Journal: Oncotarget

Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine

doi:

Figure Lengend Snippet: A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.

Article Snippet: Proteins were separated by SDS–PAGE, transferred to PVDF membrane (Millipore) and probed with the following primary antibodies: anti-IDH1 R132H (Dianova, DIA-H09), anti-DNMT1 (New England Biolabs, M0230S), anti-GFAP (Cell Signaling Tech, 2118S) and anti-β-actin (Sigma, A5316).

Techniques: Western Blot, DNA Methylation Assay, Expressing

Journal: Nature

Article Title: DNMT1-interacting RNAs block gene specific DNA methylation

doi: 10.1038/nature12598

Figure Lengend Snippet: a , Diagram showing position of qRT-PCR primers used in RIP, double-headed arrow; RNA and DNA oligonucleotides used in EMSA and REMSA. Asterisks indicate position of methylated cytosines; umDNA, hmDNA, and mDNA refer to unmethylated, hemimethylated, and methylated DNA probes, respectively; b , ecCEBPA is immunoprecipitated with anti-DNMT1 antibody. qRT-PCR, bars indicate mean ± s.d.; c , RNA- DNMT1 binding is not affected by the absence of CpG dinucleotides (right panel). Left and middle panels: RNA oligonucleotide R2 and its mutated form mut R2 (asterisks indicate cytosines substituted into uridines), both able to form stem-loop-structures; d , RNA oligonucleotide able to form stem-loop structure bind DNMT1 (R6); e , R5 RNA oligonucleotide forming stem-loop structure (R5) has a greater DNMT1 affinity compared to mut R5, unable to fold into stem-loop, (taken in equimolar amounts), at the same DNMT1 concentration; f , Left four panels: REMSA and EMSA performed with the fixed concentration of ssRNA and dsDNA oligonucleotides (1 nM) and increasing concentrations of DNMT1 protein; Right panel: Nonlinear regression analysis of bound RNA/DNA versus DNMT1 concentrations. Error bars indicate s.d. from two independent experiments; g , REMSA showing that RNA oligonucleotide R4, which is unable to form stem-loop structure, displays lower DNMT1 affinity as compared to R5 (Fig. 3f left panel) at the same DNMT1 concentrations; h , Left panel: Schematic diagram showing the DNMT1 domains and the GST-DNMT1 isolated fragments (F1–F5); Right panel: GST-DNMT1 pull down assay demonstrating binding of the folded RNA oligonucleotide R5 to the catalytic domain of DNMT1.

Article Snippet: transcription-methylation assays were performed on hemimethylated DNA (hmDNA; described in , legend to ) in the presence or absence of 5 U of human DNMT1 enzyme (New England Biolabs) and 5 U of T7 RNA polymerase (Promega) or 5 U of E. coli RNA polymerase sigma-saturated holoenzyme (Epicentre).

Techniques: Quantitative RT-PCR, Methylation, Immunoprecipitation, Binding Assay, Concentration Assay, Isolation, Pull Down Assay

Journal: Nature

Article Title: DNMT1-interacting RNAs block gene specific DNA methylation

doi: 10.1038/nature12598

Figure Lengend Snippet: a–d , Diagram showing the parallel in vitro transcription-methylation assays performed on a hemimethylated template containing the T7 promoter with and without combinations of RNA polymerase, DNMT1, or both; e , DNMT1 exerts enzymatic activity only in the absence of transcription. COBRA analysis of methylation patterns acquired in reactions shown in b–d; f , DNA methylation changes as are shown as the ratios of methylated to unmethylated CpGs in all clones analyzed per each construct (n=5). The same effect was observed with two different RNA polymerases: T7 and Sigma-Saturated (σ70)-Holoenzyme ( E. coli RNA Polymerase). DNA methylation changes were analyzed by Fisher’s exact test (* P <0.05; ** P <0.01; *** P <0.001); g , in vitro DNMT1 assay demonstrating DNMT1 enzymatic impairment by RNA oligonucleotides. The assay was performed using ecCEBPA -related and unrelated RNA oligonucleotides. Sequences and position of the ribooligonucleotides are shown on and . Error bars indicate mean ± s.d. (n=2).

Article Snippet: transcription-methylation assays were performed on hemimethylated DNA (hmDNA; described in , legend to ) in the presence or absence of 5 U of human DNMT1 enzyme (New England Biolabs) and 5 U of T7 RNA polymerase (Promega) or 5 U of E. coli RNA polymerase sigma-saturated holoenzyme (Epicentre).

Techniques: In Vitro, Methylation, Activity Assay, Combined Bisulfite Restriction Analysis Assay, DNA Methylation Assay, Clone Assay, Construct

Journal: Nature

Article Title: DNMT1-interacting RNAs block gene specific DNA methylation

doi: 10.1038/nature12598

Figure Lengend Snippet: a , Two-way Venn diagram showing DNMT1 specific peaks overlapping with transcribed elements identified in HL-60 total and poly(A+)-depleted RNA-Seq libraries. b , Cloud plots representing genes within DNMT1 unbound, bound and all RRBS-covered groups stratified by DNA methylation and expression levels. All genes are presented in . c , Examples of genes from the C ( CEBPA ) and B ( USP29 ) clusters. Peaks are visualized using the SSIRs . d , Model of DNMT1 sequestration. Upper panel: DNMT1 can access transcriptionally inactive hemimethylated genomic regions. Lower panel: DNMT1 cannot access transcriptionally active hemimethylated genomic regions.

Article Snippet: transcription-methylation assays were performed on hemimethylated DNA (hmDNA; described in , legend to ) in the presence or absence of 5 U of human DNMT1 enzyme (New England Biolabs) and 5 U of T7 RNA polymerase (Promega) or 5 U of E. coli RNA polymerase sigma-saturated holoenzyme (Epicentre).

Techniques: RNA Sequencing Assay, DNA Methylation Assay, Expressing

Journal: Genome Biology

Article Title: Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability

doi: 10.1186/s13059-018-1401-9

Figure Lengend Snippet: Decreased activity of the base excision repair (BER) pathway in cells with sustained p21 WAF1/Cip1 expression. a Increased reactive species (RS) levels were assessed with a DCFH-DA assay in Saos2 ( i ) and Li-Fraumeni ( ii ) cells with protracted p21 WAF1/Cip1 expression (* p < 0.05 (Saos2), * p = 0.05 (Li-Fraumeni), t -test; error bars indicate standard deviation; n = 5 experiments). As shown in the middle panel RS production can lead to generation of base/nucleotide oxidative lesions. b RNAseq analysis showed that essential factors of the BER pathway were statistically significantly down-regulated ( p ≤ 0.05) in 96-h induced Saos2- ( i ) and Li-Fraumeni- ( ii ) p21 WAF1/Cip1 Tet-ON cells (see also Additional file : Figure S3 for specific real time RT-PCR validation). Note that although in Saos2- p21 WAF1/Cip1 Tet-ON cells OGG1 expression was not found by RNAseq analysis, specific real-time RT-PCR and microarray analysis (see also Additional file : Figure S3) confirmed its decreased expression. Selective immunoblots for APEX1, LIG3, TDG, and MUTY confirmed the specificity of the RNA analysis results. Note that LIG3 participates also in mismatch repair (MMR; Additional file : Figure S3). α-Tubulin served as loading control. c Modified alkaline Comet assay demonstrated the presence of oxidized purines like 8-oxo-dG in 96-h induced Saos2- ( i ) and Li-Fraumeni- ( ii ) p21 WAF1/Cip1 Tet-ON cells, using 8-oxoguanine glycosylase (OGG1) (* p < 0.05 (Saos2), * p = 0.05 (Li-Fraumeni), t -test; error bars indicate standard deviation; n = 5 experiments). Comet data were corroborated by an 8-oxo-dG-specific assay measuring DNA incorporation of 8-oxo-dG in p21 WAF1/Cip1 -expressing cells , which indicated lower OGG1 activity (* p < 0.05 (Saos2), * p = 0.05 (Li-Fraumeni), t -test; error bars indicate standard deviation; n = 5 experiments). Consequently, as depicted in the model in the middle panel , recognition and excision of the affected nucleotide lesion is impaired in the BER process. The middle panel depicts the components and steps during BER. The BER pathway is responsible for removal of small lesions from DNA, especially oxidized, alkylated, deaminated bases and abasic sites. BER can be induced by oxidative stress and various genotoxic insults. Its specificity relies on the excision of base damage by glycosylases. In humans, the mechanism of BER involves the initial action of DNA glycosylases followed by the processing of the resulting abasic site either by the AP-lyase activity of the glycosylases or by the apurinic/apyrimidic endonucleases APE1/APE2, which incise the DNA strand. The resulting single-strand break can be processed by two BER subpathways. Either the short-patch branch is engaged, if a single nucleotide is replaced, or the long-patch branch, if 2–10 new nucleotides are synthesized. OGG1 8-oxoguanine DNA glycosylase, UNG uracil DNA glycosylase, TDG thymine DNA glycosylase, SMUG1 single-strand-selective monofunctional uracil-DNA glycosylase 1, NTH DNA glycosylase and apyrimidinic (AP) lyase (endonuclease III), MBD4 methyl-CpG binding domain 4, DNA glycosylase, MPG N-methylpurine DNA glycosylase, MUTY adenine DNA glycosylase, NEIL1/2/3 Nei-like DNA glycosylase 1/ 2/ 3, APEX1/2 apurinic/apyrimidinic endodeoxyribonuclease 1/2, POLB / POLD , DNA polymerase beta/ delta, PCNA proliferating cell nuclear antigen, RFC replication factor C, FEN1 flap structure-specific endonuclease 1, LIG1 / LIG3 DNA ligase 1/3, PARP1 poly(ADP-ribose) polymerase 1, XRCC1 X-ray repair cross complementing 1

Article Snippet: We also used as a DNA damage probe the human repair enzyme OGG1 (New England Biolabs) to detect the specific presence of 8-oxo-dGuanine (8-oxo-dG) [ ].

Techniques: Activity Assay, Expressing, DCFH-DA Assay, Standard Deviation, Quantitative RT-PCR, Microarray, Western Blot, Modification, Alkaline Single Cell Gel Electrophoresis, Synthesized, Binding Assay