers. UHRF1 is an important epigenetic regulator maintaining DNA methylation and histone code in the cell. It is highly expressed in a variety of cancers and is a well-known oncogene that can disrupt the epigenetic code and override the senescence machinery. Many studies have validated UHRF1 as a powerful diagnostic and prognostic tool to differentially diagnose cancer, predict the therapeutic response and assess the risk of tumor progression and recurrence. Highly sensitive, non-invasive and cost effective approaches are therefore needed to assess the level of UHRF1 in patients, which can be deployed in diagnostic laboratories to detect cancer and monitor disease progression.

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.

Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential.

High-resolution profiles of gene expression and DNA methylation highlight mitochondrial modifications during early embryonic development.

Well-organized mitochondrial functions and dynamics are critical for early embryonic development and are operated via a large number of mitochondria-related genes (MtGs) encoded by both the nuclear and the mitochondrial genome. However, the mechanisms underlying mitochondrial modifications during the critical window between blastocyst implantation and postimplantation organogenesis are poorly understood. Herein, we performed high-resolution dynamic profiling of MtGs to acquire a more detailed understanding of mitochondrial modifications during early development. Our data suggest that the resumption of mitochondrial mass growth is not only facilitated by increased mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication, but also by the appropriate balance between mitochondrial fission and fusion. In addition, increased levels of reactive oxygen species (ROS) resulting from enhanced mitochondrial functions may be the critical inducer for activating the glutathione (GSH)-based stress response system in early embryos. The appropriate balance between the mitochondrial stress response and apoptosis appears to be significant for cell differentiation and early organogenesis. Furthermore, we found that most MtGs undergo de novo promoter methylation, which may have functional consequences on mitochondrial functions and dynamics during early development. We also report that mtDNA methylation can be observed as early as soon after implantation. DNMT1, the predominant enzyme for maintaining DNA methylation, localized to the mitochondria and bound to mtDNA by the implantation stage. Our study provides a new insight into the involvement of mitochondria in early mammalian embryogenesis. We also propose that the epigenetic modifications during early development are significant for modulating mitochondrial functions and dynamics.

Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL) is a heterogeneous neoplasm and patients with relapsed/refractory disease exhibit resistance to standard cytotoxic therapies. We have previously demonstrated that the MUC1-C oncoprotein plays a critical role in regulating apoptosis and protection from oxidative stress in CTCL. Targeting of MUC1-C with a pharmacologic inhibitor, GO-203, was associated with late apoptosis and early necrosis and slowing of tumor growth in in vitro and in vivo models of CTCL, respectively. However, disease responses following MUC1-C inhibition were incomplete underscoring the need for combinatorial strategies that could exploit the vulnerability of CTCL cells to oxidative signals. Decitabine is a DNA methyltransferase inhibitor, previously shown to modulate oxidative stress in acute myeloid leukemia models. We hypothesized that treatment with decitabine and GO-203 would present a potent novel therapeutic combination for patients with CTCL.Method/Results: Our studies demonstrate that exposure of CTCL cells to decitabine in combination with GO-203, increased the generation of reactive oxygen species (ROS) and decreased levels of scavenger molecules, NADPH, glutathione (GSH), and TIGAR, a p53-inducible regulator of glycolysis and apoptosis, critical to intracellular redox homeostasis. This in turn led to marked increase in the ratio of stress activated proteins; phospho-p38 and phospho-JNK to Total-p38 and Total-JNK repectively. Further dual exposure to GO-203 and decitabine resulted in near abrogation of DNA methyl transferase 1 and 3b crucial for maintaining DNA methylation, demonstrating significant synergy of these agents for inducing hypomethylation. In concert with these findings, treatment with decitabine and GO-203 upregulated the ROS generating enzyme, NADPH oxidase 4 (Nox4) potentially due to their effect on epigenomic regulation of this protein. In concert with these findings, exposure to decitabine and GO-203 resulted in heightened apoptotic death of CTCL cell lines, primary patient derived samples and in a aggressive murine xenograft model.Conclusion: These findings indicate that decitabine augments MUC1-C inhibition induced redox imbalance and provides a novel combination of targeted and epigenetic agents for treatment of patients with CTCL. Accordingly an early phase multi-institution clinical trial to to investigate this combinatorial strategy based on epigenetic modulation of redox homeostasis in patients with relapsed/refractory T cell lymphomas is being planned. DisclosuresRosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding. Arnason:Gilead: Consultancy. Kufe:Genus Oncology LLC: Consultancy, Equity Ownership.

Dicer-independent RNA-directed DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.