5-azaC Treatment Research Articles

DNA demethylation is involved in nitric oxide-induced flowering in tomato

Flowering is one of the most important phenological periods, as it determines the timing of fruit maturation and seed dispersal. To date, both nitric oxide (NO) and DNA demethylation have been reported to regulate flowering in plants. However, there is no compelling experimental evidence of the relationship between NO and DNA demethylation during plant flowering. In this study, an NO donor and a DNA methylation inhibitor were used to investigate the involvement of DNA demethylation in NO-mediated tomato (Solanum lycopersicum cv. Micro-Tom) flowering. The results showed that the promoting effect of NO on tomato flowering was dose-dependent, with the most positive role observed at 10 μmol L-1 of the NO donor S-nitrosoglutathione (GSNO). Treatment with 50 μmol L-1 of the DNA methylation inhibitor 5-azacitidine (5-AzaC) also significantly promoted tomato flowering. Moreover, GSNO and 5-AzaC increased the peroxidase (POD) and catalase (CAT) activity and cytokinin (CTK) and proline contents, while they decreased the gibberellic acid (GA3) and indole-3-acetic acid (IAA) contents. Co-treatment with GSNO and 5-AzaC accelerated the positive effects of GSNO and 5-AzaC in promoting tomato flowering. Meanwhile, compared with GSNO or 5-AzaC treatment alone, co-treatment with GSNO+5-AzaC significantly increased the global DNA demethylation levels in different tissues in tomato. The results also indicate that DNA demethylation may be involved in NO-induced flowering. The expression of flowering genes was significantly altered by the GSNO+5-AzaC treatment. Among these genes, five flowering induction genes-ARGONAUTE 4 (AGO4A), SlSP3D/SINGLE FLOWER TRUSS (SFT), MutS HOMOLOG 1 (MSH1), ZINC FINGER PROTEIN 2 (ZFP2), and FLOWERING LOCUS D (FLD) were selected as candidate genes for further study. Then, McrBC-PCR analysis showed that the DNA demethylation of the SFT gene in the apex and the FLD gene in the stem might be involved in NO-induced flowering. Therefore, our study shows that NO might promote tomato flowering by mediating the DNA demethylation of flowering induction genes. This study provides direct evidence of a synergistic effect of NO and DNA demethylation in promoting tomato flowering.

Alpha 1,3 N-Acetylgalactosaminyl Transferase (GTA) Impairs Invasion Potential of Trophoblast Cells in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder associated with shallow invasion of the trophoblast cells and insufficient remodeling of the uterine spiral artery. Protein glycosylation plays an important role in trophoblast cell invasion. However, the glycobiological mechanism of PE has not been fully elucidated. In the current study, employing the Lectin array, we found that soybean agglutinin (SBA), which recognizes the terminal N-acetylgalactosamine α1,3-galactose (GalNAc α1,3 Gal) glycotype, was significantly increased in placental trophoblast cells from PE patients compared with third-trimester pregnant controls. Upregulating the expression of the key enzyme α1,3 N-acetylgalactosaminyl transferase (GTA) promoted the biosynthesis of terminal GalNAc α1,3 Gal and inhibited the migration/invasion of HTR8/SVneo trophoblast cells. Moreover, the methylation status of GTA promoter in placental tissues from PE patients was lower than that in the third trimester by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis. Elevated GTA expression in combination with the DNA methylation inhibitor 5-azacytidine (5-AzaC) treatment increased the glycotype biosynthesis and impaired the invasion potential of trophoblast cells, leading to preeclampsia. This study suggests that elevated terminal GalNAc α1,3 Gal biosynthesis and GTA expression may be applied as the new markers for evaluating placental function and the auxiliary diagnosis of preeclampsia.

DNA methylation impacts soybean early development by modulating hormones and metabolic pathways.

Genomic DNA methylation patterns play a crucial role in the developmental processes of plants and mammals. In this study, we aimed to investigate the significant effects of epigenetic mechanisms on the development of soybean seedlings and metabolic pathways. Our analyses show that 5-azaC-treatment affects radicle development from two Days After Imbibition (DAI), as well as both shoot and root development. We examined the expression levels of key genes related to DNA methylation and demethylation pathways, such as DRM2, which encodes RNA-directed DNA Methylation (RdDM) pathway, SAM synthase, responsible for methyl group donation, and ROS1, a DNA demethylase. In treated seedling roots, we observed an increase in DRM2 expression and a decrease in ROS1 expression. Additionally, 5-azaC treatment altered protein accumulation, indicating epigenetic control over stress response while inhibiting nitrogen assimilation, urea cycle, and glycolysis-related proteins. Furthermore, it influenced the levels of various phytohormones and metabolites crucial for seedling growth, such as ABA, IAA, ethylene, polyamines (PUT and Cad), and free amino acids, suggesting that epigenetic changes may shape soybean responses to pathogens, abiotic stress, and nutrient absorption. Our results assist in understanding how hypomethylation shapes soybean responses to pathogens, abiotic stress, and nutrient absorption crucial for seedling growth, suggesting that the plant's assimilation of carbon and nitrogen, along with hormone pathways, may be influenced by epigenetic changes.

Abstract 1735: DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing

Abstract Introduction: BRCA2 is one of two principal tumor suppressors responsible for hereditary breast/ovarian cancer syndrome (HBOC). Patients with strong family histories of breast/ovarian cancer may carry BRCA2 DNA sequence variants of unknown clinical significance (VUSs). To evaluate which of these VUSs may contribute to cancer risk by altering splicing patterns, naturally occurring BRCA2 mRNA alternate splicing events have been extensively characterized, but it is not yet known which if any of these alternate splicing events plays a role in BRCA2 function. One variant lacking exon 3 (∆3), which maintains the full length translational reading frame, lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of ∆3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. It is therefore of interest to characterize the factors that influence the frequency of naturally occurring BRCA2 exon 3-skipping, including cell types and therapies. DNA demethylating agents are used as cancer therapy to promote expression of tumor suppressor genes that may be epigenetically silenced in tumors. However, DNA methylation has been shown to be more frequent in exonic than intronic regions of protein-coding genes, suggesting that methylation may play a role in recruiting spliceosomal components to nascent pre-mRNA. It is therefore of interest to determine whether genomic demethylation affects alternative splicing patterns. Here, we explore whether BRCA2 exon 3 alternative splicing patterns are altered by demethylating agents. Methods: To determine whether DNA demethylating agents can alter the levels of ∆3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 2’-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. Results and Conclusions: 1) While the IC50 for both 5-AzadC and 5-AzaC in MCF 10A was similar (less than 1 μM), MCF7 showed considerably more resistance to 5-AzaC than 5-AzadC (IC50 of ≥ 5 μM vs ≤ 0.2 μM). 2) 5-AzaC treatment reduces the proportion of MCF 10A cultures in apoptosis but does not increase the proportion of viable cells, indicating it causes cell death by some other means. 3) While both 5-AzaC and 5-AzadC reduce relative levels of ∆3 in MCF7, 5-azadC does not reduce relative levels of ∆3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. Citation Format: Jordan Werner, Hiba Siddiqui, Samiha Syed, Osman Khan, Servando Casillas Garabito, James D. Fackenthal. DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing [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 1735.

Azacytidine shows potential in controlling the chilling injury of banana peel during cold storage

Banana suffers from chilling injury at low temperatures, which significantly challenges industrial banana quality control worldwide without a widely-accepted solution, to date. In this study, 5-azacytidine (5-azaC), a common DNA methylation inhibitor (DNA methylation is a conserved modification that regulates physiological processes), was used to treat banana fingers with subsequent storage at 7 °C for 9 days, which can be a novel strategy for controlling banana peel chilling injury and quality deterioration. In most sampling days, 5-azaC treatment significantly increased lightness value (L*), hue angle, and firmness while significantly decreased the browning index. Surprisingly, the chilling injury index was persistently declined by 5-azaC up to 26.8 % (p < 0.05). 5-azaC also reduced electrolyte leakage and levels of malondialdehyde and reactive oxygen species, indicating the control of chilling injury development. Further results showed that 5-azaC-inhibited DNA methylation contributed to banana chilling injury control by modulating the redox system and energy homeostasis. Collectively, this study offered new insights into fruit chilling injury development and control, as well as inspired potential strategies for postharvest quality control.

DNA demethylation by transitory 5‐azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak

AbstractSomatic embryogenesis (SE) is a crucial biotechnological tool for large‐scale mass propagation of selected material, genetic transformation and breeding, with many advantages for forest tree improvement. However, the application of SE in forest species is limited because of their recalcitrance. SE is also a valuable system for studying cell reprogramming, acquisition of totipotency and embryo development. Increasing evidence reveals that epigenetic reprogramming takes place during SE through DNA methylation, although there is scarce information on forest species. In this work, we have evaluated DNA methylation dynamics during SE and the effects of the DNA demethylating agent 5‐azacytidine (AzaC) in cork oak. After induction and early stages of SE, a reduced DNA methylation level is observed, followed by an increase of methylation during embryo differentiation. These changes in DNA methylation during SE progression were associated with expression profiles of DNA methyltransferase genes QsMET1, QsDRM2 and QsCMT3, and DNA demethylase QsDME‐like. Treatment with AzaC reduced DNA methylation, promoted SE induction rate and proembryogenic masses proliferation, and induced the expression of the SE marker gene QsSERK1‐like. However, continuous AzaC treatment hindered embryo differentiation, suggesting that DNA methylation is needed for further embryo development. Interestingly, AzaC removal from the culture medium of embryogenic masses restored embryo development and led to a significant increase in somatic embryo production compared with untreated cultures. These findings open new possibilities using transitory treatments with small molecule epigenetic modulators, as AzaC, to enhance SE yields for forestry breeding programs.

A cytosine analogue 5-azacitidine improves the accumulation of licochalcone A in licorice Glycyrrhiza inflata

Licochalcone A (LCA) is a characteristic compound of Glycyrrhiza inflata with anti-inflammatory, antioxidant and antitumor activities. However, G. inflata produces LCA in low quantities that does not meet the market demand. In this study, we found that DNA methylation inhibitor 5-azacitidine (5-azaC) successfully improved the LCA contents in G. inflata seedlings. Transcriptome analysis revealed a series of differentially expressed genes (DEGs), including transcription factors such as MYB, ERF, WRKY, and some structural genes related to flavonoid biosynthesis. However, whole genome bisulfite sequencing (BS-seq) results showed little effect of the 5-azaC treatment on the alteration of DNA methylation on these genes, indicating the possibility that 5-azaC acts as a stimulus, but not an epigenetic modulation factor to improve the LCA content in G. inflata. Additionally, we applied the 5-azaC treatment to field plants and hairy roots and successfully increased the LCA contents in both cases. This research demonstrates the feasibility of 5-azaC treatments in future applications to improve plant production of LCA.

The 5-azacytidine-induced epimutagenesis of sugarcane (Saccharum spp. hybrids) for aluminium tolerance

A protocol for generating aluminium (Al) tolerant sugarcane using 5-azacytidine (5-azaC)-induced epimutagenesis was developed. Eight (8) plantlets per gram of cultivar N51 calli were generated from the 5-azaC (100 μM) and Al (1.5–50 mM) treatment (+Az + Al) when compared with 104 plantlets from non-mutagenised, no Al control treatment. Following in vitro selection on 60 mM Al, ex vitro stress over four rounds of chimera dissolution during vegetative propagation was applied. In the final stress round, 36% of the Mut 2 epilines survived with plants showing higher relative water content (61.2%) when compared with 47.3% from the stressed control (S N51). The Mut 2 line maintained a higher green leaf area (83.4%) and longer roots (32.4 cm) under stress than S N51 (61.4% and 26.3 cm, respectively). Overall, Mut 2 had a high stress tolerance index of 85.4%, compared with 79.0% from the S N51, nearing that of the non-stressed N51 control (NS N51, 100%) when data were analysed using PCA and clustering analyses of morpho-physiological traits. Analysis of molecular variance (AMOVA) revealed high epigenetic differentiation (ɸst = 0.67) and a variation of 66.6% observed among N51 genotypes. The principal coordinate analysis (PCoA) showed that Mut 2 was epigenetically distinct from S N51. These findings support previous studies that 5-azaC can be used for novel trait creation via epimutagenesis and highlights the necessity for chimera dissolution to achieve stable traits in epibreeding of sugarcane.

Inhibition of DNA and RNA methylation disturbs root development of moso bamboo.

DNA methylation (5mC) and N6-methyladenosine (m6A) are two important epigenetics regulation, which presents profound impact on plant growth development. Phyllostachys edulis (P. edulis) is one of the fastest spreading plants due to its well-developed root system. However, the association between 5mC and m6A were seldom reported in P. edulis. Especially, the connection between m6A and several post-transcriptional regulations remains uncharacterized in P. edulis. Here, our morphological and electron microscope observation shown phenotype of increased lateral root under RNA methylation inhibitor (DZnepA) and DNA methylation inhibitor (5-azaC) treatment. RNA epitranscriptome based on Nanopore direct RNA sequencing (DRS) revealed that DZnepA treatment exhibits significantly decreased m6A level in 3' UTR, which was accompanied by increased gene expression, full-length ratio, higher proximal poly(A) site usage, and shorter poly(A) tail length, respectively. DNA methylation levels of CG and CHG were reduced in both CDS and transposable element (TE) upon 5-azaC treatment. Cell wall synthesis was impaired under methylation inhibition. Especially, differentially expressed genes (DEGs) presented high percentage of overlap between DZnepA and 5-azaC treatment, which suggested a potential correlation between two methylations. This study provides preliminary information for a better understanding of the link between m6A and 5mC in root development of moso bamboo.

Comparative analysis of the chrysanthemum transcriptome with DNA methylation inhibitors treatment and silencing MET1 lines

BackgroundAs one of the ten most famous flowers in China, the chrysanthemum has rich germplasm with a variety of flowering induction pathways, most of which are photoperiod-induced. After treatment with DNA methylation inhibitors, it was found that DNA methylation plays an important role in flowering regulation, but the mechanism of action remains unclear. Therefore, in this study, curcumin, 5-azaC, their mixed treatment, and MET1-RNAi lines were used for transcriptome sequencing to find out how different treatments affected gene expression in chrysanthemums at different stages of flowering.ResultsGenomic DNA methylation levels were measured using HPLC technology. The methylation level of the whole genome in the vegetative growth stage was higher than that in the flowering stage. The methylation level of DNA in the vegetative growth stage was the lowest in the curcumin and mixed treatment, and the methylation level of DNA in the transgenic line, mixed treatment, and curcumin treatment was the lowest in the flowering stage. The flowering rate of mixed treatment and curcumin treatment was the lowest. Analysis of differentially expressed genes in transcriptomes showed that 5-azaC treatment had the most differentially expressed genes, followed by curcumin and transgenic lines, and mixed treatment had the fewest. In addition, 5-azaC treatment resulted in the differential expression of multiple DNA methylation transferases, which led to the differential expression of many genes. Analysis of differentially expressed genes in different treatments revealed that different treatments had gene specificity. However, the down-regulated GO pathway in all 4 treatments was involved in the negative regulation of the reproductive process, and post-embryonic development, and regulation of flower development. Several genes associated with DNA methylation and flowering regulation showed differential expression in response to various treatments.ConclusionsBoth DNA methylase reagent treatment and targeted silencing of the MET1 gene can cause differential expression of the genes. The operation of the exogenous application is simple, but the affected genes are exceedingly diverse and untargeted. Therefore, it is possible to construct populations with DNA methylation phenotypic diversity and to screen genes for DNA methylation regulation.

Hypermethylation in the promoter region inhibits AJAP1 expression and activates the JAK/STAT pathway to promote prostate cancer cell migration and stem cell sphere formation

AJAP1 is down-regulated in multiple cancer types and plays a suppressive role in cancer progression. However, its molecular regulatory mechanism in prostate cancer has not been reported. Bioinformatics methods were employed to analyze AJAP1 expression in prostate cancer tissues and its association with TNM staging. MSP and qRT-PCR were used to quantify promoter methylation and AJAP1 expression after 5-aza-20-deoxycytidine (5-AzaC) treatment. Scratch healing assay and Transwell method were adopted to analyze the effects of aberrant AJAP1 expression, 5-AzaC and AG490 on cell migration and invasion. The levels of AJAP1 protein, EMT-related and JAK/STAT pathway-related proteins were determined by Western blot. The effects of AJAP1 aberrant expression and AG490 treatment on the sphere forming ability of prostate cancer cells were analyzed by sphere formation assay. This study confirmed the significant down-regulation of AJAP1 expression in prostate cancer tissues and cells, and its negative correlation with TNM staging. 5-AzaC treatment led to a significant reduction of AJAP1 methylation level and a significant upregulation of AJAP1 expression, indicating that the methylation level of AJAP1 promoter may affect the expression of AJAP1. Cell function experiments found that overexpression or decreased methylation of AJAP1 inhibited epithelial mesenchymal transition (EMT), migration, and invasion, while silencing or increased methylation of AJAP1 had the opposite functions. JAK2/STAT3 pathway inhibiting assay found that inhibition of JAK2/STAT3 pathway significantly reduced EMT, cell migration, and stem cell sphere formation in prostate cancer. Therefore, investigating the influence of aberrant AJAP1 expression on functions of prostate cancer cells is conducive to our in-depth understanding of the mechanism of prostate cancer genesis and development.

Epigenetic regulation of RARB overcomes the radio-resistance of colorectal carcinoma cells via cancer stem cells.

Cancer stem cells (CSCs) are able to survive after cancer therapies, leading to cancer progression and recurrence in colorectal carcinoma (CRC). Therapies targeting CSCs are believed to be promising strategies for efficiently eradicating cancers. This study was to investigate that how retinoic acid receptor beta (RARB) affected the biological characteristics of CSCs and radio-resistance in CRC and the epigenetic mechanism. The sensitivity of CSCs isolated from HCT116 cells to radiotherapy was reduced compared with the parental cells. Using database querying, we found that RARB was one of the most significantly downregulated gene in radio-resistant cells in CRC. Also, RARB was poorly expressed in our isolated CSCs, and overexpression of RARB inhibited the properties of CSCs and enhanced radiotherapy sensitivity. Mechanistically, the methylation of RARB was higher in CSCs compared with HCT116 cells, which was significantly reduced after the application of DNA methylation inhibitor 5-azacytidine (5-azaC). DNA methyltransferases (DNMT1) was found to be recruited into the RARB promoter. 5-AzaC treatment inhibited DNMT1 activity and improved radiotherapy sensitivity by promoting RARB expression. Our results imply that inhibition of DNMT1 can display a new mechanism for the epigenetic mediation of RARB in radio-resistant CRC.

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2’-deoxycytidine by methylating 2’-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity toward AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double-strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis efficiently in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response toward AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

Effects of 5-azaC on Iridoid Glycoside Accumulation and DNA Methylation in Rehmannia glutinosa.

Iridoid glycoside is the important secondary metabolite and the main active component in Rehmannia glutinosa. However, the mechanisms that underlie the regulation of iridoid glycoside biosynthesis remain poorly understood in R. glutinosa. Herein, the analysis of RNA-seq data revealed that 3,394 unigenes related to the biosynthesis of secondary metabolites were identified in R. glutinosa. A total of 357 unigenes were involved in iridoid glycoside synthesis, in which the highly conservative genes, such as DXS, DXR, GPPS, G10H, and 10HGO, in organisms were overexpressed. The analysis of the above genes confirmed that the co-occurrence ratio of DXS, DXR, and GPPS was high in plants. Further, our results showed that under normal and 5-azacytidine (5-azaC) treatment, the expression levels of DXS, DXR, GPPS, G10H, and 10HGO were consistent with the iridoid glycoside accumulation in R. glutinosa, in which the application of the different concentrations of 5-azaC, especially 50 μM 5-azaC, could significantly upregulate the expression of five genes above and iridoid glycoside content. In addition, the changes in the spatiotemporal specificity of degree and levels of DNA methylation were observed in R. glutinosa, in which the hemi-methylation was the main reason for the change in DNA methylation levels. Similar to the changes in 5-methyl cytosine (5mC) content, the DNA demethylation could be induced by 5-azaC and responded in a dose-dependent manner to 15, 50, and 100 μM 5-azaC. Taken together, the expression of iridoid glycoside synthesis gene was upregulated by the demethylation in R. glutinosa, followed by triggering the iridoid glycoside accumulation. These findings not only identify the key genes of iridoid glycoside synthesis from R. glutinosa, but also expand our current knowledge of the function of methylation in iridoid glycoside accumulation.

DNA Demethylation Induces Tree Peony Flowering with a Low Deformity Rate Compared to Gibberellin by Inducing PsFT Expression under Forcing Culture Conditions.

Gibberellin (GA) is frequently used in tree peony forcing culture, but inappropriate application often causes flower deformity. Here, 5-azacytidine (5-azaC), an efficient DNA demethylating reagent, induced tree peony flowering with a low deformity rate by rapidly inducing PsFT expression, whereas GA treatment affected various flowering pathway genes with strong pleiotropy. The 5-azaC treatment, but not GA, significantly reduced the methylation level in the PsFT promoter with the demethylation of five CG contexts in a 369 bp CG-rich region, and eight light-responsive related cis-elements were also predicted in this region, accompanied by enhanced leaf photosynthetic efficiency. Through GO analysis, all methylation-closer differentially expressed genes (DEGs) were located in the thylakoid, the main site for photosynthesis, and were mainly involved in response to stimulus and single-organism process, whereas GA-closer DEGs had a wider distribution inside and outside of cells, associated with 12 categories of processes and regulations. We further mapped five candidate DEGs with potential flowering regulation, including three kinases (SnRK1, WAK2, and 5PTase7) and two bioactive enzymes (cytochrome P450 and SBH1). In summary, 5-azaC and GA may have individual roles in inducing tree peony flowering, and 5-azaC could be a preferable regulation approach; DNA demethylation is suggested to be more focused on flowering regulation with PsFT playing a core role through promoter demethylation. In addition, 5-azaC may partially undertake or replace the light-signal function, combined with other factors, such as SnRK1, in regulating flowering. This work provides new ideas for improving tree peony forcing culture technology.

UVB-induced SFRP1 methylation potentiates skin damage by promoting cell apoptosis and DNA damage.

In response to the accumulation of genetic mutations and cellular changes, ultraviolet radiation B (UVB) skin lesions undergo dysplasia and transform into a cutaneous squamous cell carcinoma (CSCC). Consistent with our previous findings that secreted frizzled-related protein 1 (SFRP1), a member of the SFRP gene family, was downregulated in human CSCC tissue samples, we found a significant downregulation of SFRP1 in HaCaT, A431, and SCL-1 cells after UVB irradiation. DNA methyltransferase 1 (DNMT1) was significantly increased in CSCC tissues as well as UVB-exposed A431 and SCL-1 cells. Bisulfite genomic sequencing analysis showed that the downregulation of SFRP1 was mainly due to methylation of the SFRP1 promoter, as indicated by increased methylation rate of SFRP1 after UVB irradiation in HaCaT cells. Moreover, demethylation treatment with 5-aza'-deoxycytidine (5-AzaC) increased SFRP1 expression and reduced the methylation rate of SFRP1 in HaCaT cells. Flow cytometry analyses demonstrated that 5-AzaC treatment or overexpression of SFRP1 ameliorated UVB-induced apoptosis, while knockdown of SFRP1 promoted UVB-induced apoptosis in HaCaT cells. In addition, a comet assay confirmed that 5-AzaC treatment reduced DNA damage following UVB irradiation, while knockdown of SFRP1 enhanced DNA damage following UVB irradiation. In conclusion, our study identified DNA methylation of SFRP1 as a key mediator in the UVB-induced apoptosis of keratinocytes. These findings indicate that reinforcing SFRP1 defences by 5-AzaC may help prevent UVB-induced skin damage.

Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice.

Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human TTR transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.

Global Genome Demethylation Causes Transcription-Associated DNA Double Strand Breaks in HPV-Associated Head and Neck Cancer Cells

Simple SummaryHigh levels of global genome methylation in HPV-associated head and neck cancer prompted us to explore demethylation as a potential treatment by determining mechanisms of its toxicity in HPV-positive head and neck cancer cells. Previously, we reported that demethylating drug 5-azaC stabilizes p53 and reduces the expression of HPV genes and matrix metalloproteinases in HPV+ head and neck cancer cells and tumors from patients enrolled in a 5-azaC window clinical trial. Here, we extended our understanding of toxicity caused by global demethylation in HPV-associated head and neck cancer cells by finding that 5-azaC treatment results in formation of DNA double strand breaks that depend on transcription and replication.High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.

Long non-coding RNA levels can be modulated by 5-azacytidine in Schistosoma mansoni

Schistosoma mansoni is a flatworm that causes schistosomiasis, a neglected tropical disease that affects more than 200 million people worldwide. There is only one drug indicated for treatment, praziquantel, which may lead to parasite resistance emergence. The ribonucleoside analogue 5-azacytidine (5-AzaC) is an epigenetic drug that inhibits S. mansoni oviposition and ovarian development through interference with parasite transcription, translation and stem cell activities. Therefore, studying the downstream pathways affected by 5-AzaC in S. mansoni may contribute to the discovery of new drug targets. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein coding potential that have been involved in reproduction, stem cell maintenance and drug resistance. We have recently published a catalog of lncRNAs expressed in S. mansoni life-cycle stages, tissues and single cells. However, it remains largely unknown if lncRNAs are responsive to epigenetic drugs in parasites. Here, we show by RNA-Seq re-analyses that hundreds of lncRNAs are differentially expressed after in vitro 5-AzaC treatment of S. mansoni females, including intergenic, antisense and sense lncRNAs. Many of these lncRNAs belong to co-expression network modules related to male metabolism and are also differentially expressed in unpaired compared with paired females and ovaries. Half of these lncRNAs possess histone marks at their genomic loci, indicating regulation by histone modification. Among a selected set of 8 lncRNAs, half of them were validated by RT-qPCR as differentially expressed in females, and some of them also in males. Interestingly, these lncRNAs are also expressed in other life-cycle stages. This study demonstrates that many lncRNAs potentially involved with S. mansoni reproductive biology are modulated by 5-AzaC and sheds light on the relevance of exploring lncRNAs in response to drug treatments in parasites.

5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells.

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41 ± 1.51% and 19.43 ± 0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.