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, 358 (6370), 1617-1622

Synthetic Transcription Elongation Factors License Transcription Across Repressive Chromatin

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Synthetic Transcription Elongation Factors License Transcription Across Repressive Chromatin

Graham S Erwin et al. Science.

Abstract

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

Figures

Fig. 1.
Fig. 1.
Synthetic transcription elongation factors (Syn-TEFs) selectively activate FXN expression. (A) Cocrystal structures of JQ1 bound to BRD4 (PDB 3MXF) and polyamide bound to nucleosomal DNA (PDB 1M1A). The distance allowing interaction of these complexes is estimated. (B) Linear PA1 and Syn-TEF1 target the DNA sequence 5’-AAGAAGAAG-3’. Hairpin PA2 and Syn-TEF2 target 5’-WTACGTW-3’, where W = A or T. N-methyllmldazole is bolded for clarity. N-methylpyrrole (open circle), N-methylimidazole (filled circle), 3-chlorothlophene (square), and β-alanine (diamond) are represented in a ball and stick format. The structure of JQ1 linked to polyethylene glycol (PEG6) is represented as a blue circle. (C) Relative expression of FXN mRNA in GM15850 (left panel) and GM15851 (right panel) cell lines by quantitative RT-PCR. Results are mean ± SEM (n = 4), normalized to relative expression of FXN in GM15851 cells (see also fig. S4). All treatments are 24 hours with 1 μΜ of the indicated molecule, except DMSO (0.1%) and Syn-TEF1 (0.1, 0.5, or 1 μM). *P < 0.05; **P < 0.01. (D) Immunoblot of FXN and α-Tubulin (TUB) with treated GM15850 (left panel) and GM15851 (right panel) cells. Cells were treated as in (C). (E) Volcano plots of RNA-seq data display the change in global gene expression after 24 hours treatment of GM15850 (left panel) and GM15851 (right panel) cells with 1 μM Syn-TEF1 (n = 4). Values represent the posterior probability of equal expression (PPEE) versus fold-change in expression normalized to DMSO treated samples (n = 4). FXN and c-Myc are labeled red and blue, respectively.
Fig. 2.
Fig. 2.
Syn-TEF1 recruits BRD4 to its target sites and licenses productive Pol II elongation at FXN. All data are from GM15850 cells treated 24 hours with the indicated molecules. Signal traces are in reference-adjusted reads per million reads per base pair (rrpm/bp). (A) Summation of sites (SOS) profile of PA1 and Syn-TEF1 across the FXN locus. (B) BRD4 occupancy at the FXN locus. (C) Occupancy of phosphorylated serine 2 (phospho-Ser2) of the C-terminal domain of RNA Pol II at the FXN locus. (D) Occupancy of RNA Pol II at the 5’ region of FXN. The grey bar identifies the location of the repressive GAA repeats and blue/cyan filled regions highlight unannotated reads but do not have defined quantitative values. (E) Occupancy of FI3K4me3 and FI3K36me3 measured at several locations within the FXN locus. Results are mean (n = 3) ± SEM. (F) A model of the cascade of interactions and reactions initiated by Syn-TEF1 at FXN.
Fig. 3.
Fig. 3.
Syn-TEF1 recruits BRD4 to its target sites and selectively activates FXN. All data are from GM15850 cells treated 24 hours with the indicated molecules (1 μΜ). (A) Heatmap of the SOS profile of PA1 and Syn-TEF1 across the top 250 predicted binding sites of PA1 (9, 31). (B) Heatmaps of BRD4, Pol II, and phospho-Ser2 occupancy across the same genomic loci as in panel A. (C) Occupancy of BRD4 at BRD4 binding sites across the genome following treatment with Syn-TEF1 or PA1 + JQ1. (D) Scatterplot of the SOS score versus distance of the predicted Syn-TEF1 binding site to the transcription start site (TSS) for the top 500 genes predicted to be targeted by Syn-TEF1. Each gene is shaded according to the change in gene expression following Syn-TEF1 treatment. (E) Scatterplot of the SOS score, change in gene expression (Syn- TEF1 treatment), and licensing ratio (LR) of RNA Pol II for the top 500 genes predicted to be targeted by Syn-TEF1.
Fig. 4.
Fig. 4.
Syn-TEFs activate FXN expression in primary patient cells and patient derived fibroblasts, iPSCs, cardiomyocytes, sensory neurons, and mouse xenografts. All treatments were 24 hours except where specified. (A) Relative expression of FXN mRNA, normalized to GAPDH in three lymphoblastoid cell lines (LCLs) derived from three different FRDA patients. All treatments were 1 μΜ (results are mean ± SEM, n = 3). *P <0.05; **P < 0.01. (B) Expression of cell-type specific markers in GM23913 iPSCs or the iPSC-derived cardiomyocytes following treatment with 0.1% DMSO (results are mean n = 2). (C) Syn-TEF1 dependent induction of FXN mRNA in GM23913-derived cardiomyocytes (60 hours treatment, results are mean n = 2). (D) Immunohistochemistry of GM23913 iPSCs and the iPSC-derived cardiomyocytes. iPSCs were fixed and stained with OCT4and SOX2. iPSC-derived cardiomyocytes were fixed and stained with TNNT2 and MYL2. Scale bars represent 100 μm. (E) Immunoblot of FXN and β- actin (β-act) following treatment of three different primary FRDA fibroblasts, fibroblast-derived iPSCs, and sensory neurons with the indicated molecules. Fibroblasts were collected from patients UAB4259 (550/1000), UAB4230 (1000/1200), and UAB66 (90/1025). Cells were treated 72–96 hours. (F) Immunohistochemistry of FRDA patient-derived iPSCs and iPSC-derived sensory neurons. iPSCs were fixed and stained with OCT4 and SSEA-4. Sensory neurons were fixed and stained with neuronal markers CGRP and MAP-2. Scale bars represent 100 μm. (G) (i) Genotyped repeats and (ii) relative expression of FXN mRNA normalized to GAPDH in peripheral blood mononuclear cells (PBMCs) from 11 patients following 24 hours treatment with 1 μM Syn-TEF1. (H) Bioluminescent images of two representative mice harboring xenografts (HEK293 FXN-Luc with 6 and ~310 GAA repeats in left and right flanks, respectively) (29). Mice were treated with either vehicle (DMSO) or 0.5 nmol Syn-TEF1 subcutaneously into each tumor (1 nmol total per mouse). Mice were imaged 22 hours after treatment. (I) Relative expression of FXN-Luc with 6 or ~310 GAA repeats following Syn-TEF1 treatment of mice as described in (H). Data are mean ± SEM (n = 4 and n = 3, Syn-TEF1- and DMSO-treated mice, respectively). *P < 0.05. (J) Aconitase activity in GM16214 lymphoblastoid cells following 72 hours treatment with DMSO (0.1%), PA1 (125 nM), or Syn-TEFl (62.5 or 125 nM). Aconitase activity was normalized to GM16215 cells. Results are mean ± SEM (n = 3).

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