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αdnmt1 antibody (Millipore)

Name: αdnmt1 antibody
Brand: Millipore
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Millipore αdnmt1 antibody

<t>DNMT1</t> 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.

αdnmt1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes"

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks031

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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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.

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.

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.

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.

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.

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.

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.

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

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

Article Snippet: Antibodies utilized in western blot analysis were as follows: αDNMT1 (Sigma D4567) 1:2000, αDNMT1 (epitomics 2788-1) 1:3000, αβactin (Sigma 5441) 1:10 000, αLSD1 (Millipore 09-058) 1:10 000, αCBP (Santa Cruz sc-369) 1:1000, αLaminB (Santa Cruz) 1:2000, α-tubulin (Sigma T6074) 1:10 000 and αGAPDH (Millipore) 1:10 000.

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

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