17 research outputs found
Genome context of the EpCAM gene (indicated by a blue bar) on chromosome 2 p21.
<p>The gene is shown in blue, its CpG island in green and the amplicon in black. The amplicon sequence is given below, the ZF binding site sequence is shaded in red. This picture was generated using University of California Santa Cruz genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703-Kent1" target="_blank">[66]</a>.</p
Absence of off-target methylation in SKOV3 cells analyzed by bisulfite sequencing.
<p>Methylation of four non-target genes was analyzed (KIAA0179, DSCR3, Sumo3 and WRB). Data presentation is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone-0087703-g003" target="_blank">Fig. 3</a>. The sequences of the regions analyzed here are given in the Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087703#pone.0087703.s001" target="_blank">Information S1</a>.</p
Mapping the tRNA Binding Site on the Surface of Human DNMT2 Methyltransferase
The DNMT2 enzyme methylates tRNA-Asp at position C38.
Because there
is no tRNA–Dnmt2 cocrystal structure available, we have mapped
the tRNA binding site of DNMT2 by systematically mutating surface-exposed
lysine and arginine residues to alanine and studying the tRNA methylation
activity and binding of the corresponding variants. After mutating
20 lysine and arginine residues, we identified eight of them that
caused large (>4-fold) decreases in catalytic activity. These residues
cluster within and next to a surface cleft in the protein, which is
large enough to accommodate the tRNA anticodon loop and stem. This
cleft is located next to the binding pocket for the cofactor <i>S</i>-adenosyl-l-methionine, and the catalytic residues
of DNMT2 are positioned at its walls or bottom. Many of the variants
with strongly reduced catalytic activity showed only a weak loss of
tRNA binding or even bound better to tRNA than wild-type DNMT2, which
suggests that the enzyme induces some conformational changes in the
tRNA in the transition state of the methyl group transfer reaction.
Manual placement of tRNA into the structure suggests that DNMT2 mainly
interacts with the anticodon stem and loop
Examples of the results of the DNA methylation analysis of the EpCAM gene promoter in SKOV3 cells.
<p>The following abbreviations were used: SKOV3 cells, untreated cells; ZF, SKOV3 cells transfected with Zinc finger construct, ZF-Dnmt3aCD, cells transfected with the Zinc finger Dnmt3a catalytic domain construct; VEC cntrl, cells transfected with empty vector; Dnmt3aCD, cells transfected with a Dnmt3aCD construct without Zinc finger; CD1, stable cell line expressing ZF-Dnmt3aCD 1; CD2, stable cell line expressing ZF-Dnmt3aCD 2. The horizontal rows indicate the CpGs in the amplicon analyzed and the vertical rows represent individual clones that were sequenced. The blue and red colors represent unmethylated CpG and methylated CpG, respectively, for white colored sites, the methylation state is unknown due to technical reasons.</p
Principle of targeted DNA methylation and gene silencing using Zinc fingers (ZF) fused to the catalytic domain of the DNA methyltransferase Dnmt3a (Dnmt3aCD).
<p>The blue bar represents the ZF binding site, unfilled lollipops represent unmethylated CpGs and filled lollipops represent methylated CpGs.</p
Down regulation of EpCAM expression inhibits the proliferation of SKOV3 cells.
<p>A) Results of CCK8 cell proliferation assays conducted with the CD1 and CD2 stable cell lines and SKOV3 cells as reference. The results plotted are from four independent experiments and the error bars indicate the standard error of the mean. B) Viable cell counting performed by Trypan blue staining. The graph represents the data from two independent experiments and the error bars indicate the standard error of the mean.</p
MOESM1 of Application of recombinant TAF3 PHD domain instead of anti-H3K4me3 antibody
Additional file 1. Supplementary figures and table
Analysis of EpCAM gene expression after targeted promoter methylation in stable cell lines.
<p>A) Example of the RT qPCR analysis of EpCAM (left) and beta actin (right) mRNA amounts in SKOV3 cells and in two independent cell lines which stably express the ZF-Dnmt3a construct (CD1 and CD2). B) Quantification of the RT qPCR analysis of EpCAM expression as shown in panel A. We carried out two independent RNA preparations each analyzed in three technical repeats. The image shows the average of both results, the error bars indicate the standard error. C) Example of the Western blot analysis of EpCAM expression in SKOV3 cells and the CD1 and CD2 stable cell lines (upper panel). Beta actin was used as loading control (lower panel). The EpCAM and beta actin bands are marked with arrows. D) Quantification of the Western Blot analysis of EpCAM expression as shown in panel C. The figure shows an average of two independent experiments, the error bars indicate the standard deviation of the data.</p
Mapping the tRNA Binding Site on the Surface of Human DNMT2 Methyltransferase
The DNMT2 enzyme methylates tRNA-Asp at position C38.
Because there
is no tRNA–Dnmt2 cocrystal structure available, we have mapped
the tRNA binding site of DNMT2 by systematically mutating surface-exposed
lysine and arginine residues to alanine and studying the tRNA methylation
activity and binding of the corresponding variants. After mutating
20 lysine and arginine residues, we identified eight of them that
caused large (>4-fold) decreases in catalytic activity. These residues
cluster within and next to a surface cleft in the protein, which is
large enough to accommodate the tRNA anticodon loop and stem. This
cleft is located next to the binding pocket for the cofactor <i>S</i>-adenosyl-l-methionine, and the catalytic residues
of DNMT2 are positioned at its walls or bottom. Many of the variants
with strongly reduced catalytic activity showed only a weak loss of
tRNA binding or even bound better to tRNA than wild-type DNMT2, which
suggests that the enzyme induces some conformational changes in the
tRNA in the transition state of the methyl group transfer reaction.
Manual placement of tRNA into the structure suggests that DNMT2 mainly
interacts with the anticodon stem and loop
The SUV39H1 Protein Lysine Methyltransferase Methylates Chromatin Proteins Involved in Heterochromatin Formation and VDJ Recombination
SUV39H1
is an H3K9 methyltransferase involved in the formation
of heterochromatin. We investigated its substrate specificity profile
and show recognition of H3 residues between K4 and G12 with highly
specific readout of R8. The specificity profile of SUV39H1 is distinct
from its paralog SUV39H2, indicating that they can have different
additional substrates. Using the specificity profile, several novel
SUV39H1 candidate substrates were identified. We observed methylation
of 19 novel substrates at the peptide level and for six of them at
the protein level. Methylation of RAG2, SET8, and DOT1L was confirmed
in cells, which all have important roles in chromatin regulation.
Methylation of SET8 allosterically stimulates its H4K20 monomethylation
activity connecting SUV39H1 to the generation of increased H4K20me3
levels, another heterochromatic modification. Methylation of RAG2
alters its subnuclear localization, indicating that SUV39H1 might
regulate VDJ recombination. Taken together, our results indicate that
beyond the generation of H3K9me3, SUV39H1 has additional roles in
chromatin biology by direct stimulation of the establishment of H4K20me3
and the regulation of chromatin binding of RAG2