Methylation Targets Research Articles

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

BDNF gene hydroxymethylation in hippocampus related to neuroinflammation-induced depression-like behaviors in mice

BackgroundNeuroinflammation is a multifactorial condition related to glial cells and neurons activation, and it is implicated in CNS disorders including depression. BDNF is a crucial molecule that related to the pathology of depression, and it is the target of DNA methylation. DNA hydroxymethylation, an active demethylation process can convert 5-mC to 5-hmC by Tets catalyzation to regulate gene transcription. The regulatory function for BDNF gene in response to neuroinflammation remains poorly understood. MethodsNeuroinflammation and depressive-like behaviors were induced by lipopolysaccharide (LPS) administration in mice. The microglial activation and cellular 5-hmC localization in the hippocampus were confirmed by immunostaining. The transcripts of Tets and BDNF were examined by qPCR method. The global 5-hmC levels and enrichment of 5-hmC in BDNF gene in the hippocampus were analyzed using dot bolt and hMeDIP-sequencing analysis. ResultsLPS administration induced a spectrum of depression-like behaviors (including behavioral despair and anhedonia) and increased expression of Iba-1, a marker for microglia activation, in hippocampus, demonstrating that LPS treatment cloud provide stable model of neuroinflammation with depressive-like behaviors as expected. Our results showed that Tet1, Tet2 and Tet3 mRNA expressions and consequent global 5-hmC levels were significantly decreased in the hippocampus of LPS group compared to saline group. We also demonstrated that 5-hmC fluorescence in the hippocampus located in excitatory neurons identified by CaMK II immunostaining. Furthermore, we demonstrated that the enrichment of 5-hmC in BDNF gene was decreased and corresponding BDNF mRNA was down-regulated in the hippocampus in LPS group compared to saline group. ConclusionNeuroinflammation-triggered aberrant BDNF gene hydroxymethylation in the hippocampus is an important epigenetic element that relates with depression-like behaviors.

Targets of histone H3 lysine 9 methyltransferases.

Histone H3 lysine 9 di- and trimethylation are well-established marks of constitutively silenced heterochromatin domains found at repetitive DNA elements including pericentromeres, telomeres, and transposons. Loss of heterochromatin at these sites causes genomic instability in the form of aberrant DNA repair, chromosome segregation defects, replication stress, and transposition. H3K9 di- and trimethylation also regulate cell type-specific gene expression during development and form a barrier to cellular reprogramming. However, the role of H3K9 methyltransferases extends beyond histone methylation. There is a growing list of non-histone targets of H3K9 methyltransferases including transcription factors, steroid hormone receptors, histone modifying enzymes, and other chromatin regulatory proteins. Additionally, two classes of H3K9 methyltransferases modulate their own function through automethylation. Here we summarize the structure and function of mammalian H3K9 methyltransferases, their roles in genome regulation and constitutive heterochromatin, as well as the current repertoire of non-histone methylation targets including cases of automethylation.

Dna-Methylation Signature of Microrna Coding Genes Associated with Chronological Age in a High-Cardiovascular Risk Mediterranean Population. Sex-Specific Analysis

Background and aimsDNA-methylation has been associated with biological age and several epigenetic clocks have been reported (Hovarth) MicroRNA (miRNA) are short single-stranded RNA molecules implicated in the regulation of gene expression. As well as protein-coding genes, miRNA-coding genes may be targets for DNA methylation. However, the role of this methylation on aging and cardiovascular risk is still poorly studied. Our aim is to analyze the DNA methylation in miRNA-coding genes in a high-cardiovascular risk Mediterranean population, and its association with age considering sex-specific differences. MethodsA DNA-methylation analysis of miRNA genes was analyzed using the EPIC850K array, in high-cardiovascular risk 414 subjects aged 55-75y. We obtained Beta and M-values for CpGs located in the miRNA genes and their association with age in multivariate models adjusted for confounders (sex, leukocytes, batch-effect, diabetes, BMI). Sex-specific analyses were conducted. ResultsIn the whole population, significant associations between miRNA-gene methylation and chronological age were obtained. Outstanding the MIR34B (cg26561785; p=3,9 × 10-8; hypermethylation associated with higher age; r=0.28). Previous studies have linked miR-34a to vascular aging and arteriosclerosis. The following most significant associations (p=4,7 × 10-7-1,5 × 10-6) were obtained with CpG in MIR663(r=0.26), MIR9-3(r=0.26), MIR495(r=-0.26) and MIR203A(r=0.25), as main components of the methylation signature in the whole population. Sex-specific analysis detected sex-specific differences. The top-ranked CpGs in men were MIR34B (cg26561785;p=9.2 × 10-6) and MIR9-3 (cg12530503;p=1.8 × 10-05); whereas in women were MIR376B (cg05348084;p=4.3 × 10-7) and MIR203A (cg24454784;p=3.9 × 10-06). ConclusionsAltered methylation in MIR genes is associated with chronological age, and this association may be different between men and women with high-cardiovascular risk.

De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a

Epigenetic modifications to histone proteins serve an important role in regulating permissive and repressive chromatin states, but despite the identification of many histone PTMs and their perceived role, the epigenetic writers responsible for generating these chromatin signatures are not fully characterized. Here, we report that the canonical histone H3K9 methyltransferases EHMT1/GLP and EHMT2/G9a are capable of catalyzing methylation of histone H3 lysine 23 (H3K23). Our data show that while both enzymes can mono- and di-methylate H3K23, only EHMT1/GLP can tri-methylate H3K23. We also show that pharmacologic inhibition or genetic ablation of EHMT1/GLP and/or EHMT2/G9a leads to decreased H3K23 methylation in mammalian cells. Taken together, this work identifies H3K23 as a new direct methylation target of EHMT1/GLP and EHMT2/G9a, and highlights the differential activity of these enzymes on H3K23 as a substrate.

METTL3-mediated long non-coding RNA MIR99AHG methylation targets miR-4660 to promote bone marrow mesenchymal stem cell osteogenic differentiation

ABSTRACT Whether long non-coding RNA Mir-99a-Let-7c Cluster Host Gene (LncRNA MIR99AHG) is involved in osteoporosis (OP) remains vague, so we hereby center on its implication. Old C57BL/6J mice were injected with the silencing lentivirus of MIR99AHG and subjected to microCT analysis and immunohistochemistry on osteogenic cells. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) with or without transfection was determined by alkaline phosphatase (ALP) and Alizarin Red S staining. Total N(6)-methyladenosine (m6A) on the bone marrow mesenchymal stem cells (BMSCs) was quantified. The potential methylation site and the complementary binding sites with candidate microRNA (miR) were predicted via bioinformatic analyses, with the latter being confirmed via dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Quantitative real-time PCR and Western blot were used for quantification assays. MIR99AHG was decreased during the osteogenic differentiation of BMSCs, where increased Osterix (OSX), Collagen, Type I, Alpha 1 (Col1A1), Osteocalcin (OCN) and RUNX Family Transcription Factor 2 (RUNX2) as well as more color-stained areas were found. Also, silencing MIR99AHG relieved the OP in mice and reduced the loss of osteogenic cells. M6A methylation in undifferentiated BMSCs was low and MIR99AHG overexpression abolished the effects of overexpressed METTL3 on promoting osteogenic differentiation. MiR-4660, which was downregulated in BMSCs without differentiation but increased during osteogenic differentiation, could bind with MIR99AHG. Furthermore, miR-4660 promoted osteogenic differentiation and reversed the effects of overexpressed MIR99AHG. The present study demonstrated that METTL3-mediated LncRNA MIR99AHG methylation enhanced the osteogenic differentiation of BMSCs via targeting miR-4660.

Construction of a sensitive ratiometric electrochemical sensing platform for DNA methylation detection based on the design of multistep DNA amplification circuits

Methylated DNA is a promising epigenetic biomarker for diseases diagnosis, therapy monitoring, and prognosis. Here, we developed a sensitive ratiometric electrochemical biosensor for the detection of methylated DNA based on the design of multistep DNA amplification circuits. The methylated target DNA firstly initiates the DNAzyme-assisted amplification circuit and produces a number of single-stranded triggers that catalyze the subsequent double-step catalytic hairpin assembly circuits to release numerous four-way DNA junctions. The four-way DNA junctions displace the methylene blue (MB)-modified signal probes for intercalating doxorubicin (DOX) molecules on the sensing interface, resulting in a significantly increased value of iDOX/iMB to achieve sensitive monitoring of methylated DNA. Taking advantage of the significant signal amplification of multistep DNA amplification circuits, the proposed method exhibited high sensitivity for target analysis with a detection limit of 4 aM and a dynamic linear range of 10 aM to 20 pM. Moreover, the proof-of-concept application of the sensing platform was investigated, suggesting that the system is promising for potential applications in early cancer diagnosis and the fundamental research of epigenetics.

OR09-2 The RCAN 1.4 Metastasis Progression Suppressor Gene is Hypermethylated at Intron 1 and Downregulated in Papillary Thyroid Cancer

Abstract Progressive metastasis is the proximate cause of cancer-related mortality for the majority of patients with solid tumors. Identifying both drivers, and gatekeepers of late stage progression (metastasis progression suppressors; MPS) is crucial to improve biomarkers and treatments. We previously identified Regulator of Calcineurin 1.4 (RCAN1.4) as a MPS gene in thyroid cancer that functions in part through overexpression of the cap-n-collar transcription factor NFE2L3 (NRF3). However, the mechanisms for RCAN1.4 loss in thyroid cancer, and whether or not this is reversible, had not be addressed. Because the RCAN 1.4 promoter contains CG rich regions reported to be hypermethyalated in other tissues, we hypothesized that RCAN 1.4 downregulation in thyroid cancer was similarly regulated. Studies were performed in both cell lines and de-identified human papillary thyroid cancer (PTC) tumor and normal tissue pairs from our Endocrine Neoplasia Repository tumor bank. Five thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers were selected based on low expression of RCAN 1.4. To determine if lines display methylation-regulated expression of RCAN 1.4, cells were treated with 1-10uM decitabine, a DNA methyl transferase inhibitor. In all cell lines, RCAN1.4 mRNA and protein levels increased with decitabine treatment, consistent with hypermethylation. Analysis of the literature and in silico analysis of the RCAN 1.4 gene identified CG rich regions as putative methylation targets in the proximal promoter and in intron 1. Using two different methods; 1) isolation of MseI-digestion DNA fragments using CpG MethylQuest beads followed by qPCR and 2) methylation-specific PCR (MSP) of bisulfite-modified DNA, hypermethylation of the intron 1 CG rich region was identified. There was no evidence of hypermethylation of the CG rich region of the proximal promoter. Methylation of the two regions also was assessed on 18 pairs PTC and adjacent normal tissues. Similar to the cell lines, hypermethylation was identified at intron 1 of RCAN 1.4 in PTC versus normal tissues. QRT-PCR results confirmed lower levels of RCAN1-4 mRNA in the PTC vs normal tissue. These samples also demonstrated increased NRF3 expression versus normal tissue. Taken together, these data demonstrate that RCAN 1.4 is downregulated in thyroid cancer cells and human PTCs by hypermethylation of CG rich regions in intron 1 and that this effect is reversible using demethylating agents. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

Aberrant DNA and RNA Methylation Occur in Spinal Cord and Skeletal Muscle of Human SOD1 Mouse Models of ALS and in Human ALS: Targeting DNA Methylation Is Therapeutic.

Amyotrophic lateral sclerosis (ALS) is a fatal disease. Skeletal muscles and motor neurons (MNs) degenerate. ALS is a complex disease involving many genes in multiple tissues, the environment, cellular metabolism, and lifestyles. We hypothesized that epigenetic anomalies in DNA and RNA occur in ALS and examined this idea in: (1) mouse models of ALS, (2) human ALS, and (3) mouse ALS with therapeutic targeting of DNA methylation. Human superoxide dismutase-1 (hSOD1) transgenic (tg) mice were used. They expressed nonconditionally wildtype (WT) and the G93A and G37R mutant variants or skeletal muscle-restricted WT and G93A and G37R mutated forms. Age-matched non-tg mice were controls. hSOD1 mutant mice had increased DNA methyltransferase enzyme activity in spinal cord and skeletal muscle and increased 5-methylcytosine (5mC) levels. Genome-wide promoter CpG DNA methylation profiling in skeletal muscle of ALS mice identified hypermethylation notably in cytoskeletal genes. 5mC accumulated in spinal cord MNs and skeletal muscle satellite cells in mice. Significant increases in DNA methyltransferase-1 (DNMT1) and DNA methyltransferase-3A (DNMT3A) levels occurred in spinal cord nuclear and chromatin bound extracts of the different hSOD1 mouse lines. Mutant hSOD1 interacted with DNMT3A in skeletal muscle. 6-methyladenosine (6mA) RNA methylation was markedly increased or decreased in mouse spinal cord depending on hSOD1-G93A model, while fat mass and obesity associated protein was depleted and methyltransferase-like protein 3 was increased in spinal cord and skeletal muscle. Human ALS spinal cord had increased numbers of MNs and interneurons with nuclear 5mC, motor cortex had increased 5mC-positive neurons, while 6mA was severely depleted. Treatment of hSOD1-G93A mice with DNMT inhibitor improved motor function and extended lifespan by 25%. We conclude that DNA and RNA epigenetic anomalies are prominent in mouse and human ALS and are potentially targetable for disease-modifying therapeutics.

Electroacupuncture induces weight loss by regulating tuberous sclerosis complex 1-mammalian target of rapamycin methylation and hypothalamic autophagy in high-fat diet-induced obese rats.

Background: Obesity can be caused by abnormalities of hypothalamic autophagy, which is closely regulated by the epigenetic modification of TSC1-mTOR. However, whether the weight-reducing effect of EA may relate to the modification of TSC1-mTOR methylation and hypothalamic autophagy remain unclear. This study was conducted to reveal the possible mechanism by which EA reduces BW by measuring the levels of TSC1-mTOR methylation and hypothalamic autophagy-related components. Methods: The weight-reducing effect of EA was investigated in high-fat diet (HFD)-induced obese (DIO) rats by monitoring the BW, food consumption, and epididymal white adipose tissue (eWAT)/BW ratio. Hematoxylin and eosin staining was performed for morphological evaluation of eWAT. Immunofluorescence was utilized to observe the localization of LC3 in the hypothalamus. The expressions of autophagy components (Beclin-1, LC3, and p62) and mTOR signaling (mTOR, p-mTOR, p70S6K, and p-p70S6K) were assessed by western blot. The methylation rate of the TSC1 promoter was detected by bisulfite genomic sequencing. Results: Treatment with EA significantly reduced the BW, food consumption, and eWAT/BW ratio; attenuated the morphological alternations in the adipocytes of DIO rats. While HFD downregulated the expression levels of Beclin-1 and LC3 and upregulated those of p62, these changes were normalized by EA treatment. EA markedly decreased the methylation rate of the TSC1 gene promoter and suppressed the protein expressions of mTOR, p-mTOR, p70S6K, and p-p70S6K in the hypothalamus. Conclusion: EA could reduce BW and fat accumulation in DIO rats. This ameliorative effect of EA may be associated with its demethylation effect on TSC1-mTOR and regulation of autophagy in the hypothalamus.

Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation.

DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5'-phosphate terminuses. After treating with Lambda, the sequence with 5'-phosphate modification will be degraded, leaving the single-stranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.

The regulation and potential roles of m6A modifications in early embryonic development and immune tolerance at the maternal-fetal interface.

The immune microenvironment at the maternal-fetal interface was determined by the crosstalk between the trophoblast and maternal-derived cells, which dynamically changed during the whole gestation. Trophoblasts act as innate immune cells and dialogue with maternal-derived cells to ensure early embryonic development, depending on the local immune microenvironment. Therefore, dysfunctions in trophoblasts and maternal decidual cells contribute to pregnancy complications, especially recurrent pregnancy loss in early pregnancy. Since many unknown regulatory factors still affect the complex immune status, exploring new potential aspects that could influence early pregnancy is essential. RNA methylation plays an important role in contributing to the transcriptional regulation of various cells. Sufficient studies have shown the crucial roles of N6-methyladenosine (m6A)- and m6A-associated- regulators in embryogenesis during implantation. They are also essential in regulating innate and adaptive immune cells and the immune response and shaping the local and systemic immune microenvironment. However, the function of m6A modifications at the maternal-fetal interface still lacks wide research. This review highlights the critical functions of m6A in early embryonic development, summarizes the reported research on m6A in regulating immune cells and tumor immune microenvironment, and identifies the potential value of m6A modifications in shaping trophoblasts, decidual immune cells, and the microenvironment at the maternal-fetal interface. The m6A modifications are more likely to contribute to embryogenesis, placentation and shape the immune microenvironment at the maternal-fetal interface. Uncovering these crucial regulatory mechanisms could provide novel therapeutic targets for RNA methylation in early pregnancy.

Elucidating the role of PRMTs in prostate cancer using open access databases and a patient cohort dataset.

Protein arginine methylation is an understudied epigenetic mechanism catalyzed by enzymes known as Protein Methyltransferases of Arginine (PRMTs), while the opposite reaction is performed by Jumonji domain- containing protein 6 (JMJD6). There is increasing evidence that PRMTs are deregulated in prostate cancer (PCa). In this study, the expression of two PRMT members, PRMT2 and PRMT7 as well as JMJD6, a demethylase, was analyzed in PCa. Initially, we retrieved data from The Cancer Genome Atlas (TCGA) project and the Gene Expression Omnibus (GEO) database to explore the differential expression of various PRMT family members in patients with PCa and then applied immunohistochemistry in a patient cohort across the spectrum of PCa, including non-neoplastic prostate tissue and lymph node metastatic foci. The results from the TCGA analysis revealed that PRMT7, PRMT6 and PRMT3 expression increased while PRMT2, PRMT9 and JMJD6 levels decreased in the tumor compared to non-neoplastic prostate. Results from the GEO datasets were similar, albeit not identical with the TCGA results, with PRMT7 and PRMT3 being upregulated and PRMT2 and JMJD6 being downregulated in the tumor compared to non-neoplastic tissue in some of them. In addition, PRMT7 levels decreased with stage and grade progression in the TCGA analysis. In the patient cohort, both PRMTs and JMJD6 were overexpressed in PCa compared to non-neoplastic tissue, and nuclear PRMT2 and JMJD6 were upregulated in lymph node metastasis, too. PRMT7 and JMJD6 expression were upregulated with the progression of stage and JMJD6 was also increased with the elevation of grade. After androgen ablation therapy, nuclear expression of PRMT7 and JMJD6 were elevated compared to untreated tumors. PRMT2, PRMT7 and JMD6 were also correlated with markers of EMT and cell cycle regulators. Finally, our findings indicate that PRMTs and JMJD6 are involved in prostate cancer progression and revealed a potential interplay of PRMTs with EMT mediators, underscoring the need for therapeutic targeting of arginine methylation in prostate cancer.

DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the −2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.

Identifying Transcription Factors That Prefer Binding to Methylated DNA Using Reduced G-Gap Dipeptide Composition.

Transcription factors (TFs) play an important role in gene expression and regulation of 3D genome conformation. TFs have ability to bind to specific DNA fragments called enhancers and promoters. Some TFs bind to promoter DNA fragments which are near the transcription initiation site and form complexes that allow polymerase enzymes to bind to initiate transcription. Previous studies showed that methylated DNAs had ability to inhibit and prevent TFs from binding to DNA fragments. However, recent studies have found that there were TFs that could bind to methylated DNA fragments. The identification of these TFs is an important steppingstone to a better understanding of cellular gene expression mechanisms. However, as experimental methods are often time-consuming and labor-intensive, developing computational methods is essential. In this study, we propose two machine learning methods for two problems: (1) identifying TFs and (2) identifying TFs that prefer binding to methylated DNA targets (TFPMs). For the TF identification problem, the proposed method uses the position-specific scoring matrix for data representation and a deep convolutional neural network for modeling. This method achieved 90.56% sensitivity, 83.96% specificity, and an area under the receiver operating characteristic curve (AUC) of 0.9596 on an independent test set. For the TFPM identification problem, we propose to use the reduced g-gap dipeptide composition for data representation and the support vector machine algorithm for modeling. This method achieved 82.61% sensitivity, 64.86% specificity, and an AUC of 0.8486 on another independent test set. These results are higher than those of other studies on the same problems.

Isocitrate Dehydrogenase Mutations Are Associated with Different Expression and DNA Methylation Patterns of OLIG2 in Adult Gliomas.

Isocitrate dehydrogenase (IDH) mutant gliomas are associated with a better prognosis in comparison to adult IDH wild-type glioma and glioma-CpG island methylator phenotypes. Although OLIG2 is mainly expressed in oligodendrocytes in normal adult brain, it is expressed in both astrocytomas and oligodendrogliomas. Utilizing the clinical, DNA methylation, and RNA-sequencing data from the Cancer Genome Atlas (TCGA) for lower-grade glioma and glioblastoma cohorts, we explored the association between IDH mutation status and OLIG2 expression on transcription, DNA methylation, and gene target levels. Compared to IDH wild-type gliomas, IDH mutant gliomas showed consistently higher expression of OLIG2 transcripts. OLIG2 overexpression is a good surrogate marker for IDH mutation with an AUC of 0.90. At the DNA methylation level, IDH-mutant gliomas showed hyper- and hypomethylation foci upstream of the OLIG2 transcription start site. Underexpressed OLIG2 target genes in IDH mutant glioma were enriched in cell cycle-related pathways. Thus, the differential expression of OLIG2 between IDH mutant and wild-type gliomas reflects involvement in multiple pathways in tumorigenesis.

Pan-cancer landscape of the RUNX protein family reveals their potential as carcinogenic biomarkers and the mechanisms underlying their action

BackgroundThe RUNX family of transcription factors plays an important regulatory role in tumor development. Although the importance of RUNX in certain cancer types is well known, the pan-cancer landscape remains unclear.Materials and MethodsData from The Cancer Genome Atlas (TCGA) provides a pan-cancer overview of the RUNX genes. Hence, herein, we performed a pan-cancer analysis of abnormal RUNX expression and deciphered the potential regulatory mechanism. Specifically, we used TCGA multi-omics data combined with multiple online tools to analyze transcripts, genetic alterations, DNA methylation, clinical prognoses, miRNA networks, and potential target genes.ResultsRUNX genes are consistently overexpressed in esophageal, gastric, pancreatic, and pan-renal cancers. The total protein expression of RUNX1 in lung adenocarcinoma, kidney renal clear cell carcinoma (KIRC), and uterine corpus endometrial carcinoma (UCEC) is consistent with the mRNA expression results. Moreover, increased phosphorylation on the T14 and T18 residues of RUNX1 may represent potential pathogenic factors. The RUNX genes are significantly associated with survival in pan-renal cancer, brain lower-grade glioma, and uveal melanoma. Meanwhile, various mutations and posttranscriptional changes, including the RUNX1 D96 mutation in invasive breast carcinoma, the co-occurrence of RUNX gene mutations in UCEC, and methylation changes in the RUNX2 promoter in KIRC, may be associated with cancer development. Finally, analysis of epigenetic regulator co-expression, miRNA networks, and target genes revealed the carcinogenicity, abnormal expression, and direct regulation of RUNX genes.ConclusionsWe successfully analyzed the pan-cancer abnormal expression and prognostic value of RUNX genes, thereby providing potential biomarkers for various cancers. Further, mutations revealed via genetic alteration analysis may serve as a basis for personalized patient therapies.

Aberrant circulating tumour DNA methylations as biomarkers for early detection of pancreatic ductal adenocarcinoma: a retrospective study

Pancreatic ductal adenocarcinoma is a cancer with high mortality and low survival, the early detection of which is hampered by the absence of specific symptoms until an advanced stage is reached. No reliable early screening tool for pancreatic ductal adenocarcinoma is currently available. Circulating tumour DNA (ctDNA) methylation has emerged as a promising new type of biomarker for blood-based early detection of multiple types of cancer. As a part of the PanSeerX study, which aims to discover robust ctDNA methylation markers for multicancer early screening, a customised pancreatic ductal adenocarcinoma-specific targeted methylation sequencing panel has been preliminarily developed and validated for its accuracy in classifying pancreatic ductal adenocarcinoma from retrospective clinical plasma samples. We led a single-centre retrospective pilot study at the Changhai Hospital, Navy Medical University, Shanghai, China, where 62 adult patients with pancreatic ductal adenocarcinoma and 393 age-matched and sex-matched healthy controls were enrolled after written consent was obtained from each participant. Individuals with previous cancer diagnosis were excluded. Blood samples were drawn from all participants. A methylation target sequencing panel covering 1601 potential pancreatic ductal adenocarcinoma methylation markers, which were collected from our previous PanSeer study and other related studies, identified by mining the public methylomic datasets The Cancer Genome Atlas and Gene Expression Omnibus, or discovered from the analysis of in-house reduced-representation bisulfite sequencing data, was designed and technically validated. The panel was then applied to test the collected plasma samples at mean a sequencing depth of 1000× per target to quantify the methylation levels and patterns on the targets. A two-layer deep neural network model was built to classify cancer and healthy samples from the sequencing data. The robustness of entire approach was verified with a 3× cross-validation by randomly splitting samples into training set and test set at a 2:1 ratio. This study was approved by the Changhai Hospital, Navy Medical University (reference number CHEC2021-165). All blood samples were used in the development and validation of the pancreatic ductal adenocarcinoma panel. The average area under the curve of the training dataset during the 3× validation was 1·000 (95% CI 0·999-1·000; sensitivity 100·0% [95% CI 100·0-100·0; specificity 99·0%, [98·6-99·5]). The average area under the curve of the testing dataset during the 3× validation was 0·987 (95% CI 0·971-0·996; sensitivity 89·0% [95% CI 75·0-100·0]; specificity 96·0% [92·3-100·0]). Notably, this model is highly sensitive to pancreatic ductal adenocarcinoma of stages I and II, achieving a sensitivity of 81% for stage I and 88% for stage II, showing its ability to detect early stage pancreatic ductal adenocarcinoma. Our preliminary results show that our pancreatic ductal adenocarcinoma-detecting panel is highly accurate in classifying pancreatic ductal adenocarcinoma plasma from healthy controls using ctDNA methylation markers. Its high sensitivity for stages I and II pancreatic cancer is especially promising for further optimisation into diagnostics for blood-based, early pancreatic ductal adenocarcinoma screening. On the basis of these results, a larger, multicentre study is currently underway, which not only enrolled a higher number of patients with pancreatic ductal adenocarcinoma cases and healthy controls, but also included samples from acute and chronic pancreatitis to comprehensive evaluate our pancreatic ductal adenocarcinoma-detecting panel's accuracy in classifying pancreatic ductal adenocarcinoma plasma against non-malignant controls. The results of this study are expected to be reported later in 2022. The National Key Research and Development Project of China (grant 2019YFC1315904), the 234 Discipline Climbing Plan Project of the First Affiliated Hospital of Naval Military Medical University (grant 2019YXK033), and the Shanghai Science and Technology Committee.

Identification of DNA Methylation Signature and Rules for SARS-CoV-2 Associated with Age.

COVID-19 displays an increased mortality rate and higher risk of severe symptoms with increasing age, which is thought to be a result of the compromised immunity of elderly patients. However, the underlying mechanisms of aging-associated immunodeficiency against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unclear. Epigenetic modifications show considerable changes with age, causing altered gene regulations and cell functions during the aging process. The DNA methylation patterns among patients with coronavirus 2019 disease (COVID-19) who had different ages were compared to explore the effect of aging-associated methylation modifications in SARS-CoV-2 infection. Patients with COVID-19 were divided into three groups according to age. Boruta was used on the DNA methylation profiles of the patients to remove irrelevant features and retain essential signature sites to identify substantial aging-associated DNA methylation changes in COVID-19. Next, these features were ranked using the minimum redundancy maximum relevance (mRMR) method, and the feature list generated by mRMR was processed into the incremental feature selection method with decision tree (DT), random forest, k-nearest neighbor, and support vector machine to obtain the key methylation sites, optimal classifier, and decision rules. Several key methylation sites that showed distinct patterns among the patients with COVID-19 who had different ages were identified, and these methylation modifications may play crucial roles in regulating immune cell functions. An optimal classifier was built based on selected methylation signatures, which can be useful to predict the aging-associated disease risk of COVID-19. Existing works and our predictions suggest that the methylation modifications of genes, such as NHLH2, ZEB2, NWD1, ELOVL2, FGGY, and FHL2, are closely associated with age in patients with COVID-19, and the 39 decision rules extracted with the optimal DT classifier provides quantitative context to the methylation modifications in elderly patients with COVID-19. Our findings contribute to the understanding of the epigenetic regulations of aging-associated COVID-19 symptoms and provide the potential methylation targets for intervention strategies in elderly patients.

PRC2, Chromatin Regulation, and Human Disease: Insights From Molecular Structure and Function.

Polycomb repressive complex 2 (PRC2) is a multisubunit histone-modifying enzyme complex that mediates methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) is an epigenetic hallmark of gene silencing. PRC2 plays a crucial role in a plethora of fundamental biological processes, and PRC2 dysregulation has been repeatedly implicated in cancers and developmental disorders. Here, we review the current knowledge on mechanisms of cellular regulation of PRC2 function, particularly regarding H3K27 methylation and chromatin targeting. PRC2-related disease mechanisms are also discussed. The mode of action of PRC2 in gene regulation is summarized, which includes competition between H3K27 methylation and acetylation, crosstalk with transcription machinery, and formation of high-order chromatin structure. Recent progress in the structural biology of PRC2 is highlighted from the aspects of complex assembly, enzyme catalysis, and chromatin recruitment, which together provide valuable insights into PRC2 function in close-to-atomic detail. Future studies on the molecular function and structure of PRC2 in the context of native chromatin and in the presence of other regulators like RNAs will continue to deepen our understanding of the stability and plasticity of developmental transcriptional programs broadly impacted by PRC2.