Expression Of DNA Methyltransferase Research Articles

DNA Methyltransferase Expression (DNMT1, DNMT3a, and DNMT3b) as a Potential Biomarker in Age-Related Macular Degeneration.

Background/Objectives: Age-related macular degeneration (AMD) is a global cause of vision loss, with limited therapeutic options highlighting the need for effective biomarkers. This study aimed to characterize plasma DNA methyltransferase expression (DNMT1, DNMT3A, and DNMT3B) in AMD patients and explore divergent expression patterns across different stages of AMD. Methods: Thirty-eight AMD patients were prospectively enrolled and stratified by disease severity: eAMD, iAMD, nAMD, and aAMD. Comprehensive ophthalmological assessments were performed, including best-corrected visual acuity, digital color fundus photographs, and Spectral Domain Optical Coherence Tomography. Peripheral blood samples were collected for RNA extraction and qRT-PCR to access epigenetic effectors' transcriptional expression, namely DNMT1, DNMT3A, and DNMT3B genes. The collected data were analyzed using IBM SPSS 29. Results:DNMT1 expression was significantly downregulated in late AMD (-0.186 ± 0.341) compared to early/intermediate AMD (0.026 ± 0.246). Within late AMD, aAMD exhibited a marked downregulation of DNMT1 (-0.375 ± 0.047) compared to nAMD (0.129 ± 0.392). DNMT3A and DNMT3B showed similar divergent expression patterns, correlating with disease stage. Conclusions: This study identified stage-specific transcriptional differences in DNMT expression, emphasizing its potential as a biomarker for AMD progression and a target for future research into personalized therapeutic strategies.

Targeting of the G9a, DNMT1 and UHRF1 epigenetic complex as an effective strategy against pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options and a poor prognosis. The critical role of epigenetic alterations such as changes in DNA methylation, histones modifications, and chromatin remodeling, in pancreatic tumors progression is becoming increasingly recognized. Moreover, in PDAC these aberrant epigenetic mechanisms can also limit therapy efficacy. This study aimed to investigate the expression and prognostic significance of a key epigenetic complex encompassing DNA methyltransferase-1 (DNMT1), the histone methyltransferase G9a, and the scaffold protein UHRF1 in PDAC. We also evaluated the therapeutic potential of an innovative inhibitor targeting these epigenetic effectors.MethodsImmunohistochemical analysis of DNMT1, G9a, and UHRF1 expression was conducted in human PDAC tissue samples. Staining was semi-quantitatively scored, and overexpression was defined as moderate to strong positivity. The prognostic impact was assessed by correlating protein expression with patient survival. The antitumoral effects of the dual DNMT1-G9a inhibitor CM272 were tested in PDAC cell lines, followed by transcriptomic analyses to identify underlying mechanisms. The in vivo antitumoral efficacy of CM272 was evaluated in PDAC xenograft and syngeneic mouse models, both alone and in combination with anti-PD1 immunotherapy.ResultsDNMT1, G9a, and UHRF1 were significantly overexpressed in PDAC cells and stroma compared to normal pancreatic tissues. Simultaneous overexpression of the three proteins was associated with significantly reduced survival in resected PDAC patients. CM272 exhibited potent antiproliferative activity in PDAC cell lines, inducing apoptosis and altering key metabolic and cell cycle-related genes. CM272 also enhanced chemotherapy sensitivity and significantly inhibited tumor growth in vivo without detectable toxicity. Combination of CM272 with anti-PD1 therapy further improved antitumor responses and immune cell infiltration, particularly CD4 + and CD8 + T cells.ConclusionsThe combined overexpression of DNMT1, G9a, and UHRF1 in PDAC is a strong predictor of poor prognosis. CM272, by targeting this epigenetic complex, shows promising therapeutic potential by inducing apoptosis, reprogramming metabolic pathways, and enhancing immune responses. The combination of CM272 with immunotherapy offers a novel, effective treatment strategy for PDAC.

ALOX15-Driven Ferroptosis: The key target in Dihydrotanshinone I's epigenetic Battle in hepatic stellate cells against liver fibrosis.

It is known that ferroptosis promotes hepatic stellate cells (HSCs) inactivation. Arachidonate 15-Lipoxygenase (ALOX15), a ferroptosis driver gene, participates in disease progression. Dihydrotanshinone I (DHI), an active compound from Salvia miltiorrhiza, effectively regulates HSC inactivation. Nonetheless, there still needs to be clear understanding of how DHI affects HSC ferroptosis. This study primarily investigates DHI's protective effects on liver fibrosis in vivo and in vitro. Additionally, we explored the molecular mechanisms by which DHI promotes ferroptosis in HSCs. The relationship between ALOX15 level and methylation was examined. Molecular docking was performed to confirm the targeting between early growth response protein 1 (EGR1) and DHI. DHI exhibited a mitigating effect on liver fibrosis in vivo. DHI-induced inactivation of HSC by promoting ferroptosis, accompanied by an elevation in intracellular iron and reactive oxygen species (ROS) levels. Results of transcriptome sequencing and quantitative real-time PCR (qRT-PCR) confirmed the elevation of ALOX15 (a ferroptosis driver gene) in HSCs with DHI. Loss of ALOX15 inhibited DHI-induced ferroptosis. Interestingly, DNA methyltransferase 1 (DNMT1), an essential DNA methyltransferase, was downregulated by DHI. Overexpression of DNMT1 resulted in decreased ALOX15 expression in cells with DHI. Notably, transcription factor EGR1 was demonstrated to regulate DNMT1 expression. EGR1 deficiency led to an increase in DNMT1 expression, which inhibited DHI-induced ferroptosis. Molecular docking confirmed that EGR1 could serve as a direct pharmacological target of DHI. DHI upregulates EGR1 level, leading to decreased DNMT1 expression and increased ALOX15 demethylation, thereby promoting HSC ferroptosis and inactivation.

Combinatorial phenethyl isothiocyanate and withaferin A targets multiple epigenetics pathways to inhibit MCF-7 and MDA-MB-231 human breast cancer cells

BackgroundEpigenetic phytochemicals are considered as an efficacious and safe alternative to synthetic drugs in drug discovery. In this regard, combinatorial interventions enable simultaneously targeting various neoplastic pathways to eradicate multiple tumorigenic clones. Therefore, we evaluated the effects of the epigenetic-modifying compounds phenethyl isothiocyanate (PEITC) and withaferin A (WA) alone and in combination on cancer hallmarks and miRNome profiles of breast cancer (BC) cells in addition to their impact on multiple epigenetic regulatory pathways.MethodsWe performed MTT assay, flow cytometry-based cell cycle analysis, apoptosis assay, stem cell population analysis, and mammosphere assay on MCF-7 and MDA-MB-231 BC cells to evaluate the effect of combinatorial PEITC and WA treatment on cancer hallmarks. To assess the epigenetic effects of the combinatorial PEITC and WA treatment, we conducted HDAC activity assay, DNMT activity assay, western blot analysis, siRNA-mediated gene knockdown, and RT-qPCR analysis. Additionally, we explored the effect of the PEITC + WA combination on miRNome profiles in MCF-7 and MDA-MB-231 BC cells through miRNA-seq analysis and miRNA Real-Time PCR assay.ResultsOur results indicated a synergistic effect of PEITC and WA on inhibiting MCF-7 and MDA-MB-231 BC cells by triggering G2/M-phase arrest, apoptosis induction, tumor formation efficiency decrease, and stem cell population decline. Combinatorial PEITC and WA treatment significantly reduced global DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activity in addition to decreasing multiple Class I HDACs and de novo DNMTs expression in MCF-7 and MDA-MB-231 cells. PEITC + WA combination targets histone acetylation and DNA methylation pathways since the expressional changes of cell cycle and apoptosis-related proteins due to PEITC + WA treatment closely mimic the alterations seen when HDAC8 and DNMT3B are silenced. Furthermore, treating these cells with PEITC and WA significantly alters the expression of several BC-associated miRNAs.ConclusionOverall, our investigation demonstrated that combined PEITC and WA is effective in inhibiting MCF-7 and MDA-MB-231 BC cells by impacting multiple epigenetic regulatory pathways.

Exposure to a Titanium Dioxide Product Alters DNA Methylation in Human Cells.

The safety of titanium dioxide (TiO2), widely used in foods and personal care products, has been of ongoing concern. Significant toxicity of TiO2 has been reported, suggesting a risk to human health. To evaluate its potential epigenotoxicity, the effect of exposure to a TiO2 product to which humans could be exposed on DNA methylation, a primary epigenetic mechanism, was investigated using two human cell lines (Caco-2 (colorectal) and HepG2 (liver)) relevant to human exposure. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Gene promoter methylation was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNA methyltransferases, MBD2, and URHF1 was quantified by qRT-PCR. A decrease in global DNA methylation was observed in both cell lines. Across the cell lines, seven genes (BNIP3, DNAJC15, GADD45G, GDF15, INSIG1, SCARA3, and TP53) were identified in which promoters were methylated. Changes in promoter methylation were associated with gene expression. Results also revealed aberrant expression of regulatory genes, DNA methyltransferases, MBD2, and UHRF1. Findings from the study clearly demonstrate the impact of TiO2 exposure on DNA methylation in two cell types, supporting the potential involvement of this epigenetic mechanism in its biological responses. Hence, epigenetic studies are critical for complete assessment of potential risk from exposure.

Antimicrobial activity and cytotoxic and epigenetic effects of tannic acid-loaded chitosan nanoparticles

Tannic acid (TA) is a potent antitumor agent, but its low bioavailability and absorption limit its use. In this study, it was loaded into chitosan-based nanoparticles (Chi-NPs) to overcome these limitations and to improve its antimicrobial and anticancer activities. TA-loaded Chi-NPs (Chi-TA-NPs) were synthesized using the ionic gelation method and physicochemically characterized by FE-SEM, FTIR, XRD, PDI, DLS, and zeta potential analysis. Additionally, the antimicrobial activity of Chi-TA-NPs against two G+ bacterial strains, two G− bacterial strains, and a fungal strain (Candida albicans) was investigated using the microbroth dilution method. MTT assay was used to examine the cytotoxic effects of Chi-TA-NPs on HepG2 cells. The expression of DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B was examined in HepG2 cells using RT-qPCR. The amount of 5-methylcytosine in the HepG2 cell-derived genomic DNA was measured using ELISA. FE-SEM micrographs showed the loading of TA into the chitosan-based formulation. The peaks detected in the XRD and FTIR analyses confirmed the formation of the Chi-TA-NPs. The PDI value (0.247 ± 0.03), size (567.0 ± 25.84 nm), and zeta potential (17.0 ± 5.86 mV) confirmed the relative stability of Chi-TA-NPs. A constant release profile in line with the Korsmeyer-Peppas model was detected for Chi-TA-NPs, such that approximately 44% of TA was released after 300 min. In addition, Chi-TA-NPs exhibited effective antimicrobial activity against the studied microbial strains, as manifested by MIC values ranging from 250 to 1000 µg/mL. Chi-TA-NPs induced cytotoxicity in liver tumor cell line, with an IC50 value of 500 µg/mL. Furthermore, Chi-TA-NPs considerably decreased the expression of DNMT1 (2.52-fold; p = 0.01), DNMT3A (2.96-fold; p = 0.004), and DNMT3B (2.94-fold; p < 0.0001). However, 5-methylcytosine levels in HepG2 cells were unaffected by Chi-TA-NPs treatment (p = 0.62). Finally, the antimicrobial, cytotoxic, and epigenetic effects of Chi-TA-NPs were more pronounced than those of free TA and the unloaded Chi-NPs. In conclusion, Chi-TA-NPs exhibit promising potential for reducing microbial growth and promoting cytotoxicity in liver cancer cells.

ULK1 methylation promotes TGF-β1-induced endometrial fibrosis via the FOXP1/DNMT1 axis.

Intrauterine adhesion (IUA) is the second most common cause of secondary infertility in women and can also lead to menstrual abnormalities and multiple adverse pregnancy outcomes. Therefore, elucidating the mechanism of its development is crucial for the prevention and treatment of IUA. This study will investigate the function and mechanism of forkhead box P1 (FOXP1)/DNA methyltransferase 1 (DNMT1)/unc-51-like autophagy activating kinase 1 (ULK1) in IUA. Expression levels of key genes were detected using western blot and quantitative - real time reverse transcription polymerase chain reaction. Cell proliferation was detected by CCK-8 and EdU staining. Transcriptional regulation relationships were detected by dual luciferase reporter gene and chromatin immunoprecipitation (ChIP) assay. Methylation station of ULK1 was detected by methylmion specific PCR (MSP). Fibrosis and pathological changes in the uterine cavity tissues were detected by Masson and hematoxylin and eosin staining. It was observed that the expression of FOXP1 and DNMT1 increased in transforming growth factor (TGF)-β1-induced cells, while ULK1 expression decreased. Downregulation of FOXP1 could inhibit human endometrial stromal cells proliferation and autophagy, as well as decrease the expression of fibrogenic factors (collagen type I alpha 1 chain [COL1A1], fibronectin [FN], and alpha-smooth muscle actin [α-SMA]). The results of MSP and ChIP experiments showed that DNMT1 promotes methylation of the ULK1 promoter region and inhibits its transcription. In an animal model, knockdown of FOXP1 alleviated pathological fibrosis and uterine adhesions. Knockdown of FOXP1 can inhibit endometrial fibrosis in IUA rats; FOXP1 could be a potential target for the treatment of IUA.

The yeast genome is globally accessible in living cells.

Eukaryotic genomes are packaged into chromatin, which is composed of condensed filaments of regularly spaced nucleosomes, resembling beads on a string. The nucleosome contains ~147 bp of DNA wrapped almost twice around a central core histone octamer. The packaging of DNA into chromatin represents a challenge to transcription factors and other proteins requiring access to their binding sites. Consequently, control of DNA accessibility is thought to play a key role in gene regulation. Here we measure DNA accessibility genome wide in living budding yeast cells by inducible expression of DNA methyltransferases. We find that the genome is globally accessible in living cells, unlike in isolated nuclei, where DNA accessibility is severely restricted. Gene bodies are methylated at only slightly slower rates than promoters, indicating that yeast chromatin is highly dynamic in vivo. In contrast, silenced loci and centromeres are strongly protected. Global shifts in nucleosome positions occur in cells as they are depleted of ATP-dependent chromatin remodelers, suggesting that nucleosome dynamics result from competition among these enzymes. We conclude that chromatin is in a state of continuous flux in living cells, but static in nuclei, suggesting that DNA packaging in yeast is not generally repressive.

Enhancing antitumor activity of herceptin in HER2-positive breast cancer cells: a novel DNMT-1 inhibitor approach

HER2 antagonists remain the cornerstone of therapy for patients with HER2-positive breast cancer. This study introduces a novel small-molecule inhibitor of DNA methyltransferase 1 (DNMT-1), referred to as DI-1, designed to synergize with HER2 antagonists in treating HER2-positive breast cancer cells. Clinical data reveal a negative correlation between DNMT-1 expression and PTEN levels, and a positive correlation with the methylation rates of PTEN's promoter. In experiments with SKBR3 and BT474 cells, DI-1 effectively reduced the methylation of PTEN's promoter region, thereby upregulating PTEN expression. This upregulation, in turn, enhanced the cells' sensitivity to HER2 antagonists, indicating that DI-1’s mechanism involves inhibiting DNMT-1’s recruitment to PTEN's promoter region. Consequently, by increasing PTEN expression, DI-1 amplifies the sensitivity of HER2-positive breast cancer cells to treatment, suggesting its potential as a promising therapeutic strategy in this context.

The response of DNA methyltransferase and demethylase genes to abiotic stresses in tomato seedling

DNA methylation plays an important role in regulating plant growth, development and gene expression. However, less is known about the response of DNA methyltransferase and demethylase genes to various stresses. In this study, the effects of abiotic stresses on DNA methylation gene expression patterns in tomato seedlings were investigated. Results showed that most tomato DNA methyltransferase and demethylase genes contained stress-related elements. The expression of SlDML1 was significantly induced by cadmium (Cd) and sodium chloride (NaCl) stresses. SlDML2 was more sensitive and reached its maximum value under polyethylene (PEG) stress at 24 h. The expression of SlMET3L was repressed to varying degrees under Cd, NaCl and PEG stresses at 48 h. However, 5-aza-2′-deoxycytidine (5-azadC) treatment decreased the Cd and PEG stress tolerance by down-regulating the expression of DNA methyltransferase except for the SlMET3L, and up-regulating the expression levels of SlDML2, SlDML3 and SlDML4, cadmium transporters (SlHMA5, SlCAX3, and SlACC3) and osmoregulators (SlDREB, SlLEA and SlHSP70). Whereas 5-azadC treatment alleviated the salt stress through up-regulating DNA methyltransferase gene expression, and down-regulating the expression level of SlDML1, SlDML3, and SlDML4, SlHKT1, SlNHX1, and SlSOS1. Collectively, 5-azadC impaired Cd and PEG stress tolerance and enhanced salt stress tolerance by regulating the expression of methylation-related and stress-related genes in tomato seedlings. These results may provide useful information for further analysing function and evolution of DNA methylation methyltransferase and demethylase genes in tomato under stress conditions.

Increased DNMT1 acetylation leads to global DNA methylation suppression in follicular granulosa cells during reproductive aging in mammals

With increasing age, the reproductive performance of women and female animals declines. However, the molecular mechanisms underlying ovarian aging and age-related fertility decline remain unclear. Granulosa cells (GCs) are suspected to play an important role in reproductive aging, and their proliferation, apoptosis, and steroid hormone secretion are used to determine the fate of follicles and ovarian function. First, we found that the proliferative ability of GCs from the old mouse group (10-month-old) decreased compared with that from the young mouse group (6-week-old), and cell cycle arrest occurred in old mice. To investigate changes in protein modification, we compared the levels of protein acetylation in GCs from young and old mice. We found that the K1118, K1120, K1122, and K1124 sites of DNA methyltransferase 1 (DNMT1) were increasingly acetylated with age, resulting in a decrease in DNMT1 protein expression. Therefore, we performed whole-genome methylation sequencing of GCs in the two groups and found that the CG methylation levels in the old group were lower than those in the young group. Furthermore, the inhibition of DNMT1 expression in GCs resulted in cell cycle arrest. This study revealed the dynamics and importance of protein acetylation and DNA methylation in GCs during reproductive aging. The findings provide a theoretical basis for studying the mechanism of reproductive aging in mammals.

Epigenetic Mechanisms of Aluminum-Induced Neurotoxicity and Alzheimer's Disease: A Focus on Non-Coding RNAs.

Aluminum (Al) is known to induce neurotoxic effects, potentially contributing to Alzheimer's disease (AD) pathogenesis. Recent studies suggest that epigenetic modification may contribute to Al neurotoxicity, although the mechanisms are still debatable. Therefore, the objective of the present study was to summarize existing data on the involvement of epigenetic mechanisms in Al-induced neurotoxicity, especially AD-type pathology. Existing data demonstrate that Al exposure induces disruption in DNA methylation, histone modifications, and non-coding RNA expression in brains. Alterations in DNA methylation following Al exposure were shown to be mediated by changes in expression and activity of DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs). Al exposure was shown to reduce histone acetylation by up-regulating expression of histone deacetylases (HDACs) and impair histone methylation, ultimately contributing to down-regulation of brain-derived neurotrophic factor (BDNF) expression and activation of nuclear factor κB (NF-κB) signaling. Neurotoxic effects of Al exposure were also associated with aberrant expression of non-coding RNAs, especially microRNAs (miR). Al-induced patterns of miR expression were involved in development of AD-type pathology by increasing amyloid β (Aβ) production through up-regulation of Aβ precursor protein (APP) and β secretase (BACE1) expression (down-regulation of miR-29a/b, miR-101, miR-124, and Let-7c expression), increasing in neuroinflammation through NF-κB signaling (up-regulation of miR-9, miR-125b, miR-128, and 146a), as well as modulating other signaling pathways. Furthermore, reduced global DNA methylation, altered histone modification, and aberrant miRNA expression were associated with cognitive decline in Al-exposed subjects. However, further studies are required to evaluate the contribution of epigenetic mechanisms to Al-induced neurotoxicity and/or AD development.

Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells

Butyric acid as a short-chain fatty acid (SCFA) is one of the key microbial metabolites of ruminants. Numerous studies indicate that butyrate is crucial in muscle growth and development, and plays an important molecular regulatory role mainly by inhibiting histone deacetylation. DNA methylation, a major epigenetic modification, is involved in cell differentiation. Butyrate, in addition to its role in acetylation modifications, can alter the DNA methylation status of cells. However, the impact of butyrate on the DNA methylation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. In this study, we developed a differentiation model of SMSCs and employed RNA sequencing (RNA-seq) alongside whole genome bisulfite sequencing (WGBS) to explore the effects of butyrate treatment on DNA methylation status and its relationship with gene expression. Treatment of SMSCs with sodium butyrate (NaB) at 1.0 mM for 2 days significantly inhibited the expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A) at the mRNA and protein levels while promoting the expression of demethylases (TET1, TET2, TET3) at mRNA levels. WGBS identified 4292 differentially methylated regions (DMRs), comprising 2294 hypermethylated and 1998 hypomethylated regions. These DMRs were significantly enriched in the MAPK, cAMP, and Wnt signaling pathways, all of which are implicated in myogenesis and development. Combining RNA-seq and WGBS data revealed a total of 130 overlapping genes, including MDFIC, CREBBP, DMD, LTBP2 and KLF4. These genes are predominantly involved in regulating the FoxO, MAPK, PI3K-Akt, and Wnt signaling pathways. This study provides new insights into the effects of butyrate-mediated DNA methylation on SMSC development and enhances our understanding of butyrate as an epigenetic modifier beyond its role in acetylation.

Ceratonia siliqua L. pod Effects on Viability Gene Expression of Endometrial Mesenchymal Stromal/Stem Cells Isolated from Women with Endometriosis-Associated Infertility.

This study aims to investigate the effects of carob (Ceratonia siliqua L.) pod extract (CPE) on the viability of human endometrial mesenchymal stromal/stem cells (EnMSCs) and its impact on mRNA and protein expressions of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), histone deacetylase 1 (HDAC1), matrix metalloproteinase-2 (MMP2), and cyclooxygenase-2 (COX-2) in endometriotic patients. In this experimental study, EnMSCs were derived from endometrium of patients with ovarian endometrioma (OMA-EnMSCs group) and deep infiltrative endometriosis (DIE) samples of 10 endometriosisassociated infertility (EAI) women (E-EnMSCs group) and compared to EnMSCs derived from the endometrium of an endometriosis-free, normal woman as the control group (C-EnMSCs). The metabolic activity of the control and case groups were evaluated by treating them with different concentrations of CPE. Cell viability was analysed by MTT. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the expression of specific genes at the mRNA and protein levels, respectively. Treatment with 0.8 and 2 μg/mL of CPE downregulated COX-2 and HDAC1 in the E-EnMSC group compared to the C-EnMSCs group. Treatment with 0.8 μg/mL of CPE also decreased MMP2 and DNMT3B gene expressions. The COX-2 and DNMT3A genes were significantly upregulated after treatment with 2 μg/mL of CPE. Expressions of the COX-2, HDAC1, DNMT1, DNMT3A, and DNMT3B peptides decreased in the all three groups after treatment with 0.8 and 2 μg/mL of CPE. Gas chromatography-mass spectroscopy (GC-MS) analysis of CPE identified 14 bioactive compounds. Molecular docking showed the best position of each bioactive compound on the different target proteins that are involved in the process of apoptosis in EnMSCs. In vitro and in silico analyses of CPE bioactive compounds show that they may downregulate the cell inflammatory pathway involved in the pathophysiology of endometriosis.

8759 Mild Iodine Deficiency During Pregnancy Impairs Hypothalamus-Pituitary-Thyroid Function Programing of the Adult Male Offspring

Abstract Disclosure: M. Oliveira: None. E. Tavares: None. G. Henrique: None. J. Pinto: None. C. Serrano-Nascimento: None. Introduction: Iodine plays a crucial role in the biosynthesis of thyroid hormones and is essential for proper thyroid embryonic development. While severe iodine deficiency during pregnancy and lactation has well-documented deleterious effects, the consequences of mild iodine deficiency (MID) remain not fully understood. MID during pregnancy is a reality in numerous countries, even in regions with seemingly adequate iodine supply in the diet. Previous studies have highlighted the adverse impact of MID on neurological development in offspring, but comprehensive data on thyroid function, particularly in progeny at different developmental stages, are lacking. This study aimed to investigate the repercussions of MID during pregnancy on the programming of thyroid dysfunctions during adulthood. Methods: Female CD1 mice were subjected to iodine-deficient animal chow and iodine-supplemented water, either at normal (NI; 0.2 mg/L) or mild iodine deficiency (MID; 0.8 mg/L) levels during pregnancy. Gene and protein expression in the hypothalamus, pituitary, and thyroid of adult male offspring animals (PND90) were evaluated using qPCR and Western blotting. Additionally, this study explored MID-induced programming of offspring thyroid gene expression through epigenetic mechanisms. Results: Maternal MID exposure led to increased gene and protein expression of thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) in the hypothalamus and pituitary of the offspring animals. Thyroid protein and gene expression of sodium iodide symporter (NIS), TSH receptor (TSHR), thyroid peroxidase (TPO), thyroglobulin (TG), paired box 8 (PAX8), NK2 homeobox 1 (NKX2.1), and monocarboxylate transporter 8 (MCT8) were elevated in the offspring of MID-exposed animals. Epigenetic mechanisms appeared to contribute to the augmented thyroid gene expression in MID-exposed animals, with decreased DNA methyltransferase expression and reduced thyroid DNA global methylation. Additionally, histone 3 hypomethylation and increased acetylation of histones 3 and 4 were observed. Conclusion: The study suggests that MID exposure during pregnancy can be as harmful as severe iodine deficiency in programming the hypothalamus-pituitary-thyroid (HPT) axis, leading to HPT dysfunction during adult life. Consequently, our data suggest that monitoring iodine intake throughout pregnancy is crucial to prevent the occurrence of thyroid disorders in offspring during adulthood. Presentation: 6/1/2024

AP-2α decreases TMZ resistance of recurrent GBM by downregulating MGMT expression and improving DNA damage

AimsThe incidence of recurrent gliomas is high, exerting low survival rates and poor prognoses. Transcription factor AP-2α has been reported to regulate the progression of primary glioblastoma (GBM). However, the function of AP-2α in recurrent gliomas is largely unclear. MethodsThe expression of AP-2α and O6-methylguanine DNA-methyltransferase (MGMT) was detected in recurrent glioma tissues and cell lines by Western blots, the regulation mechanisms between AP-2α/MGMT promoter and RA/AP-2α promoter were studied by luciferase reporter assays, EMSA, and chIP assays. The effects of AP-2α and TMZ/RA treatment on cell viability in vitro and in vivo were investigated by MTT assays, γH2AX staining, comet assays and intracranial injection. Key findingsAP-2α expression negatively correlates with the expression of MGMT in glioma samples. AP-2α could directly bind with the promoter of the MGMT gene, suppresses transcriptional levels of MGMT and downregulate MGMT expression in TMZ-resistant U87MG-R and T98G cells, but TMZ treatment decreases AP-2α expression and increases MGMT expression. The extended TMZ treatment and increased TMZ concentrations reversed these effects. Moreover, AP-2α overexpression combines with TMZ to decrease cell viability, concurrently with improved DNA damage marker γH2AX. Furthermore, retinoic acid (RA) activates RAR/RXR heterodimers, which bind to RA-responsive elements (RAREs) of the AP-2α promoter, and activates AP-2α expression in recurrent glioma cells. Finally, in intracranial relapsed glioma mouse model, both RA and TMZ could retard tumor development and prolong the mouse survival. SignificanceAP-2α activation by gene overexpression or RA treatment reveals the suppressive effects on glioma relapse, providing a novel therapeutic strategy against malignant refractory gliomas.

MicroRNA-34a and promoter methylation contribute to peroxisome proliferator-activated receptor gamma gene expression in patients with type 2 diabetes

AimsThis study aimed to investigate the roles of DNA methylation and miR-34a in the regulation of peroxisome proliferator-activated receptor gamma (PPARγ) in patients with type 2 diabetes (T2D). MethodsWe investigated the methylation status of four regions of the PPARγ promoter and PPARγ expression in a panel of 84 T2D patients using methylation-specific PCR (MSP) and RT-qPCR, respectively. Moreover, we quantified DNA methyltransferases (DNMTs) expression and global DNA methylation levels by RT-qPCR and ELISA, respectively. We measured the expression levels of miR-34a and protein expression of PPARγ by stem-loop RT-qPCR and ELISA, respectively. ResultsWe found significant DNA hypermethylation in the R2 and R3 regions of the PPARγ promoter in people with diabetes. Functionally, this was associated with a significant reduction in PPARγ expression. In addition, we observed a significant increase in 5-methylcytosine levels in people with diabetes. A marked increase in circulating miR-34a in the early stages of T2D (up to 10 years) and a significant decrease in circulating miR-34a with increasing diabetes duration from 10 years after the onset of diabetes. Interestingly, upregulation of DNA methyltransferases 1 (DNMT1), DNMT3A, and DNMT3B was observed in people with diabetes, and the average expression of DNMTs was negatively correlated with circulating miR-34a levels. In contrast, the serum protein level of PPARγ, a direct target of miR-34a, increased considerably with diabetes duration and showed a negative correlation with circulating miR-34a, cholesterol, triglyceride, and low-density lipoprotein. ConclusionPPARγ promoter hypermethylation and miR-34a upregulation are associated with T2D pathogenesis through PPARγ dysregulation.

Gestational exposure to BPA alters the expression of glucose and lipid metabolic mediators in the placenta: Role in programming offspring for obesity

Bisphenol A (BPA) exposure during pregnancy is known to predispose offspring to obesity in later life. Our previous studies demonstrated obesogenic effects in BPA-exposed offspring, including excess body fat, increased feed efficiency, adipocyte hypertrophy, and altered leptin signaling. However, the role of the placenta in mediating these effects remained unclear. This study investigates the mechanisms by which BPA exposure affects placental glucose and lipid transporters and their impact on offspring adiposity in Wistar rats. Dams were orally gavaged with BPA [0.4 (low dose-LD) and 4.0 (high dose-HD) μg/kg body weight] from gestational day (gD) 4–14. Gestational exposure to LD BPA increased the expression of 11β hydroxysteroid dehydrogenase 1 (11β HSD1) and estrogen receptor alpha (ERα) proteins (p<0.05) in the placenta compared to control and HD BPA. Similar changes were observed in the expression of mTOR signaling mediators, fatty acid transporters, and intracellular fatty acid-binding proteins. There were no changes in the dam's body weight or lipid and glucose profiles. However, there was a dose dependent increase in glucose transporter (GLUT1) expression in the placenta. While LD BPA increased hexokinase 2 expression in the placenta, HD BPA had no effect. Both doses of BPA increased IL6 expression, but only LD BPA exposure increased PPAR-gamma expression. Additionally, BPA exposure induced ADRP expression and localization, suggesting potential lipid overload in the placenta. Furthermore, BPA exposure altered the placental epigenetic profile, with increased expression of DNA methyltransferases (DNMTs). Overall, gestational BPA exposure led to dose-specific alterations in placental glucose and lipid metabolic activities, possibly playing an role in increasing the supply of these macronutrients to the fetus and predisposing the offspring to obesity.

Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

The up-regulation of SYNCRIP promotes the proliferation and tumorigenesis via DNMT3A/p16 in colorectal cancer

Heterogeneous nuclear ribonucleoproteins (hnRNPs), a group of proteins that control gene expression, have been implicated in many post-transcriptional processes. SYNCRIP (also known as hnRNP Q), a subtype of hnRNPs, has been reported to be involved in mRNA splicing and translation. In addition, the deregulation of SYNCRIP was found in colorectal cancer (CRC). However, the role of SYNCRIP in regulating CRC growth remains largely unknown. Here, we found that SYNCRIP was highly expressed in colorectal cancer by analyzing TCGA and GEPIA database. Furthermore, we confirmed the expression of SYNCRIP expression in CRC tumor and CRC cell lines. Functionally, SYNCRIP depletion using shRNA in CRC cell lines (SW480 and HCT 116) resulted in increased caspase3/7 activity and decreased cell proliferation, as well as migration. Meanwhile, overexpression of SYNCRIP showed opposite results. Mechanistically, SYNCRIP regulated the expression of DNA methyltransferases (DNMT) 3A, but not DNMT1 or DNMT3B, which affected the expression of tumor suppressor, p16. More importantly, our in vivo experiments showed that SYNCRIP depletion significantly inhibited colorectal tumor growth. Taken all together, our results suggest SYNCRIP as a potent therapeutic target in colorectal cancer.