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1.
Figure 5.

Figure 5. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Methylation-specific PCR analysis of IGS-rDNA. (A) In the DNMT1 knockdown genome either by siRNA for DNMT1 (top) or G9a (bottom) and by depleting endogenous DNMT1 with zebularine (right). The expected gene-specific band is ∼110 bp. Unmethylated (U) and methylated (M) gene-specific products are shown. (B) ChIP analysis of IGS-rDNA loci. In the top left panel, a line diagram of the 110-bp loci with CpG residues is indicated as vertical filled boxes. The right panel shows the linearity PCR amplification of the knockdown input DNAs. The bottom panel shows the PCR products from the ChIP analysis. The antibodies used for IP are indicated at the top and knockdown conditions are shown at the bottom. (C) Q-PCR analysis of various histone H3 modifications in the IGS-rDNA loci. The knockout status is shown at the bottom.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
2.
Figure 3.

Figure 3. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Colocalization and interaction between DNMT1 and SUV39H1. (A) Cytochemical localization of SUV39H1 and H3K9me3 in heterochromatin. (B) Cytochemical localization of DNMT1 and H3K9me3 in heterochromatin. (C) Mapping of the SUV39H1-binding region on DNMT1 using overlapping GST fusion fragments of DNMT1. Various GST fusions of DNMT1 are indicated above with amino acids in parentheses and antibody used shown at right. (D) Schematic diagram of DNMT1 with SUV39H1-binding region on it shown below. (E) SUV39H1 and DNMT1 do not colocalize during DNA replication, but are associated in heterochromatin. On the left side of the panel cytochemical observation after synchronization in hours and percent of cells where DNMT1 and SUV39H1 fusions are colocalized are given. For each stage (at 0 time [panel I], between 2 and 4 h [panel II], between 8 and 12 h [panel III], and after 15 h [panel IV] post-aphidicolin treatment), 20–50 cells were counted.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
3.
Figure 6.

Figure 6. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Methylation activation via interaction of DNMT1 and G9a in vitro. (A) DNA methylation activation in the presence of DNMT1 and G9a. A deletion mutant G9a (delG9a) that did not contain the DNMT1-binding region was used as control. Components in the in vitro reaction are shown at the bottom. Unmethylated and hemimethylated DNA substrates are shown. The methyl group incorporation in shown at the left. Each reaction was repeated twice. (B) Histone methylation stimulated by DNMT1 on HCT116 nucleosome substrates. Reaction components are indicated at the bottom of the graph. G9a and delG9a were used for methylation reaction. Tritiated methyl group incorporation is in counts per minute at the left. Reactions were repeated twice in duplicates. (C, top) G9a recruits DNMT1 onto the nucleosomal array. Prebound G9a on the nucleosomal array is shown at the top along with cofactor AdoMet. DNMT1 concentration was increased as shown at the top along with the antibody used for DNMT1 loading detection shown at right. (Bottom) A similar experiment with delG9a abolished DNMT1 recruitment. (D) DNMT1 catalysis stimulated by G9a in a replication-independent manner. The prebound components used are indicated at the top. The subsequent incubation was carried out with DNMT1 and 3H-AdoMet as shown. Fluorography of the methylated DNA is shown at the top and Western blot of G9a loaded onto the chromatin is shown at the bottom with lane numbers.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
4.
Figure 4.

Figure 4. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

DNMT1 facilitates G9a-mediated methylation of the chromatin. (A) Concurrent DNMT1 and G9a loading onto the chromatin during S phase. HeLa cells were synchronized by thymidine and aphidicolin and released into regular medium. Time points in hours after the release is indicated at the top with antibody used for Western analysis indicated at the bottom. Chromatin fractions were used in the blot as indicated. (B) Analysis of DNMT1 and G9a recruitment on PCNA during early S phase. Time points in hours after the release is indicated at the top with antibody used for Western analysis at the bottom. Loading for Western analysis is normalized for PCNA to determine DNMT1 and G9a recruitment. (C) DNMT1 knockdown impairs G9a loading and H3K9 methylation. Western blot analysis of the total cell extract (left) and chromatin (right) are shown. The antibodies used are indicated at the bottom and the siRNA-mediated knockdown of DNMT1 (DNMT1 KD) and G9a (G9a KD) are indicated at the top. Control knockdown (Control KD) was performed with siLitmus. (D) Localization profile of G9a and BrdU in DNMT1 knockdown COS-7 cells. BrdU is stained red and GFP-G9a is visualized as green. A portion of knockdown cells was used for Western blot analysis to determine the degree of knockdown for DNMT1 as shown in the right panel. Histone H3 was used as control. (E) Same as in D, except that the cells were genetically knockout for DNMT1 in HCT116 background.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
5.
Figure 2.

Figure 2. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Colocalization of G9a and DNMT1 at the replication foci along with BrdU and H3K9me2 localization with either DNMT1 or G9a. Fusion proteins are shown at the top of the panels along with immunocytochemical detection of BrdU or H3K9me2 as indicated. Panels are designated A–H. In the left-hand corner of B–H, the identity of the fusion proteins and the antibody used for immunocytochemistry is noted. (Nu) nucleus stained by hoechst staining; (BrdU) bromodeoxyuridine; (H3K9me2) dimethylated histone H3 Lys 9. The DNMT1 and G9a fusion constructs used are either fused with DsRed (DsRed-DNMT1) or GFP (GFP-DNMT1). A fusion protein containing either a GFP or DsRed partner along with a portion of G9a or DNMT1 as indicated in bracket is used. (A) G9a and DNMT1 colocalization during cell cycle. On the left side of the panel, cytochemical observation after synchronization in hours and percent of cells where DNMT1 and G9a fusions are colocalized are given. For each stage (at 0 time [panel I], between 2 and 4 h [panel II], between 8 and 12 h [panel III], and after 15 h [panel IV] post-aphidicolin treatment), 20–50 cells were counted. (B) DNMT1 (1–446) and G9a. (C) DNMT1 (1–502) and G9a (1–598). (D) DNMT1 (1–502) and G9a (1–464). (E) G9a and BrdU. (F) DNMT1 and BrdU. (G) H3K9me2 and G9a. (H) DNMT1 and H3K9me2. Amino acids numbers are in parentheses.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
6.
Figure 7.

Figure 7. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Model for chromatin methylation by the DNMT1/G9a complex. (A) During DNA replication, the PCNA–DNMT1 complex recruits G9a. As the replication fork moves, concurrent events of maintenance DNA methylation of hemimethylated DNA takes place along with G9a-mediated H3K9 dimethylation. Unmethylated and methylated CpGs are shown as open and closed lollypops. Cofactor AdoMet is used as a methyl donor. (B) Post-replicative chromatin methylation involves DNMT1–G9a and DNMT1–SUV39H1 complexes. SUV39H1 can bind to H3K9me2 (blue-filled stars) using its chromodomain and catalyzes H3K9 trimethylation. DNMT1 can methylate unmethylated CpG sites. G9a–DNMT1 complexes can bind to H3K9me2 (an intermediate product) with a lower affinity for H3K9 trimethylation reaction (H3K9me3, filled stars). The presence of G9a in a DNMT1 complex will activate DNMT1 catalysis for additional de novo methylation (methylation spreading) as seen in silenced genes in cancer cells. (C) Methylation-independent gene repression and heterochromatic localization of SUV39H1 and DNMT1. Following post-replicative chromatin methylation, the heterochromatin components of the nucleus are enriched with H3K9me3, which serves as a platform for HP1 binding. HP1 recruits SUV39H1 and DNMT1 for repressor complex formation. DNMT1 is shown to recruit other repressor components such as HDAC1. However, the methylated chromatin may get demethylated to feed into the methylation cycle.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.
7.
Figure 1.

Figure 1. From: Direct interaction between DNMT1and G9a coordinates DNA and histone methylation during replication.

Interaction between DNMT1 and G9a. (A) Coimmunoprecipitation of DNMT1 and G9a in cell extracts from COS-7 cells transfected with GFP-G9a and 6xHis-DNMT1 constructs. The antibodies used for immunoprecipitation are indicated at the top and the molecular weight marker at the left. Each blot was probed with antibody as indicated. (B) Coimmunoprecipitation of endogenous DNMT1 and G9a in human HCT116 cells. The input nuclear extracts and immunoprecipitated antibody used are shown. Antibodies used for Western blot are indicated below each panel. (C) Chromatographic separation of DNMT1 and G9a complexes. Elution profile of the gel filtration standard proteins in kilodaltons is indicated at the top and antibodies used for detection of enzymes in various fractions are shown at the bottom. All three blots were against the same elution fractions. (D) Coimmunoprecipitation of endogenous DNMT1 and G9a in human Jurkat cells. The input (8% for DNMT1 and 50% for G9a) and antibodies used for immunoprecipitation are shown at the top. Antibodies used for Western blot are indicated below each panel. The unbound (U) and bound (B) G9a and DNMT1 are shown. Typically, 1 μg of antibodies was used for IP. Note that all G9a were immunoprecipitated by anti-G9a, as the unbound fraction did not contain any detectable amount of G9a. (E) Mapping of the DNMT1-binding region on G9a using GST fusions of G9a fragments. Various domains of G9a are indicated above the panels along with a schematic presentation of the GST fusion constructs. Ponceau-stained transferred proteins from GST pull-down experiments are shown along with the Western blot using anti-DNMT1. Positions of the fusion proteins are marked with asterisks. Amino acids in the fusion proteins are indicated in parenthesis at the top of the blot. (F) Schematic diagram depicting GST fusion constructs using various regions of DNMT1 along with amino acid numbers. Ponceau-stained transferred proteins from GST pull-down experiments are shown along with the Western blot of it with anti-G9a.

Pierre-Olivier Estève, et al. Genes Dev. 2006 November 15;20(22):3089-3103.

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