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1.
FIGURE 3.

FIGURE 3. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

Activation of DNMT1 activity by DMAP1. In vitro methylase assays were performed using either unmethylated or hemimethylated 30-bp oligonucleotides with 200 ng of purified DNMT1 with or without purified DMAP1 (0, 200, and 400 ng, DMAP1 tested). As a nonspecific negative control, purified ETS1 protein tested at the same concentration (200 and 400 ng). A, the results are expressed as pmol of incorporation of Ado-Met, corrected for backgrounds (minus DNMT1 reactions and reactions pretreated with proteinase K). To facilitate comparison of HM and unmethylated (UM) targets, the data are presented relative to DNMT1 lacking any protein additions. *, p < 0.05; statistical significance was determined using a Student's t test. B, the data presented as percentages of DNMT1 activity without DMAP1.

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
2.
FIGURE 6.

FIGURE 6. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

Influence of DMAP1 depletion on HR frequency. Top panel, the HeLa DR-GFP construct (10, 26) described for Fig. 1 (HO-1 cell line) was used in this experiment. HO-1 cells were infected with lentivirus expressing either nonhairpin control (Mock) or shRNA1-1 against DMAP1. After incubation for 4 days, the cells were transfected with either I-Sce1 or no I-Sce1 (negative control), and the percentages of GFP-positive cells were analyzed by FACS 4 days later. Under these conditions, DMAP1 expression levels are reduced by 4–6-fold (in different experiments, see Fig. 4), and cells display a slow growth phenotype (Fig. 2). B, analysis of HR-H and HR-L expression classes in DMAP1 depleted cells following I-Sce1 transfection was carried out as described for Fig. 1.

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
3.
FIGURE 4.

FIGURE 4. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

Methylation of p16 in HCT116 cells. Wild type HCT116 colorectal cancer cells were infected with lentivirus expressing shRNA (shDMAP1-1 and shDMAP1-2 constructs directed to DMAP1) or control shRNA (mock). A, transcriptional activity of DMAP1 and DNMT1 was determined by quantitative real time RT-PCR, and the data were normalized relative to GAPDH expression. B, analysis of polypeptide levels by Western blotting. Nuclear extracts were prepared as described under “Experimental Procedures,” and equivalent protein loads were analyzed by SDS-PAGE gels followed by Western blotting using anti-DMAP1, anti-DNMT1, and anti-lamin A/C antibody probes. C, bisulfite sequencing of p16 gene in mock, shDMAP1-1, shDMAP1-2, and aza-dC (5 μm). The cells were harvested for bisulfite analysis at 10 and 18 days post-transfection. In the aza-dC-treated cells, bisulfite treatment was carried out at 7 days only. D, bisulfite sequencing of LINE-1 and TIMP-3 genes. Genomic DNA from either mock or shRNA-infected HCT116 cells was extracted, sodium bisulfite-treated, and amplified using a suitable primer pair for p16 genomic DNA (−119 to +380, see “Experimental Procedures”). Amplified DNAs were subcloned into pGEM-T easy vector, and 10 independent clones were sequenced. The sequences were analyzed using BiQ Analyzer software (for p16) or QUMA software (LINE-1, TIMP-2. An open circle denotes a unmethylated CpG dinucleotide; a closed circles denotes a methylated CpG dinucleotide.

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
4.
FIGURE 2.

FIGURE 2. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

Growth behavior and influence of DMAP1 on endogenous DNMT1. A, genome wide activity of endogenous DNMT1 using the ICM assay. The in vivo activity of endogenous DNMT1 was measured in HeLa cells infected with shRNA expressing lentivirus 2 days post-infection. The cells were pulsed with 10 μm of aza-dC for 1 h and rapidly lysed with 1% sarkosyl followed by CsCl step gradient purification of genomic DNA as described under “Experimental Procedures.” DNA fractions (1.7g/cc) were pooled, and the DNA concentration was measured by absorbance at 260 nm. Either 0.5, 1, or 2 μg of genomic DNA were applied to nitrocellulose membrane using the slot blot manifold. The membrane was then probed with anti-DNMT1 antibody. Because the DNA concentrations are fixed, and the amount of DNA is constant per cell, the signals can be compared directly. The amount of DNMT1 covalently bound to cellular DNA reflects the genome wide activity. B, analysis of cell growth kinetics. After infection with the indicated lentiviruses (mock, diamonds; shRNA1-1, squares; shRNA1-2, triangles) and selection with puromycin, HeLa cells were seeded at 2 × 103 cells/well in a 96-well microtiter plate. At each time point, the cells were lysed with 0.6% Nonidet P-40, 0.4% PicoGreen in 1× PBS. Fluorescence was measured with Tecan Genios plate reader with excitation at 485 nm and emission at 530 nm. C, DMAP1 and DNMT1 mRNA levels. The cells were transduced with the indicated DMAP1 viruses, and 18 days later, expression of DMAP1 and DNMT1 were measured using quantitative real time RT-PCR as described under “Experimental Procedures.” The data were normalized with GAPDH expression.

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
5.
FIGURE 1.

FIGURE 1. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

HR repair-induced silencing using DR-GFP. A, HeLa DR-GFP construct (10, 26). HeLa cells containing a stably integrated copy of DR-GFP were used in the analysis (HO-1 cell line). There are two mutated GFP cassettes, separated by a puromycin selective marker. The 5′ cassette (GFP-I) has been inactivated by the addition of an I-SceI site containing tandem in-frame stop codons. The 3′ cassette (GFP-II) contains a partial (812 bp) internal GFP coding sequence and is therefore inactivated by 5′ and 3′ truncations. The two cassettes are separated by 3.7 kb. After transfection with a plasmid encoding the I-SceI, a unique double strand break induces homology-directed repair at the GFP integrant. Cassette II (GFP-II) acts as a homology donor to convert GFP-I into WT GFP in a short tract gene conversion event. Also shown are rec and unrec primers. Note that both primer sets are designed to analyze only cassette I because cassette II lacks the sequence for a downstream (3′) primer (because of 5′/3′ truncations of GFP). The rec and unrec primers are further distinguishable at the bold nucleotide positions. B, HO-1 cells were infected with lentivirus expressing either nonhairpin control (Mock) or shRNA against DMAP1. Expression of DMAP1 mRNA was quantified using real time RT-PCR. All of the data were normalized with GAPDH expression. C, analysis of HR-H and HR-L expression classes in DMAP1 knockdown cells. After infection with two shRNA lentiviruses or a mock (nonhairpin control), HO-1 were transfected with I-SceI. After incubation for 4 days, GFP-positive cells were analyzed by FACS and to determine the HR-H:HR-L ratios (using CellQuest software). D, analysis of HR frequency was performed by comparing the mock shRNA cells with shDMAP1 by PCR using the Rec primer (plus internal β-actin control) as previously described (10).

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
6.
FIGURE 7.

FIGURE 7. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

Model describing DMAP1 role in silencing of HR repair. A, HO-1 cells contain the DR-GFP reporter, and following expression of I-SceI, HR repair is initiated that recovers WT GFP sequences from the cassette II donor sequence at the BcgI site. The methylation state of the reporter GFP prior to HR does not alter recombination frequency or silencing outcome after HR (10). Following HR repair, methylation patterns are either reset or overlaid with new patterns. The I-SceI site is converted to a BcgI site as WT GFP is restored. B, in cells with normal DMAP1 levels, DMAP1 and DNMT1 are recruited as part the of the repair machinery (by proliferating cell nuclear antigen or other repair factors) (21, 30). DNA flanking the I-SceI site is hemimethylated because of the concerted action of DNMT1·DMAP1 on one strand (the opposing strand is protected, indicated by gray T shapes). After cell division, two populations of cells are derived that differ in methylation state in flanking DNA around the I-SceI site. The HR-H and HR-L are present at a 1:1 ratio. Because DMAP1 binds hemimethylated DNA with high avidity and poorly to unmethylated DNA, it is likely that DMAP1 recruits DNMT1 to promote conversion of hemimethylated to fully methylated DNA during the S phase; however, the daughter DNA strands derived from the unmethylated parentals are not good DNMT1 targets because DMAP1 binds poorly to unmethylated templates. C, in DMAP1 knockdown cells, DNMT1 activity is reduced because of the low levels of the DMAP1 co-repressor, which then hampers recruitment of DNMT1 to GFP recombinant chromatin. This effectively elevates the level of the HR-H (high expression class) and reduces the fraction of the HR-L. Because DMAP1 knockdowns also display genomic DNA instability (Fig. 6) (30), HR repair and methylation events are activated at sites of DNA damage in the knockdown cells; however, in the absence of DMAP1, DNMT1 action is less robust, leading to hypomethylation at sites of HR repair.

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.
7.
FIGURE 5.

FIGURE 5. From: DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair.

DNA binding activity and localization of DMAP1 in recombinant HR chromatin. A, DNA binding activity of DMAP1 was analyzed using SPR assay. Biotinylated hemimethylated, unmethylated, or fully methylated double strand oligonucleotide DNA were immobilized on the gold chip coated with NeutrAvidin. Purified DMAP1 protein was applied to the chip at a concentration of 1, 10, 25, 50, and 100 nm (flow rate of 10 μl/min). The KD values shown were derived from kinetic parameters based on the relationship KD = kd/ka, where kd is the dissociation rate constant, and ka is the association rate constant. The ka and kd rate constants (Table 1) were derived by nonlinear curve fitting of sensogram data at the concentrations indicated above. A representative SPR trace is shown for each DNA target, and the analysis was repeated four times with different oligonucleotide and protein preparations. B, effects of overexpression of DMAP1 on endogenous DNMT1 activity. The ICM method, which measures the total amount of endogenous DNMT1 trapped on genomic DNA (34), was used to examine the influence of high level expression of DMAP1 on global methylase activity mediated by DNMT1. HCT-116 cells were transfected with vector (mock) or DMAP1 plasmid (2.5 μg), and 48 h later, exponentially growing cells were incubated with 10 μm aza-dC for 1 h and immediately lysed with sarkosyl. The indicated amounts of genomic DNA were spotted on a slot blot and probed with anti-DNMT1 antibody. C, ChIP analysis. The molecular association between recombinant GFP, DMAP1, and DNMT1 in a chromatin context was analyzed by chromatin immunoprecipitation assay. After transfection of either mock or I-SceI plasmid, the cells were formaldehyde-fixed and harvested. The sonicated DNAs were mixed with antibodies as indicated. Final immunoprecipitated DNAs were amplified with Rec primers, which detects only the recombinant GFP DNA. Anti-LexA antibody was used as a negative control. D, band intensity ratios of DMAP1:DNMT1 before HR (− I-SceI) and after HR (+ I-SceI) in unrecombined chromatin (unRec primers) and recombinant chromatin (Rec primers). The analysis was repeated in three independent experiments (see Fig. 1A for details on primer construction).

Gun E. Lee, et al. J Biol Chem. 2010 November 26;285(48):37630-37640.

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