Journal: BMC Cancer

Article Title: Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells

doi: 10.1186/1471-2407-12-145

Figure Lengend Snippet: Genistein treatment induces hypermethylated Wnt-inhibitory genes via H3K9 acetylation, not CpG demethylation. (A) Wnt Inhibitory genes are methylated in prostate cancer patient samples. MSP of Wnt inhibitory genes was performed on genomic DNA derived from paraffin embedded prostate tissues in 8 prostate cancer samples. SOX7 was strongly methylated while WIF1, SFRP1, DKK3, and APC were partially methylated in multiple patient samples. U = unmethylated, M = methylated. (B) MSP analysis indicates no change in methylation status of APC, WIF1, SFRP1, SOX7, and DKK3 in DU145, ARCAPE, and PC3 cell lines after treatment with 20 μM genistein for 6 days. (C) Whole genome methylation profiling using the 27 K CpG Methylation Arrays was performed on ARCAPE and ARCAPM cells treated with DMSO and 20 μM genistein for 6 days. In addition, 5-aza-deoxy-cytidine was used as a positive control and PREC cells were used as a negative control. No significant changes in methylation were detected with genistein treatment. Unsupervised hierarchical clustering of 4,190 CpG loci with β-value > 0.5 is shown. (D) Bisulfite sequencing of 175 bp of the WIF1 CpG island across 13 CpG sites indicates high methylation in ARCAP-E and ARCAP-M cells with or without genistein treatment, and low methylation in PrEC cells. (E) Anti-acetyl histone H3-Lysine9 chromatin immunoprecipitation (acetyl-H3K9 ChIP) of ARCAP-E cells treated with DMSO control or genistein shows marked increases in acetyl-H3K9 following genistein treatment. (F) Immunoblot of HAT1 protein shows increased HAT1 protein in ARCaP-E and ARCaP-M after genistein treatment. (G) Gene expression with genistein treatment in ARCAPE, and ARCAPM cells. Gene expression of indicated Wnt inhibitory genes after treatment with genistein was determined using QPCR. The data are presented as fold change relative to DMSO control (mean ± SD, triplicate samples from five independent experiments). Significant p-values (p < 0.05) were computed using the student’s t -test with a two-tailed distribution and are indicated with an asterisk (*).

Article Snippet: We thank Emory Winship Cancer Institute Cancer Genomics Shared Resource supported by NCI Cancer Center Support Grant P30CA138292 for performing Illumina gene expression arrays, Illumina CpG methylation arrays, and next generation bisulfite sequencing.

Techniques: Methylation, Derivative Assay, CpG Methylation Assay, Positive Control, Negative Control, Methylation Sequencing, Chromatin Immunoprecipitation, Control, Western Blot, Gene Expression, Two Tailed Test

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: Identification of active enhancers in quiescent NS cells. ( A ) Heat map representation of the density of ChIP-seq reads for H3K27ac and p300 ±2 kb relative to the midpoint of enriched regions at 16,246 active enhancers in NS cells. This panel represents the merger of data obtained in proliferating and quiescent NS cells. A large fraction of the regions displayed presents active enhancer features only in proliferating NS cells or only in quiescent NS cells, and a smaller fraction presents these features in both cellular states. Intensity of color represents the normalized statistical significance of the signal versus input control sequences. ( B , C ) H3K27ac and p300 ChIP-seq signal and RNA expression level (FPKM) in quiescent (blue) and proliferating (green) NS cells in the vicinity of Id4 and Vash1, two representative genes that are up-regulated in quiescent and proliferating NS cells, respectively. Regions defined as quiescent and proliferating NS cell-specific enhancers are indicated by blue and green rectangles, respectively. ChIP-seq peak height corresponds to SICER P -value for H3K27ac and MACS Q -value for p300. ( D ) Average ChIP-seq signal profile for H3K27ac in quiescent (blue line) and proliferating (green line) NS cells and several other epigenetic marks in proliferating NS cells at regions defined as quiescent ( left ) and proliferating ( right ) NS cell-specific enhancers. Plots are centered on the p300 summit. Quiescent NS cell-specific enhancers show strong signals for the enhancer-associated H3K4me1 mark and weak signals for the open chromatin-associated H3K4me2 and H3K27ac marks in proliferating NS cells, consistent with these regions being marked as enhancers but minimally active in proliferating NS cells. Proliferating NS cell-specific enhancers have strong signals for H3K27ac, H3K4me1, and H3K4me2 but not the other nonenhancer-associated epigenetic modifications. Note the dip in the enrichment profile for H3K27ac, indicative of a localized depletion of nucleosomes characteristic of enhancers ( ; ). ( E ) Box plots of normalized transcript counts (FPKM) for all genes expressed in quiescent NS cells ( left ) and genes associated with quiescent NS cell-specific enhancers ( right ). The latter are expressed at higher levels than the transcriptomic average (Wilcoxon test, P < 2.2 × 10 −16 ). ( F ) Fraction of genes up-regulated in quiescent NS cells whose closest enhancer is quiescent NS cell-specific ( left ), pan-NS cell ( middle ), or proliferating NS cell-specific ( right ). Asterisk denotes significant P -value (Wilcoxon test). See also Supplemental Figure S3.

Article Snippet: DNA libraries were prepared from 10 ng of immunoprecipitated DNA according to the standard Illumina ChIP-seq protocol for quiescent NS cell H3K27ac ChIP, quiescent NS cell p300 ChIP, quiescent NS cell NFI ChIP, quiescent NS cell input DNA, proliferating NS cell H3K27ac ChIP, proliferating NS cell p300 ChIP, and proliferating NS cell input DNA.

Techniques: ChIP-sequencing, Control, RNA Expression

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: NFI TFs bind to the majority of quiescent NS cell enhancers. ( A ) Heat map representation of all enhancers active in quiescent NS cells sorted into quiescent-specific and pan-NS cell enhancers showing ChIP-seq signal for NFI TFs, H3K27ac, and p300. ( B ) Venn diagram showing the large overlap of enhancers in quiescent NS cells with regions of significant NFI TF binding. ( C , D ) Strong correlation of the strength of ChIP-seq signals for p300 and NFI in enhancers ( C ; correlation coefficient = 0.67) and close proximity of their summits ( D ; median intersummit distance = 35 base pairs [bp]), consistent with p300 recruitment by NFI TFs. ( E , F ) Functional annotation of quiescence-specific ( E ) and pan-NS cell ( F ) enhancers bound by NFI TFs by GREAT according to GO biological process. Enhancers bound by a NFI factor (purple) and those that are not significantly bound (green) were examined separately. The X -axis values represent the binomial FDR Q -values; the numbers in parentheses are the number of binomial region hits.

Article Snippet: DNA libraries were prepared from 10 ng of immunoprecipitated DNA according to the standard Illumina ChIP-seq protocol for quiescent NS cell H3K27ac ChIP, quiescent NS cell p300 ChIP, quiescent NS cell NFI ChIP, quiescent NS cell input DNA, proliferating NS cell H3K27ac ChIP, proliferating NS cell p300 ChIP, and proliferating NS cell input DNA.

Techniques: ChIP-sequencing, Binding Assay, Functional Assay

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: NFIX is both required and sufficient to induce aspects of quiescence in NS cell cultures. ( A ) Efficiency of Nfix silencing in NS cells exposed to BMP to induce quiescence at the time of shRNA electroporation analyzed by qPCR 1, 2, and 3 d after shRNA transfection and BMP exposure. A scrambled shRNA was used in the control experiment, and expression of the gene ActB is analyzed for comparison. Note that Nfix transcript levels increase progressively between days 1 and 3 as cells enter quiescence in both control and Nfix knockdown experiments. ( B ) Analysis of proliferation by EdU immunostaining after 4 h of exposure in NS cell cultures following 1, 2, and 3 d of BMP exposure as indicated. Cells are counterstained with DAPI (blue). ( C ) Percentages of EdU-positive NS cells in Nfix shRNA transfected and control cultures. The BMP-induced cell cycle arrest is delayed by Nfix silencing. The progressive reduction in cell proliferation of Nfix shRNA-treated cultures between days 1 and 3 might be due to the progressive increase in Nfix expression during this period (shown in A ). Error bars represent the standard deviation ( n = 3 biological replicates). ( D ) Analysis of proliferation by EdU immunostaining in NS cell cultures transfected 18 h earlier with a Nfix expression construct and GFP or with GFP alone. ( E ) Percentages of EdU-positive cells in NS cell cultures transfected with GFP or GFP and Nfix . Nfix efficiently promotes cell cycle arrest. ( F ) Venn diagram showing the large fraction of genes regulated in quiescent NS cells that are also regulated by Nfix . GO analysis of Nfix-activated genes that are also induced in quiescent NS cells ( G ) and Nfix-repressed genes that are up-regulated in proliferating NS cells ( H ). ( I ) Representative examples of putative NFI direct target genes (associated with a NFI-bound enhancer/promoter and activated by Nfix ) induced in quiescent NS cells and belonging to functionally important GO categories. ( J ) ChIP-seq signal for H3K27ac, p300, and NFI and RNA-seq signal (FPKM) for Svep1 and Bgn , two representative NFI direct target genes up-regulated in quiescent NSCs. Significant NFI binding within enhancer regions is indicated by pale blue rectangles. Peak height corresponds to SICER P -value for H3K27ac and MACs Q -value for p300 and NFI. See also Supplemental Figure S5 and Supplemental Table S2.

Article Snippet: DNA libraries were prepared from 10 ng of immunoprecipitated DNA according to the standard Illumina ChIP-seq protocol for quiescent NS cell H3K27ac ChIP, quiescent NS cell p300 ChIP, quiescent NS cell NFI ChIP, quiescent NS cell input DNA, proliferating NS cell H3K27ac ChIP, proliferating NS cell p300 ChIP, and proliferating NS cell input DNA.

Techniques: shRNA, Electroporation, Transfection, Control, Expressing, Comparison, Knockdown, Immunostaining, Standard Deviation, Construct, ChIP-sequencing, RNA Sequencing, Binding Assay