Journal: Cancers

Article Title: Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation

doi: 10.3390/cancers12020447

Figure Lengend Snippet: ( A ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to the indicated concentrations of 2b or 4c . Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as loading control. Blots are representative of two independent experiments. Right: Densitometric analysis of protein levels is reported. ( B ) Left: Western blot analysis of DNMT1 and DNMT3A protein expression levels in HCT116 cells exposed for 24 h to 4c at 1 µM and co-treated with bortezomib (when indicated) used at 10 nM. Control cells were treated with the same volume of vehicle (DMSO). GAPDH was used as a loading control. Blots are representative of three independent experiments. Right: Densitometric analysis of protein levels is reported. Data are represented as mean ± SEM. Significance is represented as * p < 0.05 related to the control.

Article Snippet: The following primary antibodies were used for immunoblotting: α-DNMT1 (Novus Biologicals, Littleton, CO, USA). α-DNMT3a (SantaCruz Bio-technologies, CA, USA) and α-GAPDH (Millipore Corp., Bedford, MA USA), used as a loading control.

Techniques: Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNMT1 target genes. ( A ) Venn diagram depicts numbers of genes upregulated in clones containing empty vector pEF1αIRESpuro (E1, E2, E3) of HCT116 DNMT1 − / − hypomorphs compared to parental HCT116. ‘ DNMT1 −/− ’ is the parental DNMT1 −/− hypomorph clone. A total of 229 genes were found upregulated 1.4-fold or greater in all four DNMT1 −/− clones. From the 229 gene list, genes upregulated 1.4-fold also in DNMT3b −/− were eliminated and yielded 135 genes. ( B ) RT–PCR validation for upregulation of genes in DNMT1 −/− (E1) cells. Fold change relative to HCT116 is calculated. cMYC was used as a negative control for a gene with unchanged expression in the DNMT1 −/− cells. Microarray fold changes for individual genes are written below. ( C ) Western blot to assess transient knockdown of DNMT1 in HCT116 cells transfected with DNMT1 siRNA (DNMT1si) or non-target siRNA (NTsi) for 72 h. αβactin serves as a loading control. ( D ) RT–PCR analysis of DSCR8 , DTX3 , MAGEA10 , TXNIP and cMYC genes in HCT116 cells transfected with non-target control siRNA (dark grey bar) or DNMT1 siRNA (light gray bar). Fold change relative to non-target control was calculated. Bars = standard error of three independent experiments. ( E ) ChIP at DSCR8 , DTX3 , MAGEA10 , TXNIP and cMYC promoter regions for αDNMT1. Samples included HCT116 and DNMT1 −/− (E1) cell lines. RT–PCR was conducted and the average levels of enrichment relative to input and standard errors were calculated for three independent ChIP experiments.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Clone Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Negative Control, Expressing, Microarray, Western Blot, Knockdown, Transfection, Control

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: Stable insertion of wildtype (wt) FLAG- DNMT1 and mut FLAG- DNMT1 into DNMT1 −/− hypomorphs. ( A ) DNMT1 −/− hypomorph cell lines were stably transfected with vector alone (E), wt FLAG - DNMT1 (wt) and FLAG - DNMT1 containing a point mutation, C1226W, in the catalytic domain (mut). The star represents the position of the point mutation introduced into the DNMT1 vector. ( B ) Whole-cell western blot analysis of DNMT1 levels in Hct116, DNMT1 −/− clones (E), wt and mut clones. βactin was used as a loading control. ( C ) Cytoplasmic ‘C’ and nuclear ‘N’ proteins were isolated. Western blot analysis of DNMT1 in HCT116, 2 wt clones and 2 mut clones. LaminB was used as nuclear loading control and GAPDH as a cytoplasmic loading control.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Clone Assay, Control, Isolation

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNMT1 catalytic activity is not required for gene repression of some genes. ( A ) RT–PCR analyses were performed in parental Hct116, E1 ( DNMT1 −/− ), wt DNMT1 (wt1, wt2, wt3) and mut DNMT1 (mut1 and mut2) replacement cells. Fold change relative to HCT116 was calculated. cMYC was used as a negative control. Numbers above bars are the average fold values relative to HCT116. Bars depict standard error from three independent experiments. ( B ) Agilent Microarray. The 135 genes that were upregulated 1.4-fold in DNMT1KOs and not DNMT3bKOs were mapped. The right four columns are parental DNMT1 −/− , E1, E2 and E3 clones versus wt HCT116 where E represents clones with empty vector pEF1αIRES puro. The middle column is the expression changes of DNMT3bKO cell lines compared to HCT116. The left five columns are E1 versus wt1, wt2, wt3, mut1 and mut2. Red depicts increased expression, green depicts decreased expression.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, Microarray, Clone Assay, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNA methylation analysis after DNMT1 wt or mut replacements. ( A ) MSP of DTX3 and TXNIP in parental HCT116 cells, DNMT1 −/− hypomorph clone E1, 3 wt DNMT1 restoration clones (wt1, wt2, wt3), 2 mut DNMT1 replacement clones (mut1, mut2) and DKO, a cell line with both DNMT1 and DNMT3b genetically disrupted in HCT116 cells. Unmethylated sequences are represented by an amplification signal in the U lanes and presence of methylation by amplification in the M lanes. Bisulfite sequencing of ( B ) DSCR8 , ( D ) MAGEA10 and ( F ) TXNIP . E2 and E3 are two subclones of DNMT1 −/− hypomorph cells containing the control empty vector. Each horizontal line is an individually sequenced TA cloned allele with each circle representing a CpG dinucleotide as distributed in the promoter region. Base pairs upstream and downstream relative to transcription start site (TSS at 0) are numbered along the ×-axis. Black circles are methylated cytosine residues, white circles are unmethylated cytosine residues. ( C, E, G ) Quantitation of results as total % methylated cytosine (black bars) and % non- methylated (white bars) relative to the total number of CpGs in the sequences.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: DNA Methylation Assay, Clone Assay, Amplification, Methylation, Methylation Sequencing, Control, Plasmid Preparation, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNMT1 recruitment to promoters correlates with loss of active histone marks. ChIP at DSCR8 , DTX3 , MAGEA10 , TXNIP and MYC promoter regions for ( A ) αFLAG vector ( B ) H3-K4me2, ( C ) H3-K4me3 and ( D ) H3-K9Ac. Samples included HCT116, DNMT1 −/− empty vector (E1), wt DNMT1 clones (wt1 and wt2) or mut DNMT1 clones (mut1, mut2). RT–PCR was conducted and the average levels of enrichment relative to input for αFLAG and histone 3 (H3) IP for histone marks were calculated for three independent ChIP experiments.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Plasmid Preparation, Clone Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: DNMT1 recruits LSD1 to target promoters. ( A ) ChIP of LSD1 at DSCR8 , DTX3 , MAGEA10 , TXNIP and MYC in HCT116 cells. Average % input was calculated. ChIP of a single locus ( B ) at DNMT1 target genes for αLSD1 in HCT116, DNMT1 −/− empty vector (E1) clones, wt DNMT1 clones (wt1 and wt2) or mut DNMT1 clones (mut1 and mut2) or multiple loci at DTX3 ( C ) and TXNIP ( D ). RT–PCR was conducted and the average levels of enrichment relative to input and then fold change relative to E1 were calculated for three independent ChIP experiments. P- value was calculated using a one-tail t- test analysis comparing HCT116 versus E1, E1 versus wt1 + wt2 and E1 versus mut1 + mut2. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Plasmid Preparation, Clone Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: LSD1 interacts with DNMT1. (A) Co-immunoprecipitation and western blot analyses were performed in HCT116 cells over-expressing HA-LSD1 and immunoprecipitation of anti-DNMT1 or anti-FLAG as a negative control and western blot for anti-HA. (B) Endogenous co-IP and western blot analyses were performed in HCT116 (WT) and DNMT1 −/− 5F (KO) using anti-DNMT1 or anti-LSD1 antibodies. ( C ) Endogenous co-IP using anti-LSD1 (LSD1), anti-DNMT1 (MT1) and negative control, anti-CBP (CBP) antibodies were performed in HCT116 cells. Western blots for all three proteins were performed.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Co-Immunoprecipitation Assay

Journal: Nucleic Acids Research

Article Title: DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes

doi: 10.1093/nar/gks031

Figure Lengend Snippet: Knocking down LSD1 induces variable increases in gene expression of some DNMT1 target genes. ( A ) LSD1 was transiently knocked down, using lentiviral infection delivery in E1, wt1, wt2, mut1 and mut2 cells with LSD1 (L) versus non-target (N) shRNA for 5 days. Successful knockdown was verified by western blot of αLSD1 with αLaminB used as a loading control. ( B ) RT–PCR analysis of DSCR8, MAGEA10 and TXNIP genes in each cells transfected with non-target control (light grey bar) or LSD1 (black bar) shRNA were measured. Fold change relative to NT was calculated. Bars = standard error of three independent experiments.

Article Snippet: Antibodies used for Chromatin Immunoprecipitation (ChIP) were as follows: αDNMT1 (Sigma), αFLAG (Sigma), αH3-K4me2(Millipore), αH3-K4me3 (Millipore), αH3-K9Ac (Millipore), αH3-K9me2 (Millipore), αH3-K9me3 (courtesy of Thomas Jenuwein), αH3-K27me3(Millipore), αLSD1 (Abcam) and αH3 (Abcam).

Techniques: Gene Expression, Infection, shRNA, Knockdown, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal:

Article Title: Characterization of Dnmt3b:thymine-DNA glycosylase interaction and stimulation of thymine glycosylase-mediated repair by DNA methyltransferase(s) and RNA

doi: 10.1016/j.jmb.2008.02.049

Figure Lengend Snippet: DNA methyltransferases stimulate base excision repair of T·G mismatches. A: Design and testing of oligodeoxyribonucleotides (ODNs) used in a mismatch repair assay. (a) Sequence of the double-stranded ODN. The sense strand contains either a normal CCGG site or a mutated CTGG site. The antisense strand contains either C or 5mC within an MspI/HpaII recognition site. Positions of variable nucleotides y and x are shown in bold. The MspI/HpaII recognition sequence is indicated with a bar. The asterisk indicates the position of the 32P label. (b) Predicted sensitivity to MspI or HpaII digestion of the control C·GC and C·GmC ODNs compared to the mismatched T·GC and T·GmC ODNs. (c) MspI digestion of the control ODNs results in the expected 26 nt long radiolabeled fragment from both controls, while a 26 nt long fragment is only observed after HpaII digestion of the unmethylated control. In contrast, T·G mismatch ODNs are refractory to digestion by either enzyme. M, MspI. H, HpaII. B: De novo DNA methyltrasnferases stimulate repair of T·G mismatches. (a) Evaluation of Dnmt and Tdg protein levels in J1 and Dnmt null ES cells. Nuclear extracts prepared from each cell line were immunoblotted using antibodies specific for Dnmt1, Dnmt3a, Dnmt3b and Tdg to verify the expression of Tdg and the absence of specific Dnmt expression in each cell line relative to wild-type J1. Immunoblot for Pcna served as a loading control. (b) Predicted T·GC ODN sensitivity to digestion by MspI or HpaII depending on extent of repair/DNA methylation. (c) Typical results from one of three independent experiments in which the T·GC ODN was incubated with wild-type J1 or Dnmt null ES cell nuclear extracts followed by digestion with either MspI or HpaII. The expected 26 nt repair product produced as a result of digestion is indicated by the arrowhead. In the absence of either Dnmt3b and/or Dnmt3a there is a reduction in repair efficiency when compared to wild-type nuclear extracts (refer to text for discussion of Dnmt1 null extracts). As a control (last lane), T·GC ODN was used in a repair assay but was not digested. Lack of a 26 nt fragment indicates the ODN is being repaired and not merely nicked 5′ to the mismatched thymine. (d) Quantitation of differences in the extent of mismatch repair by extracts from normal ES cells (J1) and ES cells nullizygous for the indicated Dnmts. Error bars represent the mean (± SD) of three independent experiments. [*] P < 0.005

Article Snippet: Protein was transferred to PVDF membrane and analyzed by standard immunoblotting with protein specific antibodies: α-Dnmt1 (Santa Cruz, K-18), α-Dnmt3a (Imgenex), α-Dnmt3b (Imgenex), α-Tdg( 37 ), α-PCNA (Santa Cruz, sc-56) with appropriate secondary antibodies conjugated to horse radish peroxidase (HRP).

Techniques: Sequencing, Expressing, Western Blot, DNA Methylation Assay, Incubation, Produced, Quantitation Assay