Combinatorial treatment shows the greatest efficacy in inhibiting cell growth and limiting colony formation.

<p>A. The expression levels of LSD1 in bladder cancers, T-cell acute lymphoblastic leukemia, colon adenoma and their normal counterparts were obtained from ONCOMINE. Numbers in the parentheses indicate number of samples used to generate box plots. B-D. Population doubling times for control (black), clorgyline treated (yellow), 5-Aza-CdR treated (red) and combinatorial treatment treated (green) in T24, HL60 and HCT116 cells. The corresponding numbers of hours are listed in the table below each graph. E. Quantification of number of colonies produced by colony formation assays in T24 and HCT116 cells after indicated treatments.</p

Combinatorial treatment elicits a synergistic effect in up-regulating genes in HL60 cells.

<p>A. Gene expression log2 difference is plotted on the x-axis, and the –log10 (p-value) is plotted on the y-axis. Probes that are identified as significantly different between two groups are colored in red. B. Venn intersects of the number of genes that are up-regulated upon the indicated treatment. C. Expression status obtained from ONCOMINE of 20 genes, whose expression was synergistically reactivated in HL60 cells, in peripheral blood mononuclear and chronic lymphocytic leukemia.</p

Gene up-regulation induced by combinatorial treatment is due to histone modifications in T24 cells.

<p>A. Density plots for each treatment across all 450,000 CpG sites. The x-axis represents beta values ranging from 0 (not methylated) to 1 (highly methylated). B. Heatmap of CpG probes belonging to genes which are synergistically up-regulated upon combinatorial treatment. The level of DNA methylation for each probe in each sample is represented by using the color scale shown in the legend. C. ChIP results of histone modifications after normalization to input. Error bars represent standard deviation from 3 independent experiments.</p

Clorgyline induces demethylation in HCT116 cells.

<p>A. Venn intersects of the genes that are up-regulated upon the indicated treatment. B. Density plots for each treatment across all 450,000 CpG sites. The x-axis represents beta values ranging from 0 (not methylated) to 1 (highly methylated). C. Venn intersect of the probes that are demethylated by the indicated treatment. The overlap of two circles represents the common probes that are demethylated by both treatments.</p

Combinatorial treatment elicits a synergistic effect in up-regulating genes in T24 cells

<p>. A. Gene expression log2 difference is plotted on the x-axis, and the –log10 (p-value) is plotted on the y-axis. Probes that are identified as significantly different between two groups are colored in red. B. Venn intersects of the number of genes that are up-regulated by the indicated treatment. C. Network enrichment diagram containing genes that are synergistically up-regulated upon combinatorial treatment. Synergistically upregulated genes are marked by red bars. The other symbols used are as seen on the Metacore website.</p

SNF5 is recruited to OCT4-activated and -repressed genes with distinctive chromatin landscape during differentiation.

<p>(A–E) Chromatin from NCCIT cells was immunoprecipitated with anti-OCT4 (A), anti-EZH2 (B), anti-SNF5 (C), anti-BRM (D), anti-BRG1 (E) or anti-H3 antibodies and their binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR. Quantitative PCR data represent the average of three biological experiments (the mean +SEM). A Mann-Whitney test was performed and the increase in recruitment of SNF5, BRM and BRG1 at OCT4 target genes during differentiation as found to be statistically significant with p-values of 0.013 (SNF5), 0.012 (BRM) and 0.029 (BRG1). (F) The protein level of EZH2, SNF5, BRG1, BRM and loading control ACTIN were subsequently analyzed by western blot. The data is representative of three biological experiments.</p

OCT4 target genes show distinctive nucleosome occupancy patterns that underlie the potential for gene expression.

<p>(A) Genome-wide studies were performed in human embryonic stem cells (H1) using ENCODE and GEO data (wgEncodeHudsonalphaMethylSeqRegionsRep1H1hesc for DNA methylation, GSM518373 for OCT4 ChIP-Seq and wgEncodeUwDnaseSeqPeaksRep1H1es for DNaseI). The data comprised 100 bp windows of OCT4 binding regions (29740 sites), DNA methylated regions (43659 sites) and DNaseI hypersensitive regions (123778 sites). (B and E) H1 and NCCIT cells were exposed to 10 uM RA for the indicated days. The expression levels of OCT4, NANOG, PAX6 and NEUROG1 were determined by quantitative PCR (normalized to PCNA). Quantitative PCR data represent the average of three biological experiments (the mean +SEM) (C, D, F and G) Nucleosome occupancy at the <i>PAX6</i> and <i>NEUROG1</i> promoters was analyzed by NOMe-seq during differentiation of H1 and NCCIT cells. Blue circles represent GpC sites of the DNA (unfilled blue circles represent GpC sites which are inaccessible to GpC methyltransferase, teal-filled circles represent cytosines accessible to GpC methyltransferase). Pink bars represent regions of inaccessibility large enough to accommodate a nucleosome (around 150 bp). The data is representative of three biological experiments.</p

Knockdown of SNF5 enhances a stem cell like state and blocks differentiation.

<p>(A) SNF5, OCT4, EZH2, and loading control histone H3 were analyzed by western blot, 72 h post-transfection with SNF5 siRNA in NCCIT cells. (B and C) To get the nucleosome footprint, we have performed at least three biological replicates of NOMe-seq at 72 h post-transfection with SNF5 siRNA in NCCIT and selected ∼10 sequences in an unbiased manner to represented in the figures. (D) Stably infected SNF5 knockdown NCCIT cells were selected for 21 days with antibiotics and SNF5, OCT4, EZH2, and loading control histone H3 were subsequently analyzed by western blot. G401 cells were used for a SNF5 knockdown control. (E) At the same time point, cell morphology micrographs (200X) were taken. The data is representative of three biological experiments. (F) Apoptosis of SNF5 knockdown NCCIT cells after RA treatment was determined by flow cytometric analysis. The X axis indicates Annexin V and the Y axis indicates Propidium iodide (PI). The data are representative of three biological experiments (the mean +SEM).</p

SNF5 controls the balance between pluripotency and differentiation.

<p>(A and B) 2D matrix and heat plots depicting gene expression changes in SNF5 knockdown/SNF5 overexpression and RA 7 d treated NCCIT cells. Axes indicate degree of fold change, from the middle of axis. The numbers indicate the median fold change of genes in each column or row. The intensity of each square represents the number of genes that fall in that square. (C) Fold change of SNF5 target genes (over two fold changes in opposite direction) among previously defined ES signature genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003459#pgen.1003459-BenPorath1" target="_blank">[51]</a>.</p

Overexpression of SNF5 alters SNF5 binding distribution, especially to OCT4 target genes.

<p>(A) Percentage distribution of ChIP-seq binding regions for SNF5 in control and overexpression state. (B) The binding plots show the localization of SNF5 bound sites relative to OCT4 bound sites. SNF5 bound sites (y axis) are displayed within a 5 kb window centered on the OCT4 bound site. Intensity at position 0 indicates that site overlap. (C) Venn diagram showing overlapping of OCT4 and SNF5 (the number of OCT4 only binding genes; 3412, the number of SNF5 only binding genes; 7185, and the number of both binding genes; 1862) bound genes after overexpression of SNF5 based on ChIP-seq data in NCCIT cells.</p