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. 2011 Aug 5;286(31):27176-82.
doi: 10.1074/jbc.C111.229567. Epub 2011 Jun 12.

Synthetic Reversal of Epigenetic Silencing

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Free PMC article

Synthetic Reversal of Epigenetic Silencing

Karmella A Haynes et al. J Biol Chem. .
Free PMC article

Abstract

Controlling cell fate-determining gene expression is key to stem cell differentiation, tissue regeneration, and cancer therapy. To date, custom-built transcription factors recognize the information encoded in specific DNA sequences. Chromatin proteins undergo covalent modifications and form complexes that encode a second layer of information that determines proximal gene activity. Here, we employ a novel gene-targeting approach that exploits a specific chromatin modification to reactivate silenced loci in human cells. We used the human Polycomb chromatin protein and homologues from other species to construct modular synthetic transcription factors, called Pc-TFs, that recognize the repressive trimethyl-histone H3 lysine 27 (H3K27me3) signal and switch silenced genes to an active state. Pc-TF expression in U2OS osteosarcoma cells leads to increased transcription of the senescence locus CDKN2A (p16) and other loci in a chromodomain- and activation module-dependent manner, a switch to a senescence phenotype, and reduced cell proliferation. These results indicate that silenced developmental regulators can be reactivated by a synthetic transcription factor that interacts with chromatin rather than DNA, resulting in an altered cell state. As such, our work extends the flexibility of transcription factor engineering and is the first example of chromatin-mediated synthetic transcription factor targeting.

Figures

FIGURE 1.
FIGURE 1.
Using the Polycomb system to engineer a chromatin-targeted transcription factor. A, model of H3K27 methylation by EZH1/2 of Polycomb repressive complex 2 (PRC2) (3, 6). B, gene repression by Polycomb repressive complex 1 (PRC1) (3, 6). Trimethylation of H3K27 acts as a docking site for the PRC1 complex. C, gene activation by Pc-TF. A conserved 62 amino acid Polycomb chromodomain from CBX8 of the PRC1 complex, fused to a transcription activation domain, binds H3K27me3. As a result, the silencing mark is translated into gene activation. A, EED, embryonic ectoderm development; EZH, Enhancer of zeste homologue; SU(Z)12, Suppressor of zeste 12; RBAP, retinoblastoma binding protein; BMI1, Polycomb ring finger oncogene; PCGF and MEL18, Polycomb group ring finger proteins; PHC, polyhomeotic homolog; RING1, Ring finger protein 1.
FIGURE 2.
FIGURE 2.
Pc-TF-mediated activation of a Polycomb-repressed reporter gene in HEK293 cells. A, fusion protein gene constructs. A ClustalW alignments of the Polycomb chromodomains from human CBX8 (hPCD), zebrafish CBX2 (fshPCD), and Drosophila Pc (flyPCD) is shown. Boxes mark conserved aromatic residues within the H3K27me3-binding pocket (19, 20). Pc-TF fusion proteins (hPCD-TF, fshPCD-TF, and flyPCD-TF) contain a red fluorescent mCh protein, VP64 activation domain, SV40 nuclear localization signal (NLS), and Myc epitope tag. VP64 deletions (hPCD-RFP, fshPCD-RFP, and flyPCD-RFP) and a PCD deletion (TF) serve as negative controls for Pc-TF activity assays. B, nuclear localization of fusion proteins in transiently transfected HEK293 cells. Dashed lines mark the periphery of representative mCherry-positive cells. Scale bar = 20 μm. C, hPCD-TF-mediated GAL4TK-Luc (luciferase) reporter gene activation requires prior exposure to Polycomb silencing. The schematic illustration (left) shows the predicted behavior of the synthetic Pc-TF in the absence or presence of histone methylation, controlled by doxycycline-induced expression of Gal4-EED. Pc-TF fusion proteins and deletion controls were expressed in cells before and after dox treatment. Error bars represent S.D., n = 3 measurements. A one-tailed t test was used to compare the values under the horizontal bracket (*, p < 0.01). TK, thymidine kinase minimal promoter; EED, embryonic ectoderm development.
FIGURE 3.
FIGURE 3.
Endogenous genes targeted by synthetic Pc-TFs in U2OS cells. A, expression of Pc-TFs and deletion variants in U2OS cells. Dotted lines mark the periphery of representative mCherry-positive cells. Scale bar = 20 μm. B, reverse transcription PCR of cDNA synthesized from total RNA of untreated or dox-treated cells. Relative expression levels are quantitative PCR values normalized to the untreated parental line (P.L., −dox). Error bars represent S.E., n = 2 RNA samples. C, ChIP analysis of Pc-TF target loci. IP DNA was amplified using primers 1–16 (supplemental Table S1). Probes n1 and n2, located at the constitutively active GAPDH promoter, are negative controls. Mean enrichments of IP DNA are shown as percentages of input minus background (nonspecific mock-IP DNA). Error bars represent S.D., n = 3 quantitative PCR reactions. A one-tailed t test was used to identify Pc-TF enrichments that were significantly higher (*, p < 0.01) than a randomly cross-linked protein that has no chromatin-binding domain (TF).
FIGURE 4.
FIGURE 4.
Senescence-associated phenotypes in Pc-TF-expressing U2OS cells. A, histograms of senescence-associated β-gal activity detected by C12FDG staining and flow cytometry (24). Rotenone treatment of the parental cell line (P.L.) was used as a positive control. B, average differences in median fluorescence. Error bars represent S.D., n = 2 independent induction trials. C, proliferation of Pc-TF-expressing cells (hPCD-TF, +dox) as compared with untreated cells and the parental line.

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