- A molecular tool targeting the base flipping activity of human UHRF1. [Journal Article]
- CChemistry 2019 Jul 19
- During DNA replication, Ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET…
During DNA replication, Ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket of UHRF1. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.
- DNA methylation and histone post-translational modifications in the mouse germline following in-vitro maturation of fresh or cryopreserved prepubertal testicular tissue. [Journal Article]
- RBReprod Biomed Online 2019 May 16
- CONCLUSIONS: Despite differences with the in-vivo situation, DNA methylation and histone methylation and acetylation occur in the mouse germline in in-vitro matured fresh or cryopreserved mouse prepubertal testicular tissue, and the expression of the enzymes catalysing these epigenetic modifications are maintained in vitro.
- Study of porphyrin-modified liquid exfoliated graphene field-effect transistors for evaluating DNA methylation degree. [Journal Article]
- AAnalyst 2019 Jul 15
- The applications of graphene field-effect transistors (FETs) for monitoring DNA hybridization have been widely accepted; however, for evaluating DNA methylation degree, an emerging requirement of epi…
The applications of graphene field-effect transistors (FETs) for monitoring DNA hybridization have been widely accepted; however, for evaluating DNA methylation degree, an emerging requirement of epigenetic research, no work has been found due to the difficulties in detecting 5-methylcytosine (5mC) sites along the genomic sequence as well as counting their amount (NmC). Herein, to achieve this, a strategy for exploiting a liquid exfoliated graphene (LEG)-based FET (LEG-FET) as a sensing platform was proposed. First, LEG-FETs were prepared and activated by tetra-4-aminophenyl-porphyrin (TAPP) for anchoring single-strand DNAs (ssDNAs). Second, the 5mC sites in ssDNA were recognized by the specifically absorbed 5mC antibody (5mCab) and transduced to the changed currents (ΔIDS) by LEG-FET according to the integration of the methylation-immuno sensing principle and FET's working mechanism. Briefly, more 5mCab molecules could be captured by more 5mC sites, resulting in larger ΔIDS. The TAPP effects on LEG-FET were analyzed by SEM, Raman, AFM, and XPS characterizations as well as electronic measurements. The validity of this LEG-FET sensing platform for evaluating DNA methylation degree was proven step by step; this included the examinations of the synthesized ssDNAs with the known NmC and real ssDNA samples, whose methylation degrees were pre-determined by the gold-standard method, which is based on tedious bisulphite sequence operations and expensive mass spectrometry technology. Moreover, theoretical explanations were also provided for the sensing mechanism in the proposed DNA methylation analytical components. In conclusion, the positive and linear relations of IDS changing ratio vs. NmC as well as the detection limit of one 5mC site indicate that TAPP-modified LEG-FET can provide an alternative analytical tool to realize fast and economical DNA methylation evaluation.
- Formation and biological consequences of 5-Formylcytosine in genomic DNA. [Review]
- DRDNA Repair (Amst) 2019 Jul 08; :102649
- 5-Formyl-2'-deoxycytidine (5fdC) is a naturally occurring nucleobase that is broadly distributed in genomic DNA. 5fdC is produced via the oxidation of 5-methylcytosine (5mdC) by ten-eleven translocat…
5-Formyl-2'-deoxycytidine (5fdC) is a naturally occurring nucleobase that is broadly distributed in genomic DNA. 5fdC is produced via the oxidation of 5-methylcytosine (5mdC) by ten-eleven translocation enzyme (TET) and can be further converted to 5-carboxylcytosine (5cadC) by TET. Both 5fdC and 5cadC can be restored to dC by TDG-mediated base excision repair and direct deformylation/decarboxylation. Thus, 5fdC is considered an intermediate in the TET-mediated DNA demethylation pathway. 5fdC also alters the structure and stability of genomic DNA and affects genetic expression. This review summarizes the recent research on 5fdC, detailing its formation, detection and distribution, biological functions and transformation in cells. The challenges and future prospects to further explore the function and metabolism of 5fdC are briefly discussed at the end.
- N-acetylcysteine reverses the decrease of DNA methylation status caused by engineered gold, silicon, and chitosan nanoparticles. [Journal Article]
- IJInt J Nanomedicine 2019; 14:4573-4587
- CONCLUSIONS: The global DNA methylation that is observed in cells exposed to ENPs is associated with methylation of the Alu elements. However, the change in DNA methylation levels following ENP exposure is specific to particular ENP and cell types and independent of ROS, being induced indirectly through disruption of the oxidative defense process.
- The domain architecture of protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity. [Journal Article]
- JBJ Biol Chem 2019 Jul 10
- J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozo…
J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosomaand Leishmania. JBP1 has two known functional domains: an N-terminal T hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins and a J-DNA-binding domain (JDBD) that resides in the middle of JBP1. Here, we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered structure. We found that this Δ-JDBD domain retains TH activity in vitro, but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA, and on Δ-JDBD, enabled us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.
- Single Molecule Fluorescence Imaging for Ultrasensitive DNA Methyltransferase Activity Measuring and Inhibitor Screening. [Journal Article]
- ACAnal Chem 2019 Jul 10
- Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase …
Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently the streptavidin-labelled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labelled methylated dsDNA molecules, the single molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001 - 1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.
- Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression. [Journal Article]
- BDBMC Dev Biol 2019 Jul 08; 19(1):16
- CONCLUSIONS: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.
- Regulation of DNA methylation levels in the process of oral mucosal regeneration in a rat oral ulcer model. [Journal Article]
- HHHistol Histopathol 2019 Jul 09; :18147
- DNA methylation is an important epigenetic mechanism for cellular maintenance. However, the methylation pattern and the key molecule regulated epigenetically in oral mucosal regeneration is unclear. …
DNA methylation is an important epigenetic mechanism for cellular maintenance. However, the methylation pattern and the key molecule regulated epigenetically in oral mucosal regeneration is unclear. In this study, we generated a rat oral ulcer model and investigated the cell proliferative activities and DNA methylation patterns immunohistochemically. We also performed immunohistochemical analysis of a regulator of epithelial stem/progenitor cell differentiation in the rat model. We demonstrated immunohistochemistry using antibodies for the molecules as follows: Ki-67, a marker of cellular proliferation; 5-methylcytosine (5-mC), a marker of DNA methylation; 5-hydroxymethylcytosine (5-hmC), a marker of DNA demethylation; Dnmt1, a maintenance DNA methyltransferase; Dnmt3a and Dnmt3b, de novo DNA methyltransferases; and Wnt5a, a regulator of stem/progenitor cell differentiation. In this model, re-epithelialization was completed at Day 4 after ulceration. Regenerating mucosal hypertrophy reached a peak at Day 5 and appeared normal at Day 14. Ki-67-positive cells increased at Day 2 and returned to normal at Day 6 after ulceration. The ratio of the expression level of 5-mC to 5-hmC declined at Day 5 and returned to normal at Day 6. The expression level of Dnmt1 had not changed compared to the normal control at every time point. On the other hand, the expression levels of Dnmt3a and Dnmt3b had decreased significantly at Day 5 and returned to normal at Day 6. Moreover, Wnt5a-positive cells increased at Day 5. In conclusion, oral mucosal regeneration was strictly regulated by DNA methylation. Moreover, Wnt5a might play a critical role in oral mucosal regeneration.
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- Health risk of childhood exposure to PCDD/Fs emitted from a municipal waste incinerator in Zhejiang, China. [Journal Article]
- STSci Total Environ 2019 Jun 26; 689:937-944
- This study aimed to evaluate the body burdens of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and their associated health impacts toward school-age children living near a municipal w…
This study aimed to evaluate the body burdens of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and their associated health impacts toward school-age children living near a municipal waste incinerator (MWI). A total of 82 children from the exposure area and 49 from the control area were recruited. We measured blood PCDD/F levels, conducted comet assays, calculated the percentage of 5-methylcytosine (%5-mC) and 5-hydroxymethylcytosine (%5-hmC), performed flow cytometry, measured hormonal levels, and analyzed hematological parameters. We also examined 17 congeners of PCDD/Fs in environmental samples, namely, eggs, rice, water, soil, and PM2.5. The mean blood levels of ΣPCDD/Fs and TEQ-ΣPCDD/Fs were statistically higher in the exposure group than in the control group (3.40 vs. 2.77 pg/g wet weight and 0.40 vs. 0.28 pg WHO-TEQ/g wet weight, respectively; p < 0.05). By contrast, the %5-mC and %5-hmC levels were statistically lower in the exposure group than in the control group (1.15% vs. 4.66% and 0.22% vs. 0.30%, respectively; p < 0.01), whereas the mean % tail DNA was statistically higher in the exposure group than in the control group (10.10% vs. 8.28%, p < 0.01). The mean blood levels of ΣPCDD/Fs and TEQ-ΣPCDD/Fs were both negatively correlated with %5-mC (r = -0.245 and r = -0.217, respectively; p < 0.01) but not with %5-hmC and % tail DNA (p > 0.05). Furthermore, the mean ΣPCDD/F levels in eggs and soil obtained from the exposure area were statistically higher than those of the samples obtained from the control area (31.08 vs. 4.32 pg/g dry weight and 1026.04 vs. 674.97 pg/g dry weight, respectively). In conclusion, children living near the MWI may suffer genetic and epigenetic modifications, such as DNA damage or global DNA hypomethylation due to the MWI-emitted PCDD/Fs and other contaminants.