Methylated Immunoprecipitation Research Articles

NSUN2 relies on ALYREF to regulate Nrf2-mediated oxidative stress and alleviate Dox-induced liver injury

BackgroundDoxorubicin (Dox) is associated with various liver injuries, limiting its clinical utility. This study investigates whether NSUN2 participates in Dox-induced liver injury and the associated molecular mechanism.MethodsIn vivo and in vitro liver cell injury models were constructed based on Dox therapy. The protein levels of NSUN2 and oxidative stress indicators Nrf2, HO-1, and NQO1 were evaluated by Western blot. The RNA binding potential was detected by RNA methylation immunoprecipitation (RIP). Additionally, the effect of NSUN2 on Nrf2 mRNA synthesis and localization was evaluated using an RNA fluorescence probe.ResultsNSUN2 was downregulated, and liver tissue suffered significant pathological damage in the Dox group. The levels of ALT and AST significantly increased. NSUN2 interference exacerbated Dox-induced liver cell damage, which was reversed by NSUN2 overexpression. RIP demonstrated that NSUN2 recognized and bound to Nrf2 mRNA. Western blot analysis showed the protein level of Nrf2 in the NSUN2-WT group was significantly higher than that of the control group, whereas there was no significant change in Nrf2 level in the mutant NSUN2 group. Luciferase analysis demonstrated that NSUN2 could recognize and activate the Nrf2 5′UTR region of LO2 cells. In addition, RIP analysis revealed that ALYREF could recognize and bind to Nrf2 mRNA and that ALYREF controls the regulatory effect of NSUN2 on Nrf2.ConclusionNSUN2 regulates Dox-induced liver cell damage by increasing Nrf2 mRNA m5C methylation to inhibit inhibiting antioxidant stress. The regulatory effect of NSUN2 on Nrf2 depends on ALYREF.

CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis

BackgroundRenal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown.MethodsHigh-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis.ResultsIn this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC.ConclusionsCollectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.

CircERCC6 Positively Regulates the Induced Activation of SHF Stem Cells in Cashmere Goats via the miR-412-3p/BNC2 Axis in an m6A-Dependent Manner.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m6A modified sites (named m6A-685, m6A-862, and m6A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m6A-modified circERCC6 (m6A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m6A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m6A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

Active Fraction of Polyrhachis Vicina Roger (AFPR) Ameliorate Depression Induced Inflammation Response by FTO/miR-221-3p/SOCS1 Axis.

Neuroinflammation is a significant etiological factor in the development of depression. Traditional Chinese medicine (TCM) has demonstrated notable efficacy in the treatment of inflammation. Our previous study surfaces that the active fraction of Polyrhachis vicina Roger (AFPR) has antidepressant and anti-neuroinflammatory effects, but the specific mechanisms remain to be elucidated. The objective of this study was to examine the impact of AFPR on inflammation in depression via the FTO/miR-221-3p/SOCS1 axis. Chronic unpredictable stress (CUMS)-induced rats and LPS-induced BV2 cells were employed to simulate depression models in vivo and in vitro. The levels of inflammatory factors were detected using the ELISA assay. The expression of genes and proteins was detected using qRT-PCR and Western blot. Gene interactions were detected using the dual luciferase reporter gene. Protein-RNA interactions were investigated using RNA methylation immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP). Neuroinflammation in the brain was examined through H&E staining, while neuronal apoptosis was assessed using TUNEL staining. The results showed that AFPR ameliorated depression induced inflammation by increasing SOCS1 expression. However, SOCS1 was identified as a target of miR-221-3p. Overexpression of miR-221-3p decreased the expression of SOCS1 and increased the levels of NF-κB, IL-7, and IL-6. In addition, we found that miR-221-3p was regulated by FTO-mediated m6A modification through MeRIP and RIP experiments. Interference with miR-221-3p and overexpression of FTO resulted in increased SOCS1 gene expression and decreased levels of NF-κB, IL-7, and IL-6, which were reversed by AFPR. AFPR inhibits the maturation of pri-miR-221-3p through FTO-mediated m6A modification, reduces the production of miR-221-3p, increases the expression of SOCS1, and reduces the level of inflammation, thereby improving depressive symptoms.

Comprehensive analysis of aflatoxin B1 biosynthesis in Aspergillus flavus via transcriptome-wide m6A methylome response to cycloleucine

Aspergillus flavus and its toxic aflatoxins secondary metabolites contaminate food and grains, posing a severe threat to human health and leading to liver cancer. Here, we demonstrated that cycloleucine blocked aflatoxin B1 synthesis by inhibiting N6-methyladenosine (m6A) methylation modification of messenger RNA (mRNA). m6A Methylation Immunoprecipitation Sequencing (m6A MeRIP-Seq)-based comprehensive transcriptome-wide m6A profiling identified 102 differentially expressed genes that underwent m6A modification, of which 22 hypermethylated genes were downregulated and 49 hypomethylated genes were upregulated, suggesting a negative correlation between m6A methylation and gene expression. Notably, cycloleucine inhibited aflatoxin B1 production via multiple targets. The m6A sites of several key genes involved in the aflatoxin B1 biosynthesis pathway were significantly enriched in the coding sequence and around the stop codon, resulting in their downregulation. Furthermore, m6A methylation on genes related to the aflatoxin B1 biosynthesis pathway led to reduced mRNA stability. Cycloleucine inhibition of aflatoxin B1 production highlights its potential as an agent for removing mycotoxins in environmental pollution. Environmental implicationAflatoxins, highly carcinogenic secondary metabolites produced by Aspergillus flavus, frequently contaminate crops such as peanut, corn, wheat and sesame leading to irreversible loss in the quality and yield of agricultural products and posing serious threats to food safety. Aflatoxins has also been linked to developmental delays and liver cancer in humans. In our study, ‘monitoring aflatoxin concentrations and its bioaccumulation in organisms’ has been conducted. The results demonstrated that aflatoxin production in A. flavus was completely blocked after cycloleucine treatment. Additionally, we demonstrated that inhibition of aflatoxin was linked to N6-methyladenosine methylation of multiple genes in aflatoxin biosynthesis pathway.

The m6A modification-mediated OGDHL exerts a tumor suppressor role in ccRCC by downregulating FASN to inhibit lipid synthesis and ERK signaling

Metabolic reprogramming is a hallmark of cancer, and the impact of lipid metabolism as a crucial aspect of metabolic reprogramming on clear cell renal cell carcinoma (ccRCC) progression has been established. However, the regulatory mechanisms underlying the relationship between metabolic abnormalities and ccRCC progression remain unclear. Therefore, this study aimed to identify key regulatory factors of metabolic reprogramming in ccRCC and provide potential therapeutic targets for ccRCC patients. Potential metabolic regulatory factors in ccRCC were screened using bioinformatics analysis. Public databases and patient samples were used to investigate the aberrant expression of Oxoglutarate dehydrogenase-like (OGDHL) in ccRCC. The function of OGDHL in ccRCC growth and metastasis was evaluated through in vitro and in vivo functional experiments. Mechanistic insights were obtained through luciferase reporter assays, chromatin immunoprecipitation, RNA methylation immunoprecipitation, and mutagenesis studies. OGDHL mRNA and protein levels were significantly downregulated in ccRCC tissues. Upregulation of OGDHL expression effectively inhibited ccRCC growth and metastasis both in vitro and in vivo. Furthermore, FTO-mediated OGDHL m6A demethylation suppressed its expression in ccRCC. Mechanistically, low levels of OGDHL promoted TFAP2A expression by inhibiting ubiquitination levels, which then bound to the FASN promoter region and transcriptionally activated FASN expression, thereby promoting lipid accumulation and ERK pathway activation. Our findings demonstrate the impact of OGDHL on ccRCC progression and highlight the role of the FTO/OGDHL/TFAP2A/FASN axis in regulating ccRCC lipid metabolism and progression, providing new targets for ccRCC therapy.

METTL3 potentiates osteogenic differentiation of bone marrow mesenchymal stem cells via IGF2BP1/m6A/RUNX2.

Maxillofacial bone defect is a critical obstacle for maxillofacial tumors and periodontal diseases. The osteogenic differentiation of bone marrow mesenchymal stem cells BMSCs is critical for maxillofacial osteogenesis and functional reconstruction. Here, our study focused on the functions and mechanism of N6-methyladenosine during BMSCs osteogenic differentiation BMSCs. Biofunctions of BMSCs were detected using ALP activity and alizarin red S staining assays. The molecular interaction within RNA/protein was identified by RNA immunoprecipitation and/or methylation immunoprecipitation. Results indicated that m6A 'writer' METTL3 upregulated during the osteogenic differentiation of BMSCs upon osteogenic induction. Functionally, assays' results revealed that METTL3 overexpression promoted the osteogenic differentiation of BMSC, while METTL3 knockdown repressed the osteogenic differentiation. Mechanistically, results revealed that RUNX2 mRNA was a m6A-methylated target by METTL3 at its 3'-UTR. Moreover, m6A reader IGF2BP1 recognized the m6A site on RUNX2 mRNA to enhance its stability. In conclusion, our findings revealed the novel roles of METTL3 in BMSCs osteogenic differentiation via the IGF2BP1/m6A/RUNX2 signaling axis of m6A-dependent manner, providing a potential therapeutic target for maxillofacial bone defects treatment.

NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

Doxorubicin (DOX) is a commonly used antitumor drug, but its application has been limited because of its strong cardiac damage. This study aims to explore the role of NSUN2 in DOX-induced heart injury. C57BL/6J mice were intraperitoneally injected with 20 mg/Kg DOX to induce heart injury. After 3 days, the cardiac function, cardiac histopathology, myocardial apoptosis, and the expression level of NSUN2 were detected. In vitro, H9C2 cells were transfected with NSUN2 siRNA or overexpressed lentivirus and then treated with 500 ng/ml DOX. After 24 h, the changes in reactive oxygen species (ROS), apoptosis, and NSUN2 expression were detected. After DOX treatment, both in vitro and in vivo experiments showed that the cardiac function decreased, the number of apoptotic cells increased, and the expression level of NSUN2 increased. Interfering the expression of NSUN2 by siRNA promoted DOX-induced heart injury, while overexpression of NSUN2 could inhibit DOX-induced heart injury. Further study showed that NSUN2 promoted antioxidative stress by upregulating the Nrf2 protein level. In addition, NSUN2 overexpression could increase the half-life of Nrf2 mRNA. m5C RNA methylation immunoprecipitation (MeRIP) also showed that the level of Nrf2 m5C mRNA was significantly increased in NSUN2 overexpressed group when compared to the GFP group. NSUN2 enhances the expression of Nrf2 by promoting Nrf2 mRNA m5C modification and enhances its antioxidative stress effect to alleviate DOX-induced myocardial injury.

Longitudinal monitoring of cell-free DNA methylation in ALK-positive non-small cell lung cancer patients

BackgroundDNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastatic ALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identify ALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring.MethodsLongitudinal plasma samples (n = 79) of 21 ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15 ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies.ResultsWe devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles of ALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g., PCDH10, TBX2, CDO1, and HOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman, ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rank p = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression.ConclusionWe demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring.

N6-Methyladenosine modification (m6A) of circRNA-ZNF638 contributes to the induced activation of SHF stem cells through miR-361-5p/Wnt5a axis in cashmere goats.

The objective of this study was to investigate the effects of N6-Methyladenosine modification-circRNA-zinc finger protein 638 (m6A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats. The m6A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m6A dependence were evaluated through the overexpression of circRNA-ZNF638/its m6Adeficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3'- untranslated region was analyzed through Dual-luciferase reporter assay. The m6A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m6A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHFstem cells. We further demonstrated that the internal m6A modification within circRNAZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m6A-deficient mutant of circRNAZNF638. The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m6A-dependent manner through miR-361-5p/Wnt5a axis.

Methylated circulating tumor DNA (cfMeDIP) as a predictive biomarker of clinical outcome in pan-cancer patients (pts) treated with pembrolizumab (P).

2550 Background: Bespoke mutation-based circulating tumor DNA (ctDNA) predicts response to P but relies on availability of tumor tissue and presence of mutations. Cell-free methylated immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) may overcome these limitations and be applied to more pts. Methods: Pts with mixed solid tumors divided into 5 cohorts received P 200 mg Q3wks in the investigator-initiated INSPIRE trial (NCT02644369). cfMeDIP-seq was performed at baseline (B), pre cycle 3 (C3) and later cycles. Methylation probability was inferred from read depths in 300 bp bins. cfMeDIP score was the probability-weighted sum of 3270 pan-cancer differentially-methylated regions in the TCGA PanCanAtlas. ctDNA concentration was assayed using tissue-informed bespoke targeted NGS (Signatera). ΔctDNA and ΔcfMeDIP denote the change in ctDNA or cfMeDIP between B and C3, respectively. Association with OS or PFS was assessed using Cox proportional hazards model, adjusting for cohort (aHR). Multivariable analysis (MVA) also included tumor mutation burden and PD-L1 status. Results: 194 plasma samples from 87 pts were analysed with cfMeDIP-seq (84 at B, 55 at C3, 55 at later cycles). Demographics: male 33%; median age = 61 yrs (34–82); Cohorts: triple negative breast (26%), ovarian (25%), head & neck (21%), melanoma (12%), others (15%). Median follow-up = 10.6m (0.6–64.4); Median PFS = 1.9m; Median OS = 10.6m. cfMeDIP at B below median was associated with better OS (aHR = 0.51, 95%CI 0.29-0.91; p = 0.02) in MVA. ΔcfMeDIP was evaluable in 53 pts; any decrease in ΔcfMeDIP was predictive for OS (aHR = 0.36, 95%CI 0.18-0.72; p < 0.01) and PFS (aHR = 0.42, 95%CI 0.22-0.82; p = 0.01). Both ΔctDNA and ΔcfMeDIP were evaluable in 51 pts; decrease in ΔctDNA and ΔcfMeDIP predicted for longer OS (aHR = 0.45, 95%CI 0.23-0.86; p = 0.02 vs aHR = 0.39, 95%CI 0.19-0.80; p = 0.01); and PFS (aHR = 0.44, 95%CI 0.23-0.83; p = 0.01 vs aHR = 0.5, 95%CI 0.25-0.99; p = 0.04), respectively. When both ΔctDNA and ΔcfMeDIP are integrated in MVA, ΔcfMeDIP was predictive for OS (aHR = 0.48, 95%CI 0.23-1; p = 0.05). A decrease in ΔcfMeDIP and/or ΔctDNA was associated with longer OS (aHR = 0.2, 95%CI 0.09-0.45) and PFS (aHR = 0.27, 95%CI 0.13-0.58) compared to an increase in both assays (p < 0.01) (Table). Conclusions: We applied for the first time cfMeDIP-seq and mutation-based ctDNA analysis concurrently in pan-cancer pts treated with checkpoint blockade. ΔcfMeDIP correlated strongly with OS and PFS, representing a promising plasma-based predictive epigenetic biomarker in pts treated with P. ΔctDNA and ΔcfMeDIP can complement each other to predict outcomes, demonstrating that they may capture different biological changes. Clinical trial information: NCT02644369. [Table: see text]

Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer

BackgroundLRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited.MethodsIn silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2’-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system.ResultsAcross breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2’-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer.ConclusionsOur study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Detecting Neuroendocrine Prostate Cancer Through Tissue-Informed Cell-Free DNA Methylation Analysis.

Neuroendocrine prostate cancer (NEPC) is a resistance phenotype that emerges in men with metastatic castration-resistant prostate adenocarcinoma (CR-PRAD) and has important clinical implications, but is challenging to detect in practice. Herein, we report a novel tissue-informed epigenetic approach to noninvasively detect NEPC. We first performed methylated immunoprecipitation and high-throughput sequencing (MeDIP-seq) on a training set of tumors, identified differentially methylated regions between NEPC and CR-PRAD, and built a model to predict the presence of NEPC (termed NEPC Risk Score). We then performed MeDIP-seq on cell-free DNA (cfDNA) from two independent cohorts of men with NEPC or CR-PRAD and assessed the accuracy of the model to predict the presence NEPC. The test cohort comprised cfDNA samples from 48 men, 9 with NEPC and 39 with CR-PRAD. NEPC Risk Scores were significantly higher in men with NEPC than CR-PRAD (P = 4.3 × 10-7) and discriminated between NEPC and CR-PRAD with high accuracy (AUROC 0.96). The optimal NEPC Risk Score cutoff demonstrated 100% sensitivity and 90% specificity for detecting NEPC. The independent, multi-institutional validation cohort included cfDNA from 53 men, including 12 with NEPC and 41 with CR-PRAD. NEPC Risk Scores were significantly higher in men with NEPC than CR-PRAD (P = 7.5×10-12) and perfectly discriminated NEPC from CR-PRAD (AUROC 1.0). Applying the predefined NEPC Risk Score cutoff to the validation cohort resulted in 100% sensitivity and 95% specificity for detecting NEPC. Tissue-informed cfDNA methylation analysis is a promising approach for noninvasive detection of NEPC in men with advanced prostate cancer.

Altered hypothalamic DNA methylation and stress-induced hyperactivity following early life stress

Exposure to early life stress (ELS) during childhood or prenatally increases the risk of future psychiatric disorders. The effect of stress exposure during the neonatal period is less well understood. In preterm infants, exposure to invasive procedures is associated with altered brain development and future stress responses suggesting that the neonatal period could be a key time for the programming of mental health. Previous studies suggest that ELS affects the hypothalamic epigenome, making it a good candidate to mediate these effects. In this study, we used a mouse model of early life stress (modified maternal separation; MMS). We hypothesised MMS would affect the hypothalamic transcriptome and DNA methylome, and impact on adult behaviour. MMS involved repeated stimulation of pups for 1.5 h/day, whilst separated from their mother, from postnatal day (P) 4–6. 3’mRNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing were performed on hypothalamic tissue at P6. Behaviour was assessed with the elevated plus, open field mazes and in-cage monitoring at 3–4 months of age. MMS was only associated with subtle changes in gene expression, but there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. ELS has marked effects on DNA methylation in the hypothalamus in early life and results in stress-specific hyperactivity in young adulthood. These results have implications for the understanding of ELS-mediated effects on brain development.

Genome-wide DNA Methylation Analysis of Mantle Edge and Mantle Central from Pearl Oyster Pinctada fucata martensii.

DNA methylation is a type of epigenetic modification that alters gene expression without changing the DNA sequence and mediates some cases of phenotypic plasticity. In this study, we identified sixDNA methyltransferase (DNMT) genes and twomethyl-CpG binding domain protein2 (MBD2) gene from Pinctada fucata martensii. We also analyzed the genome-wide DNA methylation levels of mantle edge (ME) and mantle central (MC) from P. f. martensii via methylated immunoprecipitation sequencing (MeDIP-Seq). Results revealed that both ME and MC had 122 million reads, and had 58,702 and 55,721 peaks, respectively. The obtained methylation patterns of gene elements and repeats showed that the methylation of the protein-coding genes, particularly intron and coding exons (CDSs), was more frequent than that of other genomic elements in the pearl oyster genome. We combined the methylation data with the RNA-seq data of the ME and MC of P. f. martensii and found that promoter, CDS, and intron methylation levels were positively correlated with gene expression levels except the highest gene expression level. We also identified 313 differential methylation genes (DMGs) and annotated 212 of them. These DMGs were significantly enriched in 30 pathways, such as amino acid and protein metabolism, energy metabolism, terpenoid synthesis, and immune-related pathways. This study comprehensively analyzed the methylomes of biomineralization-related tissues and helped enhance our understanding of the regulatory mechanism underlying shell formation.

Detection of DNA Methylation by MeDIP and MBDCap Assays: An Overview of Techniques.

DNA methylation has been characterized as the representative example of epigenetic modifications and implicated in numerous biological processes, such as genomic imprinting and X chromosome inactivation. It primarily occurs at CpG dinucleotides in mammals and plays a critical role in transcriptional regulations. Examination of DNA methylation patterns in gene(s) or across a genome is vital to understand the role of epigenetic modulation in the progress of development and tumorigenesis. Currently, lots of approaches have been developed to investigate DNA methylation patterns for either limited regions or genome-scale studies, but some of them rely on using restriction enzymes. In this chapter, we describe two commonly used protocols to detect enrichment of methylated DNA regions, namely methylated immunoprecipitation (MeDIP) and capture of methylated DNA by methyl-CpG binding domain-based (MBD) proteins (MBDCap). They are the most economical and effective methods to study DNA methylation in either single locus or genome-wide scale.

Effects of Zhizi Chuanxiong Capsule () on the Abnormal Methylation in Rabbits with Atherosclerosis.

To investigate the effects of Zhizi Chuanxiong Capsule (ZCC, ) on abnormal DNA methylation in a rabbit model of atherosclerosis (AS). After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group (n=12) fed with normal diet for 22 weeks; a model group (n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group (n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group (n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing (Medip-seq). The aorta tissues were collected for hematoxylin-eosin (HE) staining. Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low- and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes (including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology (GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low- and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, high-dose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. CONCLUSION: ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.

Hypermethylation of CpG island in promoter region of MEIS1 gene and its expression in HOX11(+) acute T lymphoblastic leukemia

To detect the status of methylation in promoter region of MEIS1 gene and its expression in cell lines and patients with HOX11(+) T-cell acute lymphoblastic leukemia (T-ALL) and explore the relationship between the expression level of MEIS1 gene and methylation of CpG island in promoter region. The methylation pattern in MEIS1 gene was detected with bisulfite sequencing PCR, DNA methylation immunoprecipitation and promoter oligonucleotide microarray. And the expression level of MEIS1 mRNA was detected with reverse transcription PCR in 3 T-ALL cell lines: sil-ALL, DND41 and RPMI as well as in 75 clinical bone marrow samples including 38 de novo T-ALL patients, 29 complete remission T-ALL patients and 8 normal samples from January 2009 to December 2011. The expressions of HOX11 mRNA in the patients were classified into high expression (HOX11(+)) groups (n = 14) and low expression (HOX11(-)) groups (n = 24) . There was hypermethylation of CpG island in promoter region of MEIS1 gene in patients with HOX11(+) T-ALL. The methylation rate of promoter CpG islands of MEIS1 gene in HOX11(+) T-ALL (76.8%) was significantly higher than that of HOX11(-) T-ALL (29.5%) (P < 0.01). The expression MEIS1 in complete remission T-ALL patients and normal samples was significantly lower than that in de novo T-ALL patients (both P < 0.05) . The expression MEIS1 in HOX11(+) cell lines was significantly lower than that in HOX11(-) cell lines (0.392 ± 0.226,0.378 ± 0.317 vs 1.059 ± 0.533, both P < 0.05) and the expression level of MEIS1 in HOX11(+) T-ALL patients (0.462 ± 0.281) significantly lower than that in HOX11(-) T-ALL patients (0.916 ± 0.437) (P < 0.01). The relationship between MEIS1 and HOX11 mRNA expression level had a negative correlation (rs = -0.416, P < 0.01) . The expression level of MEIS1 gene was negatively correlated with the methylation of CpG island in promoter region in T-ALL (rs = -0.383, P < 0.01) . The expression of MEIS1 becomes down-regulated or even lost by promoter methylation in T-ALL. HOX11 maybe a negative regulator of MEIS1 gene.

SAT0056 Dna methylation analysis identified TBX5 as an important transcription factor in rheumatoid arthritis

ObjectivesChanges in DNA methylation and histone marks have been associated with diseases such as cancer, rheumatoid arthritis (RA) and systemic lupus erythematosus. Previously, we performed methylation immunoprecipitation (MeDIP) in combination...

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

BackgroundThe processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription.ResultsTo allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease.ConclusionThe high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease.