Localization of INO80 and histone marks in the upstream regions.

<p>The data from ChIP followed by qPCR for HOXC11(A & B) and PAX7(C & D) are shown. A & C- Line diagram indicates the upstream region analyzed by qPCR of ChIP DNA (thick black line) mapping on chromosome12 and 1 respectively for <i>HOXC11</i> and <i>PAX7</i>, along with the genomic positions; the bent arrow indicates the direction of transcription of the gene, and unfilled box is the hINO80 binding motif present in the upstream region. HC1-HC6 and PX1-8 are the amplicons originating from the upstream region covering 1137bp and 1403bp of HOXC11 and PAX7 respectively using primers listed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159370#pone.0159370.s007" target="_blank">S2B Table</a>). B&D show the enrichment as % Input for each region HC1-HB6(B) and PX1-PX8 (C) of INO80 and the H3K9me3 and K3K27me3 marks as shown in the inset (*p value<0.05 and **p<0.001 in comparison with IgG for each primer set).</p

<i>In vivo</i> interaction of INO80 on predicted gene targets.

<p>(A) A line diagram for the region 2Kb upstream of HOXC11 gene is shown as the example of the region analyzed by ChIP. The filled rectangle denotes the INO80 binding motif and the position of the primers (arrows) are marked. (B) ChIP-PCR results for the indicated genes on the human genome. The interaction of INO80 protein was examined in the upstream sequences of these genes using INO80 antibody in HEK293T cells. IgG antibody was used as the negative control for ChIP experiment. The negative (-ve) and positive (+ve) indicate the PCR controls. Input is 20% of sonicated chromatin. (C) Quantitative PCR for putative INO80 targets following ChIP assay in HEK293T cells. The Y axis shows the enrichment as percentage input observed. NAT2 and PSEN lack the INO80 binding motif. (*p value<0.05, **p value<0.001 Two tailed Student t-test)</p

<i>In vivo</i> localization of INO80 in the upstream regions of its target genes.

<p>The data from ChIP followed by qPCR for HOXB13 (A & B) and HOXD4 (C & D) are shown. A & C- Line diagram indicating the upstream region analysed by qPCR of ChIP DNA as thick black line mapping on chromosome17 and 2 for HoxB13 and HoxD4 respectively along with the genomic positions, the bent arrow indicates the direction of transcription of the gene, unfilled box is the hINO80 binding motif present in the upstream region. HB1-HB5 and HD1-5 are the amplicons originating from the upstream region covering 884bp and 888bp of HoxB13 and HoxD4 respectively using primers listed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159370#pone.0159370.s007" target="_blank">S2B Table</a>). B&D show the enrichment as % Input for each region HB1-HB5 (B) and HD1-HD3(C), (*p value<0.05 and **p<0.001 in comparison with IgG for each primer set).</p

INO80 interaction with SELEX motif.

<p>(A) Interaction of GST-DBINO with DNA. (B) Optimization of molar ratio of oligonucleotide: nuclear extract from HEK293T cells for EMSA.</p

Specificity of interaction of INO80.

<p>(A) Interaction of INO80 binding motif with the nuclear extracts from siRNA-INO80 transfected cells (Sigma-EHU069661) compared to untransfected and control siRNA (sc-37007) transfected cells. (B) The supershift of the INO80 bound oligo following the addition of anti-INO80 antibody. The arrow heads indicate the three retarded oligo nucleotides, while the arrow points to the heavier complex formed after addition of the antibody.</p

Competitive EMSA with the mutant oligos.

<p>(A) List of mutant oligos used in the assays, (B) Results of competitive EMSA with mutant oligos at the indicated ratio, (C) Densitometric scan of gel shown in B. The intensity value of the band (shown by arrow) in the lanes shown in (B), normalized to band at the same position in lane 2, are plotted on the Y-axis. Three different ratios for each competing oligo were used as indicated in B and the mutant sequence used is mentioned below the histogram.</p

MEME-based prediction of INO80 binding motif.

<p>(A) Snapshot of MEME output for the sequences enriched after DBINO interaction, (B) Position weight matrix of the putative INO80 motif generated by MEME.</p

Interaction of INO80 binding motif with nuclear extract from HEK 293T cells.

<p>Results of EMSA with Cy3 labeled specific oligo at ratio of 1:5 in all the experiments. The unlabeled competing oligos both specific(A) and non-specific(B) were varied at different molar ratios as indicated at the top of the lanes. The arrow heads indicate the three retarded oligo nucleotides.</p

A model for interaction of INO80 with its target sites.

<p>The binding motif for YY1 and INO80 are indicated as rectangular boxes; +1 and the arrow indicate the transcription start site and the direction of transcription, A-the known hINO80-YY1 complex involved in chromatin remodeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159370#pone.0159370.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159370#pone.0159370.ref036" target="_blank">36</a>]. B- represents four possible scenario for the interaction of INO80 in the upstream region of the genes that it regulates; B1-is a complex recruited by YY1 in which INO80 could be a partner; B2 & B2*- represent genomic regions where both YY1 and INO80 binding motif is present in close vicinity and the recruitment may be through either of them and two different complexes interacting at the same genomic region is possible; B3- represents a region where only INO80 binding motif is present.</p

Effect of INO80 binding on expression of luciferase reporter gene in HEK293T cell line.

<p>(A) Diagrammatic representation of the constructs used in the luciferase reporter assays. The constructs are; pGL3 promoter vector without the INO80 binding site, pGL3INO80 BS-Up (BS-Up) where the INO80 binding motif is cloned upstream of the promoter of luciferase reporter gene and pGL3INO80 BS-Dn (BS-Dn) where binding motif is cloned down-stream of the poly(A) signal. (B) The effect of INO80 binding on the expression of reporter gene under normal and knock-down of hINO80 condition in HEK293T cells. The reporter expression is indicated as fold change with reference to expression from pGL3 vector. The firefly luciferase counts were normalized with renilla luciferase counts. (C) The effect of knock-down of PRC members on reporter expression. The constructs pGL3, pGL3 BS-up and pGL3 BS-Dn were transfected in cells treated with siRNA-YY1, siRNA-EED and siRNA-SUZ12. (D) The effect of the mutant oligos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159370#pone.0159370.g005" target="_blank">Fig 5A</a>) on reporter expression. The error bars represents the standard deviation (*p value<0.05 and **p<0.001).</p