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. 2017:2:1.
doi: 10.1038/s41525-016-0002-3. Epub 2017 Jan 9.

Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

David B Nyer  1 , Rene M Daer  1 , Daniel Vargas  1 , Caroline Hom  1 , Karmella A Haynes  1
Affiliations

Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

David B Nyer et al. NPJ Genom Med. 2017.

Erratum in

Abstract

Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the "Polycomb-based transcription factor" (PcTF) that reads histone modifications through a protein-protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

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Conflict of interest statement

COMPETING INTERESTS The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
PcTF expression stimulates upregulation of known targets of Polycomb in transiently transfected cells. a Map of the PcTF-expressing plasmid (top). The natural PRC1 complex mediates gene silencing (middle). PcTF expression leads to accumulation of PcTF at H3K27me3 and gene activation (bottom). b Transiently-transfected U-2 OS, SK-N-SH, and K562 cells were visualized via the mCherry red fluorescent protein (RFP) tag. qRT-PCR was used to determine mRNA levels of fusion protein transcripts (PcTF or ΔTF) and a panel of 14 target genes at 24, 48, and 72 h post-transfection. RFP signal was not detected in K562 after 48 h, therefore later time points were omitted for K562 in this assay and other experiments. The heat map shows scaled, average log2 fold change ratios for GAPDH-normalized expression in plasmid-transfected cells compared to cells mock-transfected with the vehicle (Lipofectamine LTX) only. Standard deviations are shown in Fig. S1
Fig. 2
Fig. 2
Genome-wide analysis of gene transcription and H3K27me3 at promoter regions in PcTF-expressing cells. a The scatter plot compares RNA-seq signals (FPKM log10) of 23,245 genes from cells that were mock transfected (Control) vs. log2 fold change of PcTF-expressing cells 96 h post-transfection for U-2 OS and SK-N-SH and 48 h for K562. Housekeeping genes GAPDH, ACTB, and CHMP2A are negative controls. b The Venn diagram shows unique and common sets of genes that became up-regulated at least twofold in the three cell types. Overall, 194 commonly up-regulated genes and 50 commonly downregulated genes are highlighted in the scatter plots. The PcTF-homologous CBX8 gene was used as a proxy for PcTF expression levels. c TSS plots (orange, top) show total H3K27me3 ChIP enrichment values mapped at 200 bp intervals with a step value of 50 bp. In the lower plots, values are stratified by the basal gene expression level in untreated cells (Control FPKM log10). Stratified TSS-plot data were normalized by the gene distribution for each category
Fig. 3
Fig. 3
Polycomb-repressed genes become activated by PcTF in a dose-dependent manner. a The illustration shows doxycycline (dox) induced PcTF expression in U2OS-PcTF cells, leading to accumulation of PcTF at H3K27me3-positive promoters and gene activation. b Flow cytometry analysis of dox-treated cells. Grey bars show average RFP signal (n = 3, Error = standard deviation). RFP signal histograms and RFP-positive cell frequency are shown in Fig. S4. c qRT-PCR analysis of PcTF and eight genes that showed PcTF-specific activation in transiently transfected U-2 OS cells. The heat map shows average (n = 3) log2 fold change ratios for GAPDH-normalized expression in dox-treated cells compared to one of the untreated replicates. R2 = Pearson correlation coefficient of average log2 fold change values for target genes vs. PcTF
Fig. 4
Fig. 4
ChIP-PCR and ChIP-seq analysis of PcTF and H3K27me3 distribution in U2OS-PcTF cells. a The map shows the location of primer pairs and amplicons (Table S3) used to analyze DNA from IP-enriched chromatin. Heat maps show averages of triplicate qPCR reactions from duplicate (PolII, H3K4me3) or triplicate (Myc, H3K27me3) immunoprecipitations. Standard deviations are shown in the bar graphs in Fig. S6. b The Venn diagram compares genes that have PcTF- and H3K27me3-marked TSS regions (−5 to +5 kb). TSS-centered plots show the total ChIP hotspot signal value within a sliding window of 200 bp (step size = 50 bp)
Fig. 5
Fig. 5
Analysis of genome-wide regulation in U2OS-PcTF cell and H3K27me3 localization. a The scatter plot compares RNA-seq signals (FPKM log10) of dox-induced ΔTF cells vs. the log2 fold change in expression compared to U2OS-PcTF cells (same color scheme as in Fig. 2a). Known Polycomb-regulated genes and control genes from Figs. 1 and 2 are highlighted for comparison with previous experiments. The volcano plot shows statistical significance vs. fold change for triplicate U2OS-PcTF samples. b TSS-centered plots show the total ChIP hotspot signal value (window = 200 bp, step size = 50 bp) stratified by log2 fold-change expression. c H3K27me3 ChIP signal distances from the TSS were compared for genes of lengths ≥2 kb in the subset of 316 genes where the 10 kb region is co-occupied by PcTF and H3K27me3. Genes were grouped by fold-change expression (as in b). Box plots show median values (solid vertical line), 25th (left box) and 75th (right box) percentiles, and minimum (left whisker) and maximum values (right whisker). TSS maps drawn to scale show the midpoints of ChIP signals for H3K27me3 (orange circle) and PcTF (red square) relative to the TSS. Genes are sorted from highest to lowest log2 fold-change value
Fig. 6
Fig. 6
Distances of nearest chromatin states to TSS’s. Genes are stratified by fold-change in expression level after PcTF expression in transiently transfected cells. Numbers of regions nearest the TSS of genes in each category are shown in tables next to each chart. The coordinates of poised promoters, repressed regions, and 11 other states (not shown) were determined using chromatin state classes from the ENCODE project (UCSC Browser HMM track for K562 cells). U2 = U-2 OS, SK = SK-N-SH, K5 = K562
Fig. 7
Fig. 7
Model for PcTF-mediated gene activation.,
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References

    1. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:1074–80. doi: 10.1126/science.1063127. - DOI - PubMed
    1. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31:27–36. doi: 10.1093/carcin/bgp220. - DOI - PMC - PubMed
    1. Venugopal B, Evans TR. Developing histone deacetylase inhibitors as anti-cancer therapeutics. Curr. Med. Chem. 2011;18:1658–71. doi: 10.2174/092986711795471284. - DOI - PubMed
    1. Schotta G, Ebert A, Dorn R, Reuter G. Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila. Semin. Cell. Dev. Biol. 2003;14:67–75. doi: 10.1016/S1084-9521(02)00138-6. - DOI - PubMed
    1. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes. Dev. 2006;20:1123–36. doi: 10.1101/gad.381706. - DOI - PMC - PubMed