Knockdown Of DNMT3B Research Articles

WITHDRAWN: Induced PSIG expression by Herbacetin contributes to suppressing the proliferation, migration, and invasion of melanoma cells

Melanoma is a very common malignant tumor with poor prognosis. Herbacetin is a flavonol compound with outstanding anti-tumor effects. Our work investigated the biological effects and mechanism of Herbacetin in melanoma. In our study, the mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. MSP was performed to evaluated PGIS promoter methylation level. Cell viability, migration and invasion were examined by MTT assay, transwell migration and invasion assay, respectively. Our results revealed that DNMT3B was markedly upregulated in melanoma, while PGIS was lowly expressed. Herbacetin treatment could not only inhibit the proliferation, migration, invasion of melanoma cells and inhibit the growth of melanoma in vivo. Herbacetin could also restore the abnormal expressions of DNMT3B and PGIS in melanoma cells and tumor tissues. PGIS silencing neutralized the inhibitory effects of Herbacetin on the malignant behaviors of melanoma cells. Besides, DNMT3B knockdown promoted PGIS expression via reducing PGIS promoter methylation level in melanoma cells, thereby inhibiting malignant behaviors of melanoma cells. And as expected, the inhibitory effects of Herbacetin on malignant behaviors of melanoma cells were all abolished by DNMT3B overexpression. Collectively, Herbacetin reduced DNMT3B expression to upregulate PGIS in melanoma cells and participated in suppressing the proliferation, migration, and invasion of melanoma cells.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

Epigenetic modifier alpha-ketoglutarate modulates aberrant gene body methylation and hydroxymethylation marks in diabetic heart

BackgroundDiabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. Hyperglycemic myocardial microenvironment significantly alters chromatin architecture and the transcriptome, resulting in aberrant activation of signaling pathways in a diabetic heart. Epigenetic marks play vital roles in transcriptional reprogramming during the development of DCM. The current study is aimed to profile genome-wide DNA (hydroxy)methylation patterns in the hearts of control and streptozotocin (STZ)-induced diabetic rats and decipher the effect of modulation of DNA methylation by alpha-ketoglutarate (AKG), a TET enzyme cofactor, on the progression of DCM.MethodsDiabetes was induced in male adult Wistar rats with an intraperitoneal injection of STZ. Diabetic and vehicle control animals were randomly divided into groups with/without AKG treatment. Cardiac function was monitored by performing cardiac catheterization. Global methylation (5mC) and hydroxymethylation (5hmC) patterns were mapped in the Left ventricular tissue of control and diabetic rats with the help of an enrichment-based (h)MEDIP-sequencing technique by using antibodies specific for 5mC and 5hmC. Sequencing data were validated by performing (h)MEDIP-qPCR analysis at the gene-specific level, and gene expression was analyzed by qPCR. The mRNA and protein expression of enzymes involved in the DNA methylation and demethylation cycle were analyzed by qPCR and western blotting. Global 5mC and 5hmC levels were also assessed in high glucose-treated DNMT3B knockdown H9c2 cells.ResultsWe found the increased expression of DNMT3B, MBD2, and MeCP2 with a concomitant accumulation of 5mC and 5hmC, specifically in gene body regions of diabetic rat hearts compared to the control. Calcium signaling was the most significantly affected pathway by cytosine modifications in the diabetic heart. Additionally, hypermethylated gene body regions were associated with Rap1, apelin, and phosphatidyl inositol signaling, while metabolic pathways were most affected by hyperhydroxymethylation. AKG supplementation in diabetic rats reversed aberrant methylation patterns and restored cardiac function. Hyperglycemia also increased 5mC and 5hmC levels in H9c2 cells, which was normalized by DNMT3B knockdown or AKG supplementation.ConclusionThis study demonstrates that reverting hyperglycemic damage to cardiac tissue might be possible by erasing adverse epigenetic signatures by supplementing epigenetic modulators such as AKG along with an existing antidiabetic treatment regimen.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Role of gga-miR-29b-3p in suppressing the proliferation, invasion and migration of MSB1 Marek’s disease tumor cells by the targeting of the DNMT3B gene

BackgroundMarek’s disease (MD), a class II infectious, lymphoproliferative disease that mainly afflicts poultry, has been shown to cause wasting, limb paralysis, and often acute death. It is a neoplastic disease caused by a cell-binding herpesvirus that leads to the formation of tumors in various organs and tissues. Our previous reports have found that the microRNA, gga-miR-29b-3p, showed abnormal expression in MD lymphoma. However, it remains unknown whether gga-miR-29b-3p affects MD tumorigenesis.MethodsThe MD tumor cell line MSB1 was chosen to analyze the characteristics of gga-miR-29b-3p in tumors. Cell proliferation and migration were assessed by Cell Counting Kit-8 (CCK-8) and Transwell, respectively, and cell apoptosis and cycle were analyzed via fluorescent staining and flow cytometry, respectively. The regulation between gga-miR-29b-3p and its potential target genes was verified by dual luciferase results and loss-of-function assays. The effect of target genes was verified by examining the degree of RNA interference on MSB1 cells.ResultsAnalysis revealed that gga-miR-29b-3p impaired the proliferation of the MSB1 MD tumor cell line, induced apoptosis without obvious effects on the cell cycle, and suppressed the expression of the invasion-associated MMP2 and MMP9 genes. It was concluded that DNMT3B is the direct target of gga-miR-29b-3p. As expected, the effects of DNMT3B knockdown with small interfering RNA (siRNA) on MSB1 cell proliferation, apoptosis, and cycle were associated with gga-miR-29b-3p overexpression. Moreover, BCL2 and BCL2L1 were downregulated and TNFSF10 was upregulated in both the gga-miR-29b-3p overexpression and DNMT3B knockdown groups. The expression levels of invasion-related genes were decreased post-DNMT3B knockdown. In both the gga-miR-29b-3p overexpression and DNMT3B knockdown conditions, a decrease in MEQ oncogene expression in MD virus was observed.ConclusionsOverall, gga-miR-29b-3p was demonstrated to have a suppressive effect in MD lymphoma progression via the targeting of the DNMT3B gene. Gga-miR-29b-3p overexpression and DNMT3B knockdown inhibited MSB1 cell proliferation through suppressing the pro-apoptotic gene expression and elevating the anti-apoptotic gene expression in the apoptosis pathway. Our study provides a theoretical basis for targeted treatment of MD.

P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse. Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response. Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients (GSE4581, E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients (GSE9782), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone. Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.

Exosomal circRNA_104948 Enhances the Progression of Glioma by Regulating miR-29b-3p and DNMT3B/MTSS1 Signaling.

Glioma is a common type of malignancy in the central nervous system. The pathogenesis of glioma is complex and the underlying mechanisms remain largely unknown. In our study, exosomes were exacted from patient samples, and the isolated exosomes were confirmed by transmission electron microscope. The expression of circRNA_104948, miR-29b-3p and DNMT3B were determined using RT-qPCR. Proliferative activity of cell was examined using CCK-8 assay. Cell apoptotic rate was evaluated by flow cytometry. The expression levels of proliferation or apop-tosis markers were determined using western blotting. Our data suggested that circRNA_104948 was upregulated in plasma exosomes/tissue samples of glioma patients and glioma cell lines. Furthermore, cell proliferation was enhanced and apoptosis was suppressed in normal astrocytes treated with exosomal circRNA_104948, and the effects were reversed by sh-circRNA_104948. In addition, miR-29b-3p is a novel target of circRNA_104948, and DNMT3B is a putative downstream molecule of miR-29b-3p. circRNA_104948 could regulate the proliferation/apoptosis of astrocytes through miR-29b-3p/DNMT3B/MTSS1 signaling, and the biological behavior changes induced by glioma-Exo were reversed by miR-29b-3p mimics; upregulated cell growth caused by miR-29b-3p inhibitors was abrogated by the knockdown of DNMT3B; the effects induced by miR-29b-3p mimics were abolished by the overexpression of DNMT3B. Our findings revealed the important roles of circRNA_104948 on the development of glioma, and circRNA_104948/miR-29b-3p/MTSS1/DNMT3B pathway may be a potential candidate for the target therapy of glioma patients.

Targeting DNMTs to Overcome Enzalutamide Resistance in Prostate Cancer.

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is an FDA-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period to enzalutamide, tumors will develop drug resistance. In this study, we uncovered that DNA methylation was deregulated in enzalutamide-resistant cells. DNMT activity and DNMT3B expression were upregulated in resistant cell lines. Enzalutamide induced the expression of DNMT3A and DNMT3B in prostate cancer cells with a potential role of p53 and pRB in this process. The overexpression of DNMT3B3, a DNMT3B variant, promoted an enzalutamide-resistant phenotype in C4-2B cell lines. Inhibition of DNA methylation and DNMT3B knockdown induced a resensitization to enzalutamide. Decitabine treatment in enzalutamide-resistant cells induced a decrease of the expression of AR-V7 and changes of genes for apoptosis, DNA repair, and mRNA splicing. Combination treatment of decitabine and enzalutamide induced a decrease of tumor weight, Ki-67 and AR-V7 expression and an increase of cleaved-caspase3 levels in 22Rv1 xenografts. The collective results suggest that DNA methylation pathway is deregulated after enzalutamide resistance onset and that targeting DNA methyltransferases restores the sensitivity to enzalutamide in prostate cancer cells.

Reciprocal epigenetic remodeling controls testicular cancer hypersensitivity to hypomethylating agents and chemotherapy.

Testicular germ cell tumors (TGCTs) are aggressive but sensitive to cisplatin‐based chemotherapy. Alternative therapies are needed for tumors refractory to cisplatin with hypomethylating agents providing one possibility. The mechanisms of cisplatin hypersensitivity and resistance in TGCTs remain poorly understood. Recently, it has been shown that TGCTs, even those resistant to cisplatin, are hypersensitive to very low doses of hypomethylating agents including 5‐aza deoxy‐cytosine (5‐aza) and guadecitabine. We undertook a pharmacogenomic approach in order to better understand mechanisms of TGCT hypomethylating agent hypersensitivity by generating a panel of acquired 5‐aza‐resistant TGCT cells and contrasting these to previously generated acquired isogenic cisplatin‐resistant cells from the same parent. Interestingly, there was a reciprocal relationship between cisplatin and 5‐aza sensitivity, with cisplatin resistance associated with increased sensitivity to 5‐aza and 5‐aza resistance associated with increased sensitivity to cisplatin. Unbiased transcriptome analysis revealed 5‐aza‐resistant cells strongly downregulated polycomb target gene expression, the exact opposite of the finding for cisplatin‐resistant cells, which upregulated polycomb target genes. This was associated with a dramatic increase in H3K27me3 and decrease in DNMT3B levels in 5‐aza‐resistant cells, the exact opposite changes seen in cisplatin‐resistant cells. Evidence is presented that reciprocal regulation of polycomb and DNMT3B may be initiated by changes in DNMT3B levels as DNMT3B knockdown alone in parental cells resulted in increased expression of H3K27me3, EZH2, and BMI1, conferred 5‐aza resistance and cisplatin sensitization, and mediated genome‐wide repression of polycomb target gene expression. Finally, genome‐wide analysis revealed that 5‐aza‐resistant, cisplatin‐resistant, and DNMT3B‐knockdown cells alter the expression of a common set of polycomb target genes. This study highlights that reciprocal epigenetic changes mediated by DNMT3B and polycomb may be a key driver of the unique cisplatin and 5‐aza hypersensitivity of TGCTs and suggests that distinct epigenetic vulnerabilities may exist for pharmacological targeting of TGCTs.

Abstract 2127: Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents

Abstract Testicular germ cell tumors (TGCTs) are the most frequent solid cancers of males between the ages of 16 to 39 and have the highest mortality in this age group. Testicular cancer is highly curable with platinum-based chemotherapeutics. However, 15-20% of patients are resistant and succumb to their disease. The mechanisms for cisplatin sensitivity/resistance in testicular cancer is unclear. Previously we showed that cisplatin refractory testicular cancer is highly sensitive to the DNA methyltransferase inhibitors (DNMTi), 5-Aza-2'-deoxycytidine (5-AZA-DC) and guadecitabine in vivo and in vitro. High expression of DNMT3B correlates with sensitivity to DNMTi in TGCT cells. Based on these preclinical findings we recently completed a phase 1 clinical trial which demonstrated the combination of guadecitabine and cisplatin was tolerable and effective in treating patients with platinum refractory germ cell cancer. To better understand the mechanism of chemotherapy sensitivity/resistance in testicular cancer we generated a series of cisplatin resistant cell models. Genome wide methylome array and de novo transcriptional profiling of cisplatin resistant TGCT cells revealed decreased global H3K27 methylation due to suppression of the polycomb repressive complex 2 (PRC2 ). To better understand the relationship between cisplatin and 5-AZA-DC sensitivity, 5-AZA-DC resistant testicular cancer cells were also generated. Remarkably, there was a reciprocal relationship between cisplatin and 5-AZA-DC resistance, with cisplatin resistance associated with increased sensitivity to 5-AZA-DC and 5-AZA-DC resistance associated with increased sensitivity to cisplatin. Transcriptomics profiling and other studies in 5-AZA-DC resistant cells revealed increased H3K27me3, repression of polycomb target genes and a dramatic decreased in DNMT3B expression, the exact opposite of the situation in cisplatin resistant cells. Further knockdown of DNMT3B resulted in induction of H3K27me3, decreased sensitivity to 5-AZA-DC and increased sensitivity to cisplatin. These data suggest that DNA methylation and polycomb signaling are interconnected drivers of chemotherapy and epigenetic therapy responses in TGCTs. Ongoing H3K27 me3 ChIP-Seq, global methylation analysis, and validation studies will be described to further elucidate these mechanisms with the goal of devising novel epigenetic-based therapies for testicular and potentially other cancers. Citation Format: Ratnakar Singh, Emmanuel Bikorimana, Zeeshan Fazal, Cliff Yerby, Hannah Baldwin, Aleyah Destiny Hattab, Megan Tomlin, Andrea K. Corbet, Raya Iman Boyd, Khadeeja Shahid, Doha Naguib Ahmed Mohamed Shokry, Sarah Joy Spinella, Michael J. Spinella. Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2127.

MiR-498/DNMT3b Axis Mediates Resistance to Radiotherapy in Esophageal Cancer Cells.

Objective: To explore the role of miR-498 in the radiotherapy resistance of esophageal cancer (EC) and its underlying mechanism. Methods: In vivo models of EC tissues with radioresistance or radiosensitivity were isolated from 72 EC patients who received radiotherapy. In vitro models were established after irradiation of KYSE30 cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were employed to measure the expression levels of miR-498 and DNMT3b in EC cells sensitive or resistant to irradiation. Then, protein expression of DNMT3b was verified by immunohistochemistry. The cell viability, colony formation rate, and cell apoptotic rate of EC were correspondingly assessed by CCK-8, colony formation assay, and Annexin V/PI (propidium iodide) double staining. Western blot was utilized to perform the expression levels of PI3K, p-PI3K, AKT, and p-AKT in EC cell lines after irradiation. Results: Highly expressed DNMT3b and lowly expressed miR-498 were found in EC tissues. EC tissues with radiosensitivity had higher miR-498 level and lower DNMT3b expression than EC tissues with radioresistance. Overexpression of miR-498 or knockdown of DNMT3b enhanced the radiosensitivity of EC cells. DNMT3b was a target gene of miR-498. DNMT3b diminished the radiosensitization of miR-498 in EC cells. Conclusions: MiR-498 enhances the sensitivity of EC cells to radiation by DNMT3b inhibition, and exerts biological functions by inactivating the PI3K/AKT signaling pathway.

Impact of miRNAs expression modulation on the methylation status of breast cancer stem cell-related genes.

Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes. MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting. DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24- subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24- subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c. The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A.

Dnmt3b regulates DUX4 expression in a tissue-dependent manner in transgenic D4Z4 mice

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown.MethodsTo determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation in Dnmt3b (Dnmt3bMommeD14 mice). Additionally, siRNA knockdowns of Dnmt3b were performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model.ResultsIn mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown of Dnmt3b. In D4Z4-2.5/Dnmt3bMommeD14 mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3bMommeD14 mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3bMommeD14 mice or Smchd1MommeD1 mice. Only in D4Z4-2.5/Smchd1MommeD1 mice the immune cell populations were disturbed.ConclusionsOur data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3bMommeD14 mice. In addition, the Smchd1MommeD1 variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD.

Role of DNA methylation on human CTSG in dermatomyositic myoideum.

Dermatomyositis (DM) is a multifactorial chronic autoimmune disorder with characteristic skin and muscle pathological changes and involvement of other organ systems. Cathepsin G (CTSG) contributes to the risk of developing DM, which is likely to be associated with inflammatory cytokines. Differential DNA methylation on CTSG has been determined to be implicated in DM in vivo. However, the underlying mechanism of this epigenetic regulation on CTST in DM is poorly explored. In this study, we investigated DNA methylation signature on CTSG at single-nucleotide resolution in quadriceps femoris of six DM patients and paracancerous muscles of three patients with rhabdomyosarcoma on inner thigh using pyrosequencing and observed that the overall DNA methylation level of CTSG was increased in DM compared with control, in which CpG loci at third and fourth exons but not promoter contributed to the significant hypermethylation. Furthermore, we observed that transcription and DNA methylation of CTSG were both declined in DNMT3a knockdown compared with DNMT1 and DNMT3b knockdown in human skeletal muscle SJCRH30 and A-204 cell lines exposed to tumor necrosis factor-α. Furthermore, Bortezomib (NF-κB inhibitor) and Brevilin A (JAK/STAT inhibitor) were employed to treat SJCRH30 and A-204 cells, respectively, and we observed that CTSG was hypomethylated and silenced after Bortezomib treatment compared with untreatment and Brevilin A. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction indicated that DNMT3a could bind to the coding regions of CTSG and the interaction was dependent on NF-κB activity. Taken together, our results determined a novel regulatory mechanism of DNA methylation on CTSG in DM.

Estrogen-induced epigenetic silencing of FTH1 and TFRC genes reduces liver cancer cell growth and survival

ABSTRACT Estrogen (E2) regulates hundreds of genes involved in cell metabolism and disrupts iron homoeostasis in various cell types. Herein, we addressed whether E2-induced epigenetic modifications are involved in modulating the expression of iron-regulatory genes. Epigenetic status of FTH1 and TFRC genes was assessed in E2-treated cancer cells. E2-induced DNA methylation was associated with decreased FTH1 and TFRC expression in Hep-G2 and Huh7 cells, but not in AGS or MCF7 cells. Demethylation with 5-Aza-2-deoxycytidine upregulated the expression of both these genes in Hep-G2 cells. The expression of DNMT3B, PRMT5, and H4R3me2s increased in E2-treated cells. Chromatin immunoprecipitation showed that E2 treatment recruited PRMT5 and H4R3me2s on FTH1 but not on TFRC. Knockdown of PRMT5, DNMT3B, and Estrogen-receptor alpha rescued FTH1 from E2-induced silencing. However, knockdown of DNMT3B alone blocked the inhibitory effects of E2 on TFRC. Analysis of human liver tissues in publicly available datasets showed that FTH1 and TFRC are highly expressed in primary liver tumours, but a lower expression is associated with better survival. Interestingly, we showed that the silencing of FTH1 and/or TFRC inhibited carcinogenesis in Hep-G2 cells. For the first time, our findings uncovered the novel signalling pathway involved in the protective effects of E2 against liver cancer.

Identification and validation of mRNA 3'untranslated regions of DNMT3B and TET3 as novel competing endogenous RNAs of the tumor suppressor PTEN

PTEN inactivation is a frequent event in oncogenesis. Multiple regulatory mechanisms such as promoter hypermethylation, antisense regulation, histone modifications, targeting by microRNAs (miRNAs/miRs) and regulation by transcription factors have all been shown to affect the tumor suppressor functions of PTEN. More recently, the functional involvement of competing endogenous RNAs (ceRNAs) in miRNA-dependent and coding-independent regulation of genes shed light on the highly nuanced control of PTEN expression. The present study has identified and validated DNA methyltransferase 3β (DNMT3B) and TET methylcytosine dioxygenase 3 (TET3) as novel ceRNAs of PTEN, with which they share multiple miRNAs, in HCT116 colorectal cancer cells. miR-4465 was identified and characterized as a miRNA that directly targets and regulates all 3 transcripts via their 3′untranslated regions (3′UTRs) through a combination of luciferase reporter assays, abrogation of miRNA response elements (MREs) via site-directed mutagenesis, target protection of MREs with locked nucleic acids, RT-qPCR assays and western blot analysis. Competitive miRNA sequestration was demonstrated upon reciprocal 3′UTR overexpression and siRNA-mediated knockdown of their respective transcripts. Overexpression of DNMT3B or TET3 3′UTR promoted apop-tosis and decreased migratory capacity, potentially because of shared miRNA sequestration and subsequent activation of PTEN expression. Knockdown of TET3 and DNMT3B decoupled their protein-coding from miRNA-dependent, coding-independent functions. Furthermore, the findings suggested that the phenotypic outcome of ceRNAs is dictated largely by the number of shared miRNAs, and predictably, by the existence of other ceRNA networks in which they participate. Taken together, the findings of the present study identified DNMT3B and TET3 as novel ceRNAs of PTEN that may impact its dose-sensitive tumor suppressive function.

P.48The role of Dnmt3b in DUX4 repression in transgenic mice

Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in muscle cells. DUX4 is encoded in the D4Z4 macrosatellite repeat array, consisting of 8-100 repeat units in non-affected individuals. The most common form of FSHD, FSHD type 1, is caused by a contraction of the D4Z4 repeat to 1-10 repeat units, leading to somatic derepression of DUX4. FSHD type 2 (FSHD2) is often caused by mutations in SMCHD1, an epigenetic repressor of the D4Z4 repeat. Recently, DNMT3B mutations were identified in FSHD2 patients that lack a mutation in SMCHD1. DNMT3B is a de novo DNA methyltransferase, but the exact role of DNMT3B on repression of DUX4 remains unknown. To study the role of Dnmt3b on DUX4 repression, we crossed mice hemizygous for a FSHD-sized D4Z4 repeat (D4Z4-2.5 mice) with mice carrying a heterozygous mutation in Dnmt3b leading to exon 13 skipping (Dnmt3bMommeD14 mice). While we did not find any significant changes in DUX4 expression for several skeletal muscles and non-muscle tissues, we observed a significantly higher DUX4 expression in the spleen and lymph nodes of D4Z4-2.5/Dnmt3bMommeD14 mice compared with D4Z4-2.5 mice. In addition, we found lower DNA methylation levels at the D4Z4 repeat in D4Z4-2.5/Dnmt3bMommeD14 mice. Dnmt3b siRNA knockdown experiments in murine embryonic stem cells (mESCs) carrying either a long D4Z4 repeat (12.5 repeat units) or a short D4Z4 repeat (2.5 repeat units) confirmed that in mESCs with a short D4Z4 repeat high DUX4 expression levels were further enhanced after Dnmt3b knockdown. In contrast, DUX4 expression was not affected after Dnmt3b knockdown in mESCs with a long D4Z4 repeat that normally do not express DUX4. In short, our data suggest that Dnmt3b is involved in DUX4 repression in some somatic tissues and mESCs of transgenic FSHD mice.

Human mitochondrial DNA is extensively methylated in a non-CpG context.

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.

Abstract 2613: MYC controls DNA methylation on a global scale through DNMT3b upregulation in T-ALL and Burkitts Lymphoma

Abstract Background: Oncogenic MYC is implicated in 60-70% of all human cancers, including T-cell acute lymphoblastic leukemia (T-ALL) and Burkitts lymphoma (BL). Genetic inactivation of MYC can cause tumor regression through cellular senescence in mouse models (exhibiting oncogene addiction), making it a promising therapeutic target. However, a pharmacological MYC inhibitor remains elusive, thus these findings cannot be translated into clinic. We are the first to demonstrate that MYC can control DNA methyl-transferase (DNMT) expression and thus, genome-wide DNA methylation; as an alternative strategy to disrupt the MYC network indirectly, we have chosen to target MYC-dependent DNMTs as a tumor-cell specific strategy. Experimental Procedures and Results: Utilizing expression profiling in patient-derived cell lines and clinical specimens, we found DNMT 1, 3a, and 3b to be overexpressed in MYC-driven T-ALL and BL compared to non-malignant tissue. Using transgenic inducible “tet-off” MYC-models in mouse T-ALL (EµSRα-tTA;tet-o-MYC) and human Burkitt’s lymphoma (P493-6), we have shown that DNMT1 and DNMT3b expression depend on high MYC expression levels in T-ALL and BL. Using patient-derived cell lines, we’ve shown that depletion of endogenous MYC caused a reduction of both DNMT1 and DNMT3b expression. Chromatin Immunoprecipitation (ChIP) profiling for MYC binding, further establishes the DNMT1 and DNMT3b promoters as bona fide targets of the MYC oncogene. Furthermore, shRNA-mediated knockdown of DNMT3b or pharmacological inhibition using NanaomycinA, indicates a tumor promoter function in the context of MYC-driven tumor maintenance in T-ALL. To further elucidate the consequence on DNA methylation and gene expression upon DNMT3b loss of function, Reduced Representation Bisulfite Sequencing and RNA-sequencing was performed to analyze alterations in DNA methylation and gene expression, respectively. We found that DNMT3b maintains CpG island DNA methylation near promoters, responsible for gene silencing during tumor maintenance. Accompanying changes in DNA methylation, we identified approximately one thousand differentially expressed genes upon DNMT3b knockdown, with upregulated genes responsible for controlling differentiation, transport, and development; while downregulated genes controlled processes including signal transduction, apoptosis, and regulation of cellular proliferation. Conclusion: Together, we provide novel evidence that MYC directly controls global DNA methylation through deregulation of DNMT1 and DNMT3b expression to maintain gene programs for T-ALL tumor maintenance. Furthermore, we provide evidence that targeted inhibition of individual DNMTs may reveal alternative tumor-specific therapies for T-ALL, BL, and other MYC-driven cancers. Citation Format: Candace J. Poole, Atul Lodh, Jan van Riggelen. MYC controls DNA methylation on a global scale through DNMT3b upregulation in T-ALL and Burkitts Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2613.

MiR-29b suppresses proliferation and mobility by targeting SOX12 and DNMT3b in pancreatic cancer.

Pancreatic cancer is one of the leading causes of solid carcinoma with the worst survival rate. The reasons for the worst survival rate include the lack of biomarkers for early detection, diagnosis at a late stage, and the limitation of the current therapy. Further study to investigate the underlying molecular mechanism in pancreatic cancer patients is necessary. A previous study showed that the miR-29b expression level is dysregulated, suggesting that it may serve an important function in pancreatic cancer. The CCK8 assay and the colony formation assay were used to detect the proliferation ability of the treated pancreatic cancer cells; a wound-healing assay and a transwell assay were used to test the migration and invasion ability and the interactive action of miR-29b and SOX12 or DNMT3b was examined by a luciferase assay. Cell proliferation, migration, and invasion were attenuated by miR-29b, whereas knockdown of SOX12 and DNMT3b could block SW1990 malignant activity. Further, the double luciferase assay showed that miR-29b can target SOX12 and DNMT3b directly by binding to their 3'-untranslated region. Finally, a rescue experiment was conducted by transfecting miR-29b and SOX12 overexpressed plasmid into cells. Cell proliferation, migration, and invasion inhibition induced by miR-29b were reversed by SOX12 overexpression, and revail of the expression of DNMT3b. MiR-29b suppressed proliferation, migration, and invasion by directly targeting SOX12 and DNMT3b in pancreatic cancer cells, and DNMT3b might be a target gene of SOX12.