Summary of the EMSA and MST results.

<p>The respective MST binding curves are plotted on the left, the fluorescence channel depicting the EMSA of R-loop substrate is shown in the middle panel and the merge of the EMSA fluorescence channels revealing the substrate and the controls is shown on the right. The concentration of S9.6 antibody used for the individual experiments is shown on top of the gel. The nucleic acid-antibody complexes are indicated by a triangle and control R-loops (ctrl1 and ctrl2) are indicated on the right side of the gel. <b>(A)</b> Analysis of the R-loop 23GC52. The double stranded DNA D23GC17L served as negative control (ctrl1, red). <b>(B)</b> Analysis of the R-loop 15GC0U. The R-loop 23GC52L served as positive control (ctrl, red). <b>(C)</b> Analysis of the R-loop 15GC0A. The double stranded DNA D23GC17L (ctrl1, red) served as negative control and the R-loop 23GC52L (ctrl2, blue) served as positive control. <b>(D)</b> Analysis of the R-loop 29GC21. The R-loop 15GC0A (ctrl, red) served as positive control. <b>(E)</b> Analysis of the R-loop 16GC80. The double stranded DNA D23GC17L (ctrl, red) served as negative control. <b>(F)</b> Analysis of the R-loop 22GC90. The double stranded DNA D23GC17L (ctrl, red) served as negative control. <b>(G)</b> Analysis of the R-loop 22GC10. The double stranded DNA D23GC17L (ctrl, red) served as negative control.</p

Overview to the experimental results obtained by this study and comparable studies.

<p>The name of the molecules, their sequence and their binding affinities towards the S9.6 antibody determined by microscale thermophoresis assays and EMSA are given.</p

Analysis of R-loop-antibody-interaction by electromobility shift assays.

<p>To directly compare the binding properties of different R-loops and to provide internal controls for the assay we use three different fluorescent labels. Oligonucleotides are either labelled with Cy5, Cy3 or FAM dyes, mixed in stoichiometric amounts and incubated with increasing concentrations of the S9.6 antibody. The channels of the fluorescence scans are given individually and as a merge (bottom-left panel). The R-loops and the DNA control used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178875#pone.0178875.g001" target="_blank">Fig 1</a> were tested in EMSA. The mixture of substrates was incubated for 30 min with the increasing concentrations of S9.6 antibody (4nM to 1066nM) as indicated. Samples were separated on 8% native polyacrylamide gels. Lane 23 (47) reveals the migration behaviour of the single-stranded DNA (O: Oligonucleotide) for the R-loop substrate that was prepared from two individual molecules (22GC75). The triangle marks nucleic acid-antibody complexes. The positions of DNA and R-loop substrates is indicated on the right of the gel (D: DNA strand; R: RNA strand).</p

Determination of R-loop-antibody interactions by MST.

<p><b>(A)</b> The Technical setup of the MST technology is shown. The optics focuses in the center of the glass capillaries, thereby detecting the fluorescence signal of the labelled molecule. An IR-laser is used to establish a temperature gradient in the observation window of the optical system. Changes in fluorescence intensity are used to monitor thermophoretic movement of the molecules in solution. The concentration of the R-loop sequence is kept constant in our assays and the concentration of the antibody was varied. <b>(B)</b> A single MST time trace, showing the changes in fluorescence due to the movement molecules in a temperature gradient. After an initial cold phase (5 sec, laser off), the laser is switched on and instantly establishes the temperature gradient. After the T-Jump phase, in which the fluorescent dye decreases its signal yield due to heat induction, the thermophoretic movement starts. After 30 sec the IR-laser is turned off and the molecules diffuse back. <b>(C)</b> Interpretation of the results of a typical MST experiment. The MST time traces of 16 capillaries containing the same concentration of fluorescently labelled R-loop and an increasing concentration of the unlabelled S9.6 antibody used in our study are recorded and plotted in one graph (left panel). The normalized fluorescence of the MST traces is plotted against the concentration of the ligand (right panel). The data points are fitted to obtain the binding affinity. <b>(D)</b> MST data analysis of the double stranded DNA oligonucleotide D23GC17L, serving as a no binding control. The top-left panel shows the capillary scan to monitor potential sticking effects and absolute fluorescence signals. The overlay of the 16 capillary scan reveals a homogeneous curve shape, indicating no sticking (top-right). The bottom-left panel shows an overlay of 16 the recorded, normalized thermophoresis curves. As the DNA is not expected to bind the curves do perfectly overlap over the antibody concentration range of 640nM to 20pM. The bottom-right plot shows the normalized fluorescence Fnorm (‰) from T-Jump and Thermophoresis vs. the concentration of antibody. STDEV derives from two repeats. The signal does not significantly change, indicating no binding. <b>(E)</b> MST data analysis of the oligonucleotide 23GC52L, forming an R-loop. Two individual experiments performed at different MST power conditions (20% and 40%), creating a temperature gradient of either about 1.5°C or 3°C, are plotted (left panel). The right plot shows fraction bound calculation and the corresponding K_d fit. <b>(F)</b> MST data analysis of the oligonucleotide 22GC75, forming an R-loop. One set of thermophoresis curves of three independent experiments is plotted (left panel). The calculated fraction bound of two experiments is plotted, showing that full binding is not achieved. The binding affinity is estimated from the binding curves and given.</p

DNA binding properties of Dnmt1.

<p><b>(A)</b> DNA fragments of different length (size range from 15 to 250 bp) were incubated with increasing concentrations of Dnmt1 (lanes 2–5). DNA and nucleoprotein complexes were separated on native polyacrylamide gels and stained with ethidium bromide. The asterisk indicates the positions of the Dnmt1-DNA complexes. <b>(B)</b> Quantification of the Dnmt1 binding assay shown in <b>(C)</b>, using an equimolar mixture of fluorescently labeled DNA fragments from 15 to 60 bp in length. The remaining free DNA was quantified and plotted. <b>(C)</b> Competitive electromobility shift assay with a mixture of differently sized fluorescently labeled double stranded (ds) oligonucleotides (4 pmol each). The DNA fragments (15–60 bp; 30 bp: lane 2–7, 45 bp: lane 8–14; 15 bp and 60 bp: lane 15–21) were incubated with increasing concentrations of Dnmt1 (0.1 μM- 0.5 μM, and complex formation was analyzed on a 15% native polyacrylamide gel.</p

Nucleosomes and pRNA compete for the binding of NoRC.

<p>(<b>A</b>) ATPase assay. NoRC was incubated with the indicated pRNAs and radioactive ATP as a tracer. Hydrolysed phosphate was separated via thin layer chromatography and analysed on a PhosphoImager. The quantification of three independent reactions is plotted. Error bars show the standard deviations. (<b>B</b>) Competitive binding assays using NoRC, pRNA and nucleosomes. Nucleosomes assembled on the rDNA promoter (−190 to +90) (lane 1) were incubated with NoRC (lanes 2 to 12), resulting in quantitative complex formation (lanes 2 and 8). These complexes were incubated with increasing concentrations of pRNA as indicated and analysed by EMSA. The nucleosome occupying the position −120/+27 is indicated. Lanes 13 to 24 shows the experiment, but performed with Snf2H. (<b>C</b>) Model describing the putative roles of NoRC and pRNA in rRNA gene silencing.</p

Dnmt1 requires symmetric linker DNA in order to bind to nucleosomal DNA.

<p><b>(A)</b> DNA fragments containing the nucleosome positioning sequence (NPS) located either in the center or at the border of the DNA molecules were assembled into mono-nucleosomes using the salt dialysis method. The DNA templates are named according to the size and location of the DNA linkers with respect to the NPS. <b>(B, C)</b> Different combinations of nucleosomal substrates (50 nM each) were mixed in a 1:1 ratio (lanes 2 and 6) and incubated with increasing concentrations of Dnmt1 (100 nM– 500 nM; lanes 3–5 and 7–9). Reactions were analyzed by native polyacrylamide gel electrophoresis next to a molecular weight marker (M). <b>(D)</b> Monitoring the Dnmt1 binding affinity to mono-nucleosomes containing asymmetrical and symmetrical DNA linkers (77-NPS-0 and 77-NPS-77). Reactions were analyzed on 6% (left panel) and on 4.5% (right panel) native polyacrylamide gels. The position of the Dnmt1-nucleosome complex is indicated (lanes 7–10). <b>(E)</b> Binding of Dnmt1 does not disrupt the nucleosome. The 77-NPS-77 nucleosome (lane 3) was incubated with Dnmt1 to form the stable nucleosome–Dnmt1 complex (lane 4). This complex was incubated with increasing concentrations of competitor DNA to compete Dnmt1 off the nucleosome (lane 5–7). Reaction products were analyzed on a native polyacrylamide gel. The positions of nucleosomes and Dnmt1-nucleosome complexes are indicated. Lane 2 shows the 6 kb competitor plasmid DNA. <b>(F)</b> (upper part) Summary of the results of the Dnmt1 band shift assays and (lower part) a cartoon showing how Dnmt1 (grey box) could bind on the entry/exit sites of the nucleosome (red: DNA; bluish: histones).</p

Nucleosomes inhibit the Dnmt1 dependent DNA methylation <i>in vitro</i>.

<p><b>(A)</b> Experimental setup of the bisulfite sequencing and the radioactive methyltransferase assay. Black triangles indicate CpG sites and the oval indicates the position of the nucleosome. <b>(B)</b> Analysis of the C91-NPS2-C104 template (342 bp fragment harbouring 27 CpG sites) as free (lane 2) and nucleosomal DNA (lane 3) on a native polyacrylamide gel. <b>(C)</b> Enzymatic activity of Dnmt1 on the NPS2 (no linker DNA in nucleosomal form) and the C91-NPS2-C104 template both as free and nucleosomal DNA analyzed in the radioactive methyltransferase assay using [³H]-SAM (360 nM) as substrate. The incorporation of the [³H]-modified CH₃ group was quantified, indicated as counts per minutes (cpm). <b>(D)</b> Bisulfite analysis of the DNA methylation reaction of Dnmt1, using the C91-NPS2-C104 DNA as free DNA and nucleosomal substrate. The DNA methylation efficiency of Dnmt1 at individual CpG sites of the free (white bars) and mononucleosomal DNA (black bars) is given. The (+) and (-) strands are shown and CpG sites are marked as arrows. The ellipse illustrates the position of the nucleosome. In both assays, the bisulfite sequencing and the radioactive methyltransferase assay, the C91-NPS2-C104 and NPS2 templates were use as as non-methylated substrates.</p

Dnmt1 binds to the DNA linkers at the entry/exit sites of the nucleosome.

<p><b>(A)</b> Experimental setup (left) and quality analysis (right) of the DNaseI protection assay. The 77-NPS-77 template (301 bp long) was fluorescently labeled by PCR using 5’ labeled oligonucleotides (5'FAM and 5'HEX) and was assembled into nucleosomes by salt gradient dialysis. Free DNA (lanes 2, 3 and 5) and nucleosomes (Nuc; lanes 6 and 8) and nucleosome-Dnmt1 complexes (Nuc-D1; lane 9) were partially digested with 0.1U DNaseI (DN; lanes 3, 6 and 9). The reaction was stopped by the addition of 5 mM EDTA to inactivate the DNaseI and subsequently separated on a native polyacrylamide gel. DNaseI treated complexes were extracted from the gel and analyzed by capillary electrophoresis. <b>(B)</b> Comparison of the electropherograms of DNaseI treated free DNA (red line) with nucleosomal DNA (blue line). The position of the nucleosome core is indicated (grey bar). The electropherograms for both directions (5’HEX: on top and 5’FAM: on bottom) are shown. <b>(C)</b> As in <b>(B)</b>, the electropherograms of the DNase I treated nucleosome (blue line) are compared with the Dnmt1-nucleosome complex (red line). The electropherograms for both directions (5’HEX: on top and 5’FAM: on bottom) are shown. The protected regions are highlighted in boxes. RFU: relative fluorescence units.</p

NoRC binds to the entry/exit sites of the nucleosome.

<p>(<b>A</b>) Overview of the experimental approach. (<b>B</b>) Analytical EMSA of the DNase I footprinting reaction. For further analysis of the DNase I digestion pattern the nucleosome and nucleosome/NoRC complexes were isolated from the gel. The arrow indicates the NoRC/nucleosome complexes. (<b>C</b>) DNase I footprinting of DNA and centrally positioned nucleosomes. A 247 bp rDNA promoter fragment (−231 to +16 respective to the start site) was radioactively labelled either at the 5′ or 3′ end. The free DNA (bar) and the centrally positioned nucleosome (gray ellipse) were treated with DNase I and after 10 sec and 30 sec the reactions were stopped with EDTA. Nucleosomes and DNA were resolved by EMSA and the bands were isolated. Purified DNA was subsequently analysed on 7% sequencing gels. A scheme of the central positioned nucleosome is shown on the right. (<b>D</b>) Recombinant NoRC was incubated with a purified nucleosome positioned at the center of the DNA fragment and partially digested with DNase I (10 and 30 sec). The reaction was stopped by the addition of EDTA and the nucleoprotein complexes were separated by native gel electrophoresis. Nucleosomes and NoRC/nucleosome complexes were isolated, DNA purified and analysed on 7% sequencing gels. The nucleosome position (gray ellipse) and the radioactive end-labeling (³²P) are indicated. Changes in the digestion pattern upon NoRC treatment are marked with a gray bar, significant changes are highlighted with stars.</p