DNA Methylation Enzymes Research Articles

Tibolone treatment after traumatic brain injury exerts a sex-specific and Y chromosome-dependent regulation of methylation and demethylation enzymes and estrogen receptors in the cerebral cortex

Tibolone, a synthetic steroid used for the treatment of climacteric symptoms, displays sex-specific protective actions in experimental models of brain diseases. Previous in vitro findings suggest that tibolone reduces oxidative stress and neuroinflammation through the regulation of DNA methylation and the activation of estrogen receptors (ERs) α and β. In this study, we assessed whether tibolone regulates the expression of genes coding for DNA methylation and demethylation enzymes and ERs in the injured cerebral cortex of animals suffering a traumatic brain injury. The four-core genotype mouse model was used to determine whether the effect of tibolone on gene regulation was influenced by gonads or by cell-autonomous actions of sex chromosomes. Tibolone treatment resulted in sex-specific modification in the expression of genes coding for DNA methyl transferases (Dnmt) 3a, and 3b, for growth arrest and DNA-damage-inducible proteins (Gadd) 45β and 45γ, and for ERα and ERβ. In contrast, tibolone did not affect the expression of genes coding for Dnmt1, Gadd45α, and ten-eleven translocation methylcytosine dioxygenases 1–3. The sex-specific effect of tibolone on Dnmt3a expression depended on gonadal sex. In contrast, the presence or absence of the Y chromosome determined the effect of tibolone on Dnmt3b, Gadd45β, Gadd45γ, ERα and ERβ expression. These findings suggest that tibolone exerts a sex-specific regulation of DNA methylation and ER expression in the injured cerebral cortex that is determined by a combination of gonadal effects and cell-autonomous actions of sex chromosome genes.

Berberine Inhibits KLF4 Promoter Methylation and Ferroptosis to Ameliorate Diabetic Nephropathy in Mice.

Inflammation, oxidative stress, fibrosis, and ferroptosis play important roles in diabetic nephropathy development. Krüppel-like factor 4 (KLF4) is a transcriptional factor, which regulates multiple cell processes and is involved in diabetic nephropathy. Berberine has various biological activities, including anti-inflammation, antioxidative stress, and antiferroptosis. Berberine has been shown to inhibit diabetic nephropathy, but whether it involves KLF4 and ferroptosis remains unknown. We established a diabetic nephropathy mice model and administered berberine to the mice. The kidney function, renal structure and fibrosis, expression of KLF4 and DNA methylation enzymes, DNA methylation of the KLF4 promoter, mitochondria structure, and expression of oxidative stress and ferroptosis markers were analyzed. Berberine rescued kidney function and renal structure and prevented renal fibrosis in diabetic nephropathy mice. Berberine suppressed the expression of DNMT1 and DNMT2 and upregulated KLF4 expression by preventing KLF4 promoter methylation. Berberine inhibited the expression of oxidative stress and ferroptosis markers, maintained mitochondria structure, and prevented ferroptosis. Berberine ameliorates diabetic nephropathy by inhibiting Klf4 promoter methylation and ferroptosis.

Cost-effective solutions for high-throughput enzymatic DNA methylation sequencing.

Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)-which profiles ∼4 million CpG sites-for miniaturization, flexibility, and multispecies use at a cost of ∼$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R² = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R² = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations-the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia-and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.

PATH-11. FEASIBILITY OF BRAIN TUMOR CLASSIFICATION BY ENZYMATIC DNA METHYLATION SEQUENCING ANALYSIS OF CELL-FREE DNA OBTAINED FROM CEREBROSPINAL FLUID

Abstract BACKGROUND Array-based DNA methylation profiling is the gold standard for molecular classification of brain tumors. However, it relies on significant amount of input DNA extracted from surgical tissue, thus limiting its use for tumors where biopsy is challenging or limited in quantity. Cell-free tumor DNA (cfDNA) in cerebrospinal fluid (CSF) presents alternative opportunities for brain tumor diagnosis and disease monitoring following treatment. Novel enzymatic DNA methylation sequencing (EM-seq) methods may allow us to overcome input DNA limitations and accurately quantify methylation from cfDNA. METHODS We performed methylation sequencing using the NEBNext EM-seq kit on cfDNA from archival CSF samples collected from three brain tumor patients with confirmed histopathological diagnoses. Variable amounts of input cfDNA (0.1ng-10ng) were tested. We utilized the methylseq pipeline for data processing. For tumor classification, data was limited to CpG sites overlapping the MethylationEPIC array before analysis using MNP-Flex, a modified platform-agnostic version of the Heidelberg methylation classifier RESULTS Using the EM-seq method, genomic coverage for 10 and 1ng input DNA samples (average: 46x and 26x, respectively) was sufficient for generating global methylation profiles. Samples with 0.1ng input showed an average coverage of only 5.32x due to high levels of read duplication. However, methylation levels for CpG sites with at least 5x coverage were highly correlated across varying input DNA amounts, suggesting that lower input cfDNA could still be used for tumor classification based on relevant CpG sites. CONCLUSIONS Using the MNP-Flex classifier, which was originally trained with methylation array data from tumor tissue, we successfully predicted brain tumor types (both high-grade glioma and medulloblastoma) with cfDNA methylation data down to only 0.1ng of input cfDNA, matching diagnosis based on tissue methylation and histopathology in this pilot study. Further classification of additional tumor types using CSF cfDNA is required to confirm the clinical utility of this platform.

Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Abstract 5242: In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors

Abstract CAR T cells are donor/patient-derived T cells genetically engineered to express a chimeric receptor against a specific tumor antigen (TA), which allows an MHC-independent recognition and targeting of tumor cells. While CART cell therapies have been tremendously successful against hematological cancers, with 6 T cell products being approved up to date from the FDA (Sengsayadeth S etal, EJHaem. 2022), the activity on solid tumors is more difficult. Solid tumors remain a unique challenge in part due to trafficking problems of CAR T cells, a hostile TME and heterogenous antigen expression, which limits CART cell capacity to find their target. Several strategies are being developed at the moment with the aim to overcome these hurdles, such as multispecific CART cells, co-therapy with checkpoint inhibitors, or inhibition of DNA methylation enzymes to prevent exhaustion. To offer an in vitro platform to assess the effectiveness of upcoming cellular therapies we developed several pipelines to allow the investigation of cytotoxic potential in 2D and 3D formats. We show here our results using a CART cell product expressing a coreceptor to enhance its activation potential upon recognition of the target. We next tested the capacity of these cells to kill solid tumor in vitro by coculturing with potential target cells cultured in a monolayer (2D). By measuring the luciferase expression of the target cells, we could track the cytotoxicity exerted by the effector cells. In vitro, the CART cells were able to kill in a dose dependent manner different solid tumor cell lines expressing the cognate antigen for the co-receptor. At Reaction Biology we have developed spheroid-based 3D killing assays in which target cells from solid tumor entities are pre-cultured to form spheroids prior to their coculture with effector cells. Using this technique, we challenged the capacity of these CART cells to recognize their antigen in a 3D format. This 3D assay system can be used as well in combination with co therapies such as PD1 inhibitors, in a sensitive and high throughput manner. Finally, we evaluated the therapeutic capacity of these CART cells in vivo in a subcutaneous THP-1 tumor model. Mice treated with these CART cells showed a marked reduction of tumor volume. In summary we show here a pipeline to characterize in vitro the effectivity of new T cell products, in particular those designed to overcome some of the hurdles against solid tumors. Citation Format: Carla N. Castro, Veronica Bergo, Holger Weber, Philipp Metzger. In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5242.

Activation of Hypoxia Inducible Factor-1 Alpha-Mediated DNA Methylation Enzymes (DNMT3a and TET2) Under Hypoxic Conditions Regulates S100A6 Transcription to Promote Lung Cancer Cell Growth and Metastasis.

Aims: This research was aimed at investigating the effects of hypoxia inducible factor-1 alpha (HIF-1α)-mediated DNA methylation enzymes (ten-eleven translocase-2 [TET2] and DNA methyltransferase-3a [DNMT3a]) under hypoxic conditions on S100A6 transcription, thereby promoting the growth and metastasis of lung cancer cells. Methods: The expression of HIF-1α or S100A6 in lung cancer cells was interfered with under normoxic and hypoxic conditions, and the cell proliferative, migratory, and invasive properties were assessed. The mechanism of HIF-1α-regulated TET2 and DNMT3 effects on S100A6 transcription under hypoxic conditions was further investigated. Results: Functionally, S100A6 over-expression promoted lung cancer cell proliferation and metastasis. S100A6 over-expression reversed the inhibitory effects of HIF-1α interference on the proliferation and metastasis of lung cancer cells. S100A6 was induced to express in an HIF-1α-dependent manner under hypoxic conditions, and silencing S100A6 or HIF-1α suppressed lung cancer cell proliferation and metastasis under hypoxic conditions. Further, The Cancer Genome Atlas-lung adenocarcinoma database analysis revealed that S100A6 mRNA levels had a negative correlation with methylation levels. Mechanistically, CpG hypomethylation status in the S100A6 promoter hypoxia response element had an association with HIF-1α induction. TET2 was enriched in S100A6 promoter region of lung cancer cells under hypoxic conditions, whereas DNMT3a enrichment was reduced in S100A6 promoter region. HIF-1α-mediated S100A6 activation was linked to DNMT3a-associated epigenetic inactivation and TET2 activation. Innovation: The activation of HIF-1α-mediated DNA methylation enzymes under hypoxic conditions regulated S100A6 transcription, thereby promoting lung cancer cell growth and metastasis. Conclusion: In lung cancer progression, hypoxia-induced factor HIF-1α combined with DNA methylation modifications co-regulates S100A6 transcriptional activation and promotes lung cancer cell growth and metastasis.

Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state.

JOURNAL/nrgr/04.03/01300535-202410000-00024/figure1/v/2024-02-06T055622Z/r/image-tiff Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

Identification of Chromatin Regulators Required for Enucleation

Erythropoiesis involves several distinct steps as precursor cells to differentiate and mature into erythrocytes which follows differentiation to proerythroblasts, transformation into basophilic, polychromatic, and orthochromatic erythroblasts, followed by enucleation to form reticulocytes and erythrocytes. Indeed, erythropoiesis and enucleation are complex processes that involve the coordinated action of multiple regulators. Various transcription factors, signaling pathways, and epigenetic modifiers play significant roles in controlling gene expression, cell differentiation, and cellular events during erythropoiesis. As an example, BCL11A is necessary for nucleus expulsion and the progression of erythropoiesis. Similarly, other transcription factors, histone deacetylases, DNA methylation enzymes, and chromatin remodeling complexes also contribute to the enucleation process. However, the exact role of chromatin regulators and how they are involved in the enucleation process is still not fully understood. To identify novel chromatin factors that regulate both enucleation and proliferation of erythroid cells, we conducted a pooled epigenome-wide CRISPR-Cas9 knockout screen using the human erythroid cell line BEL-A. The epigenetic knockout library consists of 719 genes associated with chromatin-related pathways and 35 essential genes, which include those encoding ribosomal proteins. Each gene is targeted by 10 single guide RNAs (sgRNAs). The primary objective of this screen was to determine essential chromatin regulators that play a critical role in the process of enucleation. To achieve this, we planned the screening groups as followed: (i) cells collected immediately after antibiotic selection (time zero), (ii) undifferentiated, expanded cells grown for 13 days, (iii) expelled nuclei (pyrenocytes), and (iv) orthochromatic erythroblasts that have not undergone enucleation. To isolate the distinct cellular stages mentioned above, we utilized fluorescence-activated cell sorting (FACS). The pyrenocyte group was compared with all other groups to identify depleted gRNAs, which represent factors that are necessary for enucleation. Setd8, which is an important epigenetic factor that is required for enucleation was determined to be depleted only in pyrenocytes (β score -0,433), validating our screen approach. In addition, we identified more than 15 other genes that showed even greater depletion than Setd8, and could be essential for enucleation. Among the identified genes, EP300, PCGF1, Setd1B, CDY2B, and TLK2 were found to be the most depleted in pyrenocytes. Additionally, TRIM28, KAT5, and EXOSC7 were identified as enriched genes in the study. On the other hand, we identified a group of genes that exhibited significant depletion in pyrenocytes compared to orthochromatic erythroblasts. Among these genes, UHRF1, DPF2, KEAP1, and TAF10 were found to be most depleted.To better understand the role of KEAP1 in enucleation, KEAP1 knockout (KO) BEL-A cells were generated. The enucleation ratio of the KEAP1 KO cells decreased significantly, and their survival also dramatically decreased after differentiation on day 12 compared to the wild-type cells. Live cell imaging of enucleation of the KEAP1 KO cells further confirmed the decrease of the enucleation rate. The significant decrease of enucleation observed in KEAP1 KO cells has triggered questions about the specific pathways and processes required for enucleation. Given that KEAP1 is the main regulator of NRF2, our future studies will mainly focus on understanding the importance of oxidative stress in enucleation.

Mechanistic Insights into Harmine-Mediated Inhibition of Human DNA Methyltransferases and Prostate Cancer Cell Growth.

Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 μM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of ∼14 μM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.

Single-cell Transcriptome Landscape of DNA Methylome Regulators Associated with Orofacial Clefts in the Mouse Dental Pulp.

Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. The biomedical research institution. This study did not include patients. This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes.

Altered Brain Expression of DNA Methylation and Hydroxymethylation Epigenetic Enzymes in a Rat Model of Neuropathic Pain.

The role of epigenetics in chronic pain at the supraspinal level is yet to be fully characterized. DNA histone methylation is crucially regulated by de novo methyltransferases (DNMT1-3) and ten-eleven translocation dioxygenases (TET1-3). Evidence has shown that methylation markers are altered in different CNS regions related to nociception, namely the dorsal root ganglia, the spinal cord, and different brain areas. Decreased global methylation was found in the DRG, the prefrontal cortex, and the amygdala, which was associated with decreased DNMT1/3a expression. In contrast, increased methylation levels and mRNA levels of TET1 and TET3 were linked to augmented pain hypersensitivity and allodynia in inflammatory and neuropathic pain models. Since epigenetic mechanisms may be responsible for the regulation and coordination of various transcriptional modifications described in chronic pain states, with this study, we aimed to evaluate the functional role of TET1-3 and DNMT1/3a genes in neuropathic pain in several brain areas. In a spared nerve injury rat model of neuropathic pain, 21 days after surgery, we found increased TET1 expression in the medial prefrontal cortex and decreased expression in the caudate-putamen and the amygdala; TET2 was upregulated in the medial thalamus; TET3 mRNA levels were reduced in the medial prefrontal cortex and the caudate-putamen; and DNMT1 was downregulated in the caudate-putamen and the medial thalamus. No statistically significant changes in expression were observed with DNMT3a. Our results suggest a complex functional role for these genes in different brain areas in the context of neuropathic pain. The notion of DNA methylation and hydroxymethylation being cell-type specific and not tissue specific, as well as the possibility of chronologically differential gene expression after the establishment of neuropathic or inflammatory pain models, ought to be addressed in future studies.

DNA methylation enzymes in the kidneys of male and female BTBR ob/ob mice.

Diabetic kidney disease (DKD) is the leading cause of the end-stage renal disease. Recent studies have shown that epigenetic modifications contribute to alterations in gene expression and the development of DKD. This study aimed to show an expression profile of key DNA (de)methylation enzymes (DNMT, TET proteins) and their differences between sexes under obesity and diabetic condition. Male and female black and tan brachyury (BTBR) ob/ob mice and their corresponding wild-type littermates (BTBR WT) were studied until 16 weeks of age. Metabolic parameters, kidney morphophysiology and the expression of fibrotic markers and epigenetic enzymes were studied in whole kidney tissue or specifically in the glomerulus. The results showed sexual dimorphism in the development of metabolic disease and in kidney morphophysiology. Female mice have a different profile of DNMTs expression in both WT and obese/diabetic condition. Furthermore, metabolic condition negatively modulated the glomerular expression of TET1 and TET3 only in females. To our knowledge, this is the first study that shows a kidney profile of the expression of key (de)methylation enzymes, DNMTs and TETs, in the BTBR ob/ob experimental model of DKD and its association with sex. The knowledge of this epigenetic profile may help future research to understand the pathophysiology of DKD in males and females.

Deficits in odor discrimination versus odor identification in patients with schizophrenia and negative correlations with GABAergic and DNA methyltransferase mRNAs in lymphocytes.

People with schizophrenia have been reported to show deficits in tests of olfactory function. DNA methylation and GABAergic input have been implicated in biochemical processes controlling odor in animal studies, but this has not been investigated in human studies. In a study of measures of DNA methylation and GABAergic mRNAs in lymphocytes, we also measured odor identification and discrimination with the Sniffin' Sticks battery in 58 patients with chronic schizophrenia (CSZ) and 48 controls. mRNAs in lymphocytes were assessed by qPCR using TaqManTM probes. Cognition was assessed by the MATRICS battery (Measurement and Treatment Research to Improve Cognition in Schizophrenia) in CSZ and controls, and symptoms in CSZ were assessed by PANSS scale (Positive and Negative Symptom Scale). The relationships of odor deficits with mRNA, cognition, and symptoms were explored by correlation analysis. Variables which significantly differentiated CSZ from controls were explored by logistic regression. Overall, CSZ showed significantly (P≤.001) lower scores on odor discrimination compared to controls, with a moderate effect size, but no difference in odor identification. Deficits in odor discrimination, which has not been standardly assessed in many prior studies, strongly differentiated CSZ from controls. In logistic regression analysis, odor discrimination, but not odor identification, was a significant variable predicting schizophrenia versus control class membership. This is the first study to report relationship between odor deficits and DNA methylation and GABAergic mRNAs in blood cells of human subjects. There were negative correlations of odor identification with DNA methylation enzymes mRNAs and significant negative correlations with odor discrimination and GABAergic mRNAs. Lower odor scores were significantly associated with lower cognitive scores on the MATRICS battery in CSZ but not control subjects. In CSZ, lower odor scores were significantly associated with negative symptom scores, while higher odor identification scores were associated with PANNS Excitement factor. Odor discrimination was a more powerful variable than odor identification in discriminating CSZ from controls and should be used more regularly as an odor measure in studies of schizophrenia. The substantive meaning of the negative correlations of odor discrimination and GABAergic mRNA variables in peripheral lymphocytes of CSZ needs more investigation and comparison with results in neural tissue.

The HP1α protein is mandatory to repress the circadian clock and its output genes during the 12 h period of transcriptional repression

The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.

Induction of cytotoxic effects and changes in DNA methylation-related gene expression in a human fibroblast cell line by the metal-organic framework [H2NMe2]3 [Tb(III)(2,6 pyridinedicarboxylate)3] (Tb-MOF).

Lanthanide metal-organic frameworks (lanthanide MOFs) may be utilized for a variety of environmental and human health applications due to their luminescent properties and high thermal and water stability. However, the cytotoxic and epigenetic effects produced in human cells are not known. Therefore, we evaluated the cytotoxic effects, internalization, and changes in the mRNA abundance of DNA methylation and demethylation enzymes by exposing human fibroblast cells to a metal-organic framework [H2NMe2]3 [Tb(III)(2,6 pyridinedicarboxylate)3] (Tb-MOF). For this purpose, the cells were exposed to six concentrations (0.05 to 1.6mg/mL) of Tb-MOF for 48h. Field emission electron microscopy coupled to linear energy dispersive spectroscopy (FESEM‒EDS) and confocal microscopy analysis were performed. The cytotoxicity was determined with crystal violet and MTT assays. The results demonstrated the internalization of Tb-MOF at concentrations as low as 0.05mg/mL, as well as concentration-dependent toxicity. Additionally, we detected significant changes in the gene expression levels of DNA methyltransferases and demethylases due to the presence of Tb-MOF, suggesting that Tb-MOF could generate epigenetic changes even at low concentrations. The results of our study may establish a foundation for future research attempting to develop and apply secure nanomaterials (e.g., MOFs) to minimize damage to the environment and human health.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels.

Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

High-fat diet induced obesity alters Dnmt1 and Dnmt3a levels and global DNA methylation in mouse ovary and testis.

Obesity impairs reproductive capacity, and the link between imprinting disorders and obesity has been discussed in many studies. Recent studies indicate that a high-fat diet may cause epigenetic changes in maternal and paternal genes, which may be transmitted to offspring and negatively affect their development. On this basis, our study aims to reveal the changes in DNA methylation and DNA methyltransferase enzymes in the ovaries and testes of C57BL/6 mice fed a high-fat diet and created a model of obesity, by comparing them with the control group. For this purpose, we demonstrated the presence and quantitative differences of DNA methyltransferase 1 and DNA methyltransferase 3a enzymes as well as global DNA methylation in ovaries and testis of C57BL/6 mice fed a high-fat diet by using immunohistochemistry and western blot methods. We found that a high-fat diet induces the levels of Dnmt1 and Dnmt3a proteins (p < 0.05). We observed increased global DNA methylation in testes but, interestingly, decreased global DNA methylation in ovaries. We think that our outcomes have significant value to demonstrate the effects of obesity on ovarian follicle development and testicular spermatogenesis and may bring a new perspective to obesity-induced infertility treatments. Additionally, to the best of our knowledge, this is the first study to document dynamic alteration of Dnmt1 and Dnmt3a as well as global DNA methylation patterns during follicle development in healthy mouse ovaries.

Single-cell DNA methylation sequencing by combinatorial indexing and enzymatic DNA methylation conversion

BackgroundDNA methylation is a critical molecular mark involved in cellular differentiation and cell-specific processes. Single-cell whole genome DNA methylation profiling methods hold great potential to resolve the DNA methylation profiles of individual cell-types. Here we present a method that couples single-cell combinatorial indexing (sci) with enzymatic conversion (sciEM) of unmethylated cytosines.ResultsThe sciEM method facilitates DNA methylation profiling of single-cells that is highly correlated with single-cell bisulfite-based workflows (r2 > 0.99) whilst improving sequencing alignment rates, reducing adapter contamination and over-estimation of DNA methylation levels (CpG and non-CpG). As proof-of-concept we perform sciEM analysis of the temporal lobe, motor cortex, hippocampus and cerebellum of the human brain to resolve single-cell DNA methylation of all major cell-types.ConclusionTo our knowledge sciEM represents the first non-bisulfite single-cell DNA methylation sequencing approach with single-base resolution.

Pre-reproductive stress in adolescent female rats alters maternal care and DNA methylation patterns across generations

Stress during development affects maternal behavior and offspring phenotypes. Stress in adolescence is particularly consequential on brain development and maturation, and is implicated in several psychiatric disorders. We previously showed that pre-reproductive stress (PRS) in female adolescent rats affects behavior and corticotropin releasing hormone receptor 1 (CRHR1) expression in first- (F1) and second- (F2) generation offspring. We further showed that offspring phenotypes are partially reversed by post-stress treatment with fluoxetine (FLX) or the CRHR1 antagonist NBI27914 (NBI). Epigenetic processes, such as DNA methylation, are implicated in the stress response and interact with maternal care quality across generations. Here, we asked whether PRS and FLX or NBI exposure would affect maternal care and global DNA methylation in the brains of exposed dams and their adult F1 and paternally-derived F2 offspring. We found that PRS decreased self-care while increasing pup-care behaviors. PRS also increased DNA methylation in the amygdala of dams and their F1 male offspring, but decreased it in F2 females. Drug treatment had no effect on maternal care, but affected DNA methylation patterns in F0 and F1 generations. Furthermore, PRS altered the expression of DNA methylating enzymes in brain, blood and oocytes. Finally, maternal care variables differentially predicted methylation levels in PRS and control offspring. Thus, the effects of adolescent stress are long-lasting and impact methylation levels across three generations. Combined with our findings of epigenetic changes in PRS-exposed oocytes, the present data imply that biological changes and social mechanisms act in concert to influence adult offspring phenotypes.