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.
Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases
. However, repeat expansions are often not explored beyond neurological and ...neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer
. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.
Targeting the genome with sequence‐specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole‐based polyamides can be rationally ...designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high‐affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.
Siteseeing: The first mapping of pyrrole/imidazole polyamides across several genomic sites with distinct chromatin characteristics in live cells is reported. Bioinformatic analysis of the data shows that clustered binding sites best predict binding in cells. The results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.
Abstract Background The segment involvement score (SIS) is a semiquantitative measure of the extent of atherosclerosis burden by coronary computed tomography angiography (-CTA). We sought to evaluate ...by meta-analysis the prognostic value of SIS, and to compare it with other CTA measures of coronary artery disease (CAD). Methods Electronic databases from 1946 to January 2016 were searched. Studies reporting SIS, or an equivalent measure by coronary CTA, and clinical outcomes were included. Maximally adjusted hazard ratios (HR), predominantly for clinical variables, were extracted for SIS, obstructive CAD, Agatston coronary artery calcium score, and plaque composition. These were pooled using DerSimonian-Laird random effects models. Results Eleven nonrandomized studies with good methodological quality enrolling 9777 subjects (mean age 61 ± 11 years, 57% male, mean follow up 3.3 years) who had 472 (4.8%) MACE (cardiac or all cause death, non-fatal myocardial infarction or late revascularization), were included. SIS (per segment increase) had pooled HR of 1.25 (95% CI: 1.16,1.35; I2 = 71.4%, p < 0.001) for MACE. HR for MACE was 1.37 (95% CI: 1.32,1.42; I2 = 95.6%, p < 0.001) for number of segments with stenosis (per segment increase), 3.39 (95% CI: 1.65,6.99; I2 = 87.8%, p = 0.001) for obstructive CAD (binary variable) and 1.00 (95% CI: 1.00,1.01; I2 = 75.0%, p = 0.490) for Agatston score (per unit increase). HRs by plaque composition (calcified, non-calcified and mixed; per segment change) were 1.24 (95% CI: 1.10,1.39; I2 = 81.6%, p = 0.001), 1.20 (95% CI: 0.97,1.48; I2 = 92.9%, p = 0.093) and 1.27 (95% CI: 1.03,1.58; I2 = 89.8%, p = 0.029), respectively. Conclusion Despite heterogeneity in endpoints, extent of CAD as quantified by SIS on coronary CTA is a strong, independent predictor of cardiovascular events.
Transcription factors control the fate of a cell by regulating the expression of genes and regulatory networks. Recent successes in inducing pluripotency in terminally differentiated cells as well as ...directing differentiation with natural transcription factors has lent credence to the efforts that aim to direct cell fate with rationally designed transcription factors. Because DNA-binding factors are modular in design, they can be engineered to target specific genomic sequences and perform pre-programmed regulatory functions upon binding. Such precision-tailored factors can serve as molecular tools to reprogramme or differentiate cells in a targeted manner. Using different types of engineered DNA binders, both regulatory transcriptional controls of gene networks, as well as permanent alteration of genomic content, can be implemented to study cell fate decisions. In the present review, we describe the current state of the art in artificial transcription factor design and the exciting prospect of employing artificial DNA-binding factors to manipulate the transcriptional networks as well as epigenetic landscapes that govern cell fate.
Sliding on DNA: From Peptides to Small Molecules Xiong, Kan; Erwin, Graham S.; Ansari, Aseem Z. ...
Angewandte Chemie (International ed.),
November 21, 2016, Volume:
55, Issue:
48
Journal Article
Peer reviewed
Open access
Many DNA binding proteins utilize one‐dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a ...quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA—even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure–function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small‐molecule synthetic sleds, and molecular sled activity in N‐methylpyrrole/N‐methylimidazole polyamides that helps explain how these molecules locate rare target sites.
Synthetic small molecules with a wide range of chemical functionality can slide on DNA, and specific modifications can be used to tune crucial properties like DNA affinity and sliding speed. The picture shows a polyamide bound to DNA and its trajectory when sliding on DNA.
Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and ...N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to “read” specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.
Maximally adjusted hazard ratios (HR) for SIS, obstructive CAD, CAC, and plaque composition were extracted from the included studies and pooled using DerSimonian-Laird random effects models.
Background Stem cell therapy is a promising treatment modality for injured cardiac tissue. A novel mechanism for this cardioprotection may include paracrine actions. Our lab has recently shown that ...gender differences exist in mesenchymal stem cell (MSC) paracrine function. Estrogen is implicated in the cardioprotection found in females. It remains unknown whether 17β-estradiol (E2) affects MSC paracrine function and whether E2-treated MSCs may better protect injured cardiac tissue. We hypothesize that E2-exposed MSCs infused into hearts prior to ischemia may demonstrate increased vascular endothelial growth factor (VEGF) production and greater protection of myocardial function compared to untreated MSCs. Materials and methods Untreated and E2-treated MSCs were isolated, cultured, and plated and supernatants were harvested for VEGF assay (enzyme-linked immunosorbent assay). Adult male Sprague-Dawley rat hearts ( n = 13) were isolated and perfused via Langendorff model and subjected to 15 min equilibration, 25 min warm global ischemia, and 40 min reperfusion. Hearts were randomly assigned to perfusate vehicle, untreated male MSC, or E2-treated male MSC. Transcoronary delivery of 1 million MSCs was performed immediately prior to ischemia in experimental hearts. Results E2-treated MSCs provoked significantly more VEGF production than untreated MSCs (933.2 ± 64.9 versus 595.8 ± 10.7 pg/mL). Postischemic recovery of left ventricular developed pressure was significantly greater in hearts infused with E2-treated MSCs (66.9 ± 3.3%) than untreated MSCs (48.7 ± 3.7%) and vehicle (28.9 ± 4.6%) at end reperfusion. There was also greater recovery of the end diastolic pressure with E2-treated MSCs than untreated MSCs and vehicle. Conclusions Preischemic infusion of MSCs protects myocardial function and viability. E2-treated MSCs may enhance this paracrine protection, which suggests that ex vivo modification of MSCs may improve therapeutic outcome.
The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. ...Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.
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