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Results: 4

1.
FIGURE 3.

FIGURE 3. From: The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation.

Subnuclear distribution of wild type EYFP-Dnmt3a and EYFP-Dnmt3a-D329A mutant in NIH3T3 cells. The subnuclear localization patterns were analyzed by laser scanning microscopy and assigned into one of the following categories: localization mainly homogenous, homogenous localization with some large spots, and localization almost exclusively in large spots. The figure shows examples of each type of localization patterns and gives the distribution of occurrence of the different patterns in both experiments in 100 arbitrarily chosen cells.

Arunkumar Dhayalan, et al. J Biol Chem. 2010 August 20;285(34):26114-26120.
2.
FIGURE 2.

FIGURE 2. From: The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation.

Native chromatin and histone binding of the Dnmt3a PWWP domain and its variants. A, GST pulldown assay interaction analysis of the PWWP domain and the D329A variant with mononucleosomes purified from human cells. The bound proteins were separated and immunoblotted with anti-H3K36me3 antibody. B, for specificity analysis, the bound fractions were separated and immunoblotted with anti-H3K36me3 antibody, anti-H3K4me3 antibody, anti-H3K9me3 antibody, and H3K27me3 antibody. C, GST pulldown interaction analysis of PWWP domain and D329A mutant domain with native histone proteins, purified from human cells. The bound fractions were separated and immunoblotted with anti-H3K36me3 antibody. In A–C, all of the bands correspond to histone H3.

Arunkumar Dhayalan, et al. J Biol Chem. 2010 August 20;285(34):26114-26120.
3.
FIGURE 4.

FIGURE 4. From: The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation.

A, methylation of demethylated native nucleosomes by Dnmt3a enzymes. Relative methylation activity of Dnmt3a2, Dnmt3a2-D329A, and Dnmt3a-CD on demethylated native nucleosomes in the absence (dark gray bars) and the presence (light gray bars) of anti-H3K36me3 antibody. The error bars indicate standard deviations derived from at least three independent experiments. B, schematic picture showing how the H3 tail interactions of the Dnmt3a ADD domain (dark grey rectangle) binding to the end of the H3 tail (12) and the PWWP domain (light grey hexagon) binding to Lys36 (black triangle) may anchor the catalytic domain of the enzyme (dark grey oval) to methylate DNA preferentially in linker region (12).

Arunkumar Dhayalan, et al. J Biol Chem. 2010 August 20;285(34):26114-26120.
4.
FIGURE 1.

FIGURE 1. From: The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation.

Peptide binding by the Dnmt3a wild type PWWP domain and its variants. A, interaction of the wild type PWWP domain with histone tail arrays containing different combinations of 107 known and hypothetical modifications at the H3, H4, H2A, H2B, and H1b histone tails. The isolated GST domain does not interact with peptide arrays (data not shown). The spot corresponding to the H3 29–46 K36me3 peptide is indicated with an arrow; some other spots that do not interact with the PWWP domain are labeled with numbers and annotated below the image. B, interaction of wild type PWWP domain, D329A, and E290A variants with modified histone tails. The H3K36me3 and H3K36me2 spots on the different arrays used for this study are highlighted with black and gray arrows, respectively. C, binding of the H3K36me3 peptide to PWWP domain studied by fluorescence depolarization (black diamonds). The line represents a fit of the data to a binary binding equilibrium, which revealed a Kd of 64 μm. The gray squares display changes in fluorescence depolarization observed after adding same volumes of buffer. D, weak competition of H3K36me3 peptide binding to the PWWP domain by the addition of unmethylated H3K36 peptide confirms specific binding.

Arunkumar Dhayalan, et al. J Biol Chem. 2010 August 20;285(34):26114-26120.

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