DNA methylation and gene expression changes globally after 5-Aza-CdR treatment and SRCAP knockdown.

<p>A. Density plots for each sample across all 27,578 CpG sites analyzed. The X-axis represents beta values ranging from 0 (no DNA methylation) to 1 (high DNA methylation). Black colored lines represent samples treated with PBS and red lines are samples treated with 5-Aza-CdR. Solid lines are NC siRNA treated samples and dashed lines are SRCAP siRNA treated samples. B,C. Scatter plot overlayed with histogram and density distribution. Each dot represents an interrogated CpG probes beta value. Colored dots represents density or number of probes as indicated in the adjacent axis as illustrated as a both a histogram and density distribution. Contour lines are drawn to further illustrate the number of probes for a specified region. B. Represents a scatter plot between 5-Aza-CdR vs PBS treatment (control) in NC siRNA treated RKO cells. C. Represents a scatter plot between SRCAP siRNA vs NC siRNA treated cells. D,E . The gene expression log2 fold difference is plotted on the x-axis, and the q-value which accesses significance is plotted on the y-axis (−1* log10 scale). Probes that are identified as significantly different between two groups are colored in red. D. NC 5-Aza-CdR vs NC PBS; E. NC vs SRCAP siRNA F. Scatter plot of all transcript probes assayed on the Illumina Human expression beadchip. Probes identified as differentially expressed and demethylated after 5-Aza-CdR treatment in non-target siRNA treated cells (x-axis) are colored red. Inset shows the distribution (box plots) of the demethylation induced gene reactivation fold changes in non-target siRNA and SRCAP siRNA treated cells. G. One-dimensional hierarchical clustering of the demethylated CpG probes which interrogate the promoters of genes significantly upregulated by 5-Aza-CdR treatment. Each row represents a probe; each column represents a sample. The level of DNA methylation (beta value) for each probe in each sample is represented by using the color scale shown in the legend; white indicates missing data. H. Box plot of DNA methylation levels distribution for genes which were significantly upregulated and demethylated by 5-Aza-CdR.</p

Dynamic nucleosome occupancy changes on symmetrically and asymmetrically methylated DNA duplexes after 5-Aza-CdR treatment.

<p>A. The nucleosome occupancy changes at the <i>MLH1</i> promoter after 5-Aza-CdR treatment were investigated by NOMe-seq assay. The GpC methyltransferase accessibility on each strand is shown. B. Nucleosome depleted regions were detected at the <i>CDKN2A</i> promoter five days after 5-Aza-CdR treatment. C. Nucleosome depleted regions were also detected in the <i>MYOD1</i> promoter five days after 5-Aza-CdR treatment. D,E. The nucleosome occupancy on asymmetrically methylated DNA duplexes was analysis by NOMe-seq after Hpa II digestion at the <i>MLH1</i> and <i>CDKN2A</i> promoters. Green bars presents regions of 250 bp in length, which covers the −1 nucleosome plus 100 bp downstream of that nucleosome.</p

Simplified schematic of 5-Aza-CdR–induced DNA demethylation and chromatin configuration changes.

<p>During DNA replication, 5-Aza-CdR is incorporated into DNA, sequesters DNMTs immediately and this results in demethylation. After a cycle of cell doubling, the majority of DNA molecules are hemimethylated and occupied by nucleosomes. Subsequent DNA replications produce more symmetrically demethylated DNA molecules, possessing NDRs around the TSS. In addition, DNA demethylation results in SRCAP-mediated H2A.Z deposition at gene promoter, which promote gene reactivation.</p

5-Aza-CdR induces dynamic changes in gene expression, DNA methylation, and histone marks.

<p>A, D, G, RT-PCR results showing the expression levels of <i>MLH1</i>, <i>CDKN2A</i> and <i>MYOD1</i> at the indicated days (D) after 1 uM 5-Aza-CdR treatment. The mRNA levels were normalized to <i>GAPDH</i>. Error bars represent the standard deviation of biological triplicates. B, E, H, DNA methylation levels at promoters were measured by Ms-SNuPE. Error bars represent the range between biological duplicates. C, F, I, ChIP results of histone variants and modifications were normalized to histone H3 at the indicated time points after treatment. Arrows indicate TSSs and the upper vertical bars represent CpG sites. Underlying bold horizontal lines indicate the ChIP regions amplified by PCR. Results from three independent biological experiments of H2A.Z and two independent biological experiments of H3K4me3 (acH3K9/14) are shown.</p

DNA methylation influences nucleosome occupancy.

<p>A. Nucleosome occupancy and DNA CpG methylation levels at the <i>MLH1</i> promoter in LD419 cells (upper) and RKO cells (lower) were investigated by NOMe-seq. B. Nucleosome occupancy and DNA CpG methylation levels at the <i>CDKN2A</i> promoter in LD419 cells (upper) and RKO cells (lower) were examined by NOMe-seq. The upper vertical bars represent CpG sites and the lower vertical bars indicate GpC sites. Open and filled circles represent unmethylated and methylated CpG sites respectively. The teal filled circles indicate GpC sites which are methylated and therefore accessible to GpC methyltransferase. Pink areas represent regions which are resistant to GpC methyltransferase and longer than 146 bp.</p

SRCAP knockdown prevents 5-Aza-CdR–induced H2A.Z deposition and nucleosome remodeling.

<p>A, RKO cells were treated with the indicated siRNA for 24 hours followed by treatment of 1 uM 5-Aza-CdR (+) or PBS (−) for another 24 hours. RNA was isolated 72 hours after drug treatment. The gene expression levels were measured by RT-PCR and the data represent the means of biological triplicates. The enrichment of histone marks after the indicated treatment was measured by ChIP and normalized to the Histone H3 level. The data represent biological duplicates. B. NOMe-seq results show the nucleosome occupancy at the <i>MLH1</i>, <i>CDKN2A</i> and <i>MYOD1</i> promoter after the indicated treatment.</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

SNF5 is a key executor of epigenetic regulation in pluripotency and differentiation.

<p>(A and B) Differentiation signals cause recruitment of SNF5 to both OCT4 activated and repressed target genes with distinctive roles (closing or generating NDRs) dependent on cellular context. Changes in exogenous SNF5 levels disrupt the balance between pluripotent and differentiated states. (Refer to Discussion for a detailed explanation).</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

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