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Chromosoma Feb 2009Gene silencing by DNA methylation is well documented and known to be essential for various biological phenomena in many organisms. In contrast, the processes that... (Review)
Review
Gene silencing by DNA methylation is well documented and known to be essential for various biological phenomena in many organisms. In contrast, the processes that convert the silent state of a gene whose DNA is methylated and predicted to form facultative heterochromatin to the actively transcribed state remain elusive. In Arabidopsis, recent studies have shown that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING1 (ROS1) participate in DNA demethylation. DME is necessary for genomic imprinting in the endosperm, while ROS1 is involved in pruning DNA methylation patterns in transposons and genic regions of vegetative tissues. These findings provide us with molecular clues for understanding the underlying mechanisms of DNA demethylation and gene activation. In this review, we will consider and discuss the processes of controlling gene activation through DNA demethylation, which are predicted to include the recognition of target sequences, DNA demethylation, the transformation of the chromatin to the active state, and transcription. Many of these processes remain poorly understood at this stage.
Topics: Arabidopsis; Arabidopsis Proteins; DNA Glycosylases; DNA Methylation; DNA, Plant; Gene Expression Regulation, Plant; Genome, Plant
PubMed: 18839198
DOI: 10.1007/s00412-008-0183-3 -
Journal of Genetics and Genomics = Yi... Aug 2022Plants recognize microbe-associated molecular patterns (MAMPs) to activate immune responses and defense priming to defend against pathogen infections. Transcriptional...
Plants recognize microbe-associated molecular patterns (MAMPs) to activate immune responses and defense priming to defend against pathogen infections. Transcriptional regulation of gene expression is crucial for plant immunity and is mediated by multiple factors, including DNA methylation. However, it remains unknown whether and how DNA demethylation contributes to immune responses in MAMP-triggered immunity. Here, we report that active DNA demethylation is required for MAMP-triggered immunity to bacterial pathogens. The rdd-2 triple mutant carrying mutations in ROS1, DML2, and DML3 that encode DNA glycosylases, which are key DNA demethylation enzymes, exhibits compromised immune responses triggered by the MAMPs flg22 and elf18. Genome-wide methylome analysis reveals that flg22 induces rapid and specific DNA demethylation in an RDD-dependent manner. The expression levels of salicylic acid signaling-related and phytoalexin biosynthesis-related genes are tightly associated with the flg22-induced promoter demethylation. The compromised accumulation of priming compounds and antimicrobial metabolites ultimately leads to a defense priming defect in the rdd-2 mutant. Our results reveal the critical role of active DNA demethylation in the MAMP-triggered immune response and provide unique insight into the molecular mechanism of flg22-modulated DNA demethylation.
Topics: Arabidopsis; Arabidopsis Proteins; DNA Demethylation; DNA Glycosylases; Gene Expression Regulation, Plant; Plant Diseases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins
PubMed: 35288370
DOI: 10.1016/j.jgg.2022.02.021 -
Advances in Experimental Medicine and... 2022Growth arrest and DNA damage 45 (Gadd45) family genes, Gadd45A, Gadd45B, and GADD45 G are implicated as stress sensors that are rapidly induced upon...
Growth arrest and DNA damage 45 (Gadd45) family genes, Gadd45A, Gadd45B, and GADD45 G are implicated as stress sensors that are rapidly induced upon genotoxic/physiological stress. They are involved in regulation of various cellular functions such as DNA repair, senescence, and cell cycle control. Gadd45 family of genes serve as tumor suppressors in response to different stimuli and defects in Gadd45 pathway can give rise to oncogenesis. More recently, Gadd45 has been shown to promote gene activation by demethylation and this function is important for transcriptional regulation and differentiation during development. Gadd45 serves as an adaptor for DNA repair factors to promote removal of 5-methylcytosine from DNA at gene specific loci. Therefore, Gadd45 serves as a powerful link between DNA repair and epigenetic gene regulation.
Topics: Cell Cycle Checkpoints; Cell Cycle Proteins; DNA Damage; DNA Demethylation; DNA Repair
PubMed: 35505162
DOI: 10.1007/978-3-030-94804-7_4 -
Cancers Feb 2022DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many... (Review)
Review
DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of and the onset of tumorigenesis. In this review, we summarize the recent findings on TDG's function as a transcriptional regulator as well as the physiological relevance of TDG and active DNA demethylation in cancer.
PubMed: 35159032
DOI: 10.3390/cancers14030765 -
Journal of Integrative Plant Biology Dec 2022Active DNA demethylation effectively modulates gene expression during plant development and in response to stress. However, little is known about the upstream regulatory...
Active DNA demethylation effectively modulates gene expression during plant development and in response to stress. However, little is known about the upstream regulatory factors that regulate DNA demethylation. We determined that the demethylation regulator 1 (demr1) mutant exhibits a distinct DNA methylation profile at selected loci queried by methylation-sensitive polymerase chain reaction and globally based on whole-genome bisulfite sequencing. Notably, the transcript levels of the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1) were lower in the demr1 mutant. We established that DEMR1 directly binds to the ROS1 promoter in vivo and in vitro, and the methylation level in the DNA methylation monitoring sequence of ROS1 promoter decreased by 60% in the demr1 mutant. About 40% of the hyper-differentially methylated regions (DMRs) in the demr1 mutant were shared with the ros1-4 mutant. Genetic analysis indicated that DEMR1 acts upstream of ROS1 to positively regulate abscisic acid (ABA) signaling during seed germination and seedling establishment stages. Surprisingly, the loss of DEMR1 function also caused a rise in methylation levels of the mitochondrial genome, impaired mitochondrial structure and an early flowering phenotype. Together, our results show that DEMR1 is a novel regulator of DNA demethylation of both the nuclear and mitochondrial genomes in response to ABA and plant development in Arabidopsis.
Topics: Arabidopsis Proteins; Genome, Mitochondrial; Protein-Tyrosine Kinases; DNA Demethylation; Nuclear Proteins; Proto-Oncogene Proteins; Arabidopsis; DNA Methylation; Gene Expression Regulation, Plant
PubMed: 36223079
DOI: 10.1111/jipb.13386 -
Nature Protocols Dec 2022DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally... (Review)
Review
DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; RNA, Guide, CRISPR-Cas Systems; DNA Methylation; Gene Editing; CRISPR-Cas Systems; DNA Demethylation; Gene Expression
PubMed: 36207463
DOI: 10.1038/s41596-022-00741-3 -
Molecular Human Reproduction Jul 2012DNA methylation and demethylation are crucial for modulating gene expression and regulating cell differentiation. Functions and mechanisms of DNA... (Review)
Review
DNA methylation and demethylation are crucial for modulating gene expression and regulating cell differentiation. Functions and mechanisms of DNA methylation/demethylation in mammalian embryos are still far from being understood clearly. In this review we firstly describe new insights into DNA demethylation mechanisms, and secondly introduce the differences in active DNA methylation patterns in zygotes and early embryos in various mammalian species. Thirdly, we attempt to clarify the functions of DNA demethylation in early embryos. Most importantly we summarize the importance of active DNA demethylation and its possible relevance to human IVF clinics. Finally research perspectives regarding DNA demethylation are also discussed.
Topics: Animals; Blastocyst; DNA Methylation; Humans
PubMed: 22447119
DOI: 10.1093/molehr/gas014 -
Gastroenterology Dec 2020Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to α-ketoglutarate and other molecules via glutaminase... (Observational Study)
Observational Study
BACKGROUND & AIMS
Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to α-ketoglutarate and other molecules via glutaminase and SLC25A22. This results in inhibition of demethylases and epigenetic alterations in cells that increase proliferation and stem cell features. We investigated whether mutant KRAS-mediated glutaminolysis affects the epigenomes and activities of colorectal cancer (CRC) cells.
METHODS
We created ApcKras mice with intestine-specific knockout of SLC25A22 (ApcKrasSLC25A22 mice). Intestine tissues were collected and analyzed by histology, immunohistochemistry, and DNA methylation assays; organoids were derived and studied for stem cell features, along with organoids derived from 2 human colorectal tumor specimens. Colon epithelial cells (1CT) and CRC cells (DLD1, DKS8, HKE3, and HCT116) that expressed mutant KRAS, with or without knockdown of SLC25A22 or other proteins, were deprived of glutamine or glucose and assayed for proliferation, colony formation, glucose or glutamine consumption, and apoptosis; gene expression patterns were analyzed by RNA sequencing, proteins by immunoblots, and metabolites by liquid chromatography-mass spectrometry, with [U-C]-glutamine as a tracer. Cells and organoids with knocked down, knocked out, or overexpressed proteins were analyzed for DNA methylation at CpG sites using arrays. We performed immunohistochemical analyses of colorectal tumor samples from 130 patients in Hong Kong (57 with KRAS mutations) and Kaplan-Meier analyses of survival. We analyzed gene expression levels of colorectal tumor samples in The Cancer Genome Atlas.
RESULTS
CRC cells that express activated KRAS required glutamine for survival, and rapidly incorporated it into the tricarboxylic cycle (glutaminolysis); this process required SLC25A22. Cells incubated with succinate and non-essential amino acids could proliferate under glutamine-free conditions. Mutant KRAS cells maintained a low ratio of α-ketoglutarate to succinate, resulting in reduced 5-hydroxymethylcytosine-a marker of DNA demethylation, and hypermethylation at CpG sites. Many of the hypermethylated genes were in the WNT signaling pathway and at the protocadherin gene cluster on chromosome 5q31. CRC cells without mutant KRAS, or with mutant KRAS and knockout of SLC25A22, expressed protocadherin genes (PCDHAC2, PCDHB7, PCDHB15, PCDHGA1, and PCDHGA6)-DNA was not methylated at these loci. Expression of the protocadherin genes reduced WNT signaling to β-catenin and expression of the stem cell marker LGR5. ApcKrasSLC25A22 mice developed fewer colon tumors than ApcKras mice (P < .01). Organoids from ApcKrasSLC25A22 mice had reduced expression of LGR5 and other markers of stemness compared with organoids derived from ApcKras mice. Knockdown of SLC25A22 in human colorectal tumor organoids reduced clonogenicity. Knockdown of lysine demethylases, or succinate supplementation, restored expression of LGR5 to SLC25A22-knockout CRC cells. Knockout of SLC25A22 in CRC cells that express mutant KRAS increased their sensitivity to 5-fluorouacil. Level of SLC25A22 correlated with levels of LGR5, nuclear β-catenin, and a stem cell-associated gene expression pattern in human colorectal tumors with mutations in KRAS and reduced survival times of patients.
CONCLUSIONS
In CRC cells that express activated KRAS, SLC25A22 promotes accumulation of succinate, resulting in increased DNA methylation, activation of WNT signaling to β-catenin, increased expression of LGR5, proliferation, stem cell features, and resistance to 5-fluorouacil. Strategies to disrupt this pathway might be developed for treatment of CRC.
Topics: Animals; Colon; Colorectal Neoplasms; DNA Demethylation; Drug Resistance, Neoplasm; Female; Fluorouracil; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glutamine; Hong Kong; Humans; Intestinal Mucosa; Kaplan-Meier Estimate; Ketoglutaric Acids; Male; Mice, Knockout; Mitochondrial Membrane Transport Proteins; Neoplastic Stem Cells; Proto-Oncogene Proteins p21(ras); Wnt Signaling Pathway; Xenograft Model Antitumor Assays
PubMed: 32814111
DOI: 10.1053/j.gastro.2020.08.016 -
Experimental & Molecular Medicine Apr 2017
Topics: Animals; Chromatin; Cytosine; DNA Demethylation; DNA Methylation; Epigenesis, Genetic; Genetic Markers; Histones; Humans; Neoplasms
PubMed: 28450735
DOI: 10.1038/emm.2017.38 -
FASEB Journal : Official Publication of... Feb 2022DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven...
DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.
Topics: 3T3 Cells; Animals; Apoptosis; Biomarkers; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; DNA; DNA Demethylation; DNA Methylation; Gene Expression Regulation; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Promoter Regions, Genetic; Proto-Oncogene Proteins
PubMed: 34997955
DOI: 10.1096/fj.202101402R