Publications by authors named "Rahul M Kohli"

63 Publications

SARS-CoV-2 spike protein binding selectively accelerates substrate-specific catalytic activity of ACE2.

J Biochem 2021 Mar 27. Epub 2021 Mar 27.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has given rise to the devastating global pandemic. In most cases, SARS-CoV-2 infection results in the development of viral pneumonia and acute respiratory distress syndrome, known as "coronavirus disease 2019" or COVID-19. Intriguingly, besides the respiratory tract, COVID-19 affects other organs and systems of the human body. COVID-19 patients with pre-existing cardiovascular disease have a higher risk of death, and SARS-CoV-2 infection itself may cause myocardial inflammation and injury. One possible explanation of such phenomena is the fact that SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the receptor required for viral entry. ACE2 is expressed in the cells of many organs, including the heart. ACE2 functions as a carboxypeptidase that can cleave several endogenous substrates, including angiotensin II, thus regulating blood pressure and vascular tone. It remains largely unknown if the SARS-CoV-2 infection alters the enzymatic properties of ACE2, thereby contributing to cardiovascular complications in patients with COVID-19. Here, we demonstrate that ACE2 cleavage of des-Arg9-bradykinin substrate analog is markedly accelerated, while cleavage of angiotensin II analog is minimally affected by the binding of spike protein. These findings may have implications for a better understanding of COVID-19 pathogenesis.
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http://dx.doi.org/10.1093/jb/mvab041DOI Listing
March 2021

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites.

J Mol Biol 2021 Apr 7;433(8):166877. Epub 2021 Feb 7.

Department of Medicine, Department of Biochemistry & Biophysics, Perelman School of Medicine, Philadelphia, PA 19147, USA. Electronic address:

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.
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http://dx.doi.org/10.1016/j.jmb.2021.166877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005466PMC
April 2021

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Mol Cell 2021 02 21;81(4):859-869.e8. Epub 2020 Dec 21.

Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn Epigenetics Institute, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.
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http://dx.doi.org/10.1016/j.molcel.2020.11.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897302PMC
February 2021

Modular affinity-labeling of the cytosine demethylation base elements in DNA.

Sci Rep 2020 11 20;10(1):20253. Epub 2020 Nov 20.

Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Wake Forest School of Medicine, Winston-Salem, NC, 27101, USA.

5-methylcytosine is the most studied DNA epigenetic modification, having been linked to diverse biological processes and disease states. The elucidation of cytosine demethylation has drawn added attention the three additional intermediate modifications involved in that pathway-5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-each of which may have distinct biological roles. Here, we extend a modular method for labeling base modifications in DNA to recognize all four bases involved in demethylation. We demonstrate both differential insertion of a single affinity tag (biotin) at the precise position of target elements and subsequent repair of the nicked phosphate backbone that remains following the procedure. The approach enables affinity isolation and downstream analyses without inducing widespread damage to the DNA.
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http://dx.doi.org/10.1038/s41598-020-76544-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679407PMC
November 2020

TET-mediated 5-methylcytosine oxidation in tRNA promotes translation.

J Biol Chem 2020 Nov 16. Epub 2020 Nov 16.

University of Pennsylvania, United States.

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.
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http://dx.doi.org/10.1074/jbc.RA120.014226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949041PMC
November 2020

Discovery of an Unnatural DNA Modification Derived from a Natural Secondary Metabolite.

Cell Chem Biol 2021 Jan 13;28(1):97-104.e4. Epub 2020 Oct 13.

Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Despite widespread interest for understanding how modified bases have evolved their contemporary functions, limited experimental evidence exists for measuring how close an organism is to accidentally creating a new, modified base within the framework of its existing genome. Here, we describe the biochemical and structural basis for how a single-point mutation in E. coli's naturally occurring cytosine methyltransferase can surprisingly endow a neomorphic ability to create the unnatural DNA base, 5-carboxymethylcytosine (5cxmC), in vivo. Mass spectrometry, bacterial genetics, and structure-guided biochemistry reveal this base to be exclusively derived from the natural but sparse secondary metabolite carboxy-S-adenosyl-L-methionine (CxSAM). Our discovery of a new, unnatural DNA modification reveals insights into the substrate selectivity of DNA methyltransferase enzymes, offers a promising new biotechnological tool for the characterization of the mammalian epigenome, and provides an unexpected model for how neomorphic bases could arise in nature from repurposed host metabolites.
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http://dx.doi.org/10.1016/j.chembiol.2020.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855694PMC
January 2021

Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq).

Methods Mol Biol 2021 ;2198:349-367

Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Here, we provide a detailed protocol for our previously published technique, APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), which localizes 5-hydroxymethylcytosine at single nucleotide resolution using nanogram quantities of input genomic DNA. In addition to describing suggested troubleshooting workflows, these methods include four important updates which should facilitate widespread implementation of the technique: (1) additionally optimized reaction conditions; (2) redesigned quality controls which can be performed prior to resource-consumptive deep sequencing; (3) confirmation that the less active, uncleaved APOBEC3A (A3A) fusion protein, which is easier to purify, can be used to perform ACE-Seq ; and (4) an example bioinformatic pipeline with suggested filtering strategies. Finally, we have provided a supplementary video which gives a narrated overview of the entire method and focuses on how best to perform the snap cool and A3A deamination steps central to successful execution of the method.
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http://dx.doi.org/10.1007/978-1-0716-0876-0_27DOI Listing
March 2021

High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing.

Methods Mol Biol 2021 ;2198:321-331

Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, USA.

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.
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http://dx.doi.org/10.1007/978-1-0716-0876-0_25DOI Listing
March 2021

High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening.

Nat Commun 2020 07 13;11(1):3455. Epub 2020 Jul 13.

Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.
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http://dx.doi.org/10.1038/s41467-020-17209-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359328PMC
July 2020

Targeting evolution to inhibit antibiotic resistance.

FEBS J 2020 Oct 8;287(20):4341-4353. Epub 2020 Jun 8.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Drug-resistant bacterial infections have led to a global health crisis. Although much effort is placed on the development of new antibiotics or variants that are less subject to existing resistance mechanisms, history shows that this strategy by itself is unlikely to solve the problem of drug resistance. Here, we discuss inhibiting evolution as a strategy that, in combination with antibiotics, may resolve the problem. Although mutagenesis is the main driver of drug resistance development, attacking the drivers of genetic diversification in pathogens has not been well explored. Bacteria possess active mechanisms that increase the rate of mutagenesis, especially at times of stress, such as during replication within eukaryotic host cells, or exposure to antibiotics. We highlight how the existence of these promutagenic proteins (evolvability factors) presents an opportunity that can be capitalized upon for the effective inhibition of drug resistance development. To help move this idea from concept to execution, we first describe a set of criteria that an 'optimal' evolvability factor would likely have to meet to be a viable therapeutic target. We then discuss the intricacies of some of the known mutagenic mechanisms and evaluate their potential as drug targets to inhibit evolution. In principle, and as suggested by recent studies, we argue that the inhibition of these and other evolvability factors should reduce resistance development. Finally, we discuss the challenges of transitioning anti-evolution drugs from the laboratory to the clinic.
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http://dx.doi.org/10.1111/febs.15370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578009PMC
October 2020

Deciphering the Role of Colicins during Colonization of the Mammalian Gut by Commensal .

Microorganisms 2020 May 2;8(5). Epub 2020 May 2.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Colicins are specific and potent toxins produced by that result in the rapid elimination of sensitive cells. Colicin production is commonly found throughout microbial populations, suggesting its potential importance for bacterial survival in complex microbial environments. Nonetheless, as colicin biology has been predominately studied using synthetic models, it remains unclear how colicin production contributes to survival and fitness of a colicin-producing commensal strain in a natural environment. To address this gap, we took advantage of MP1, an strain that harbors a colicinogenic plasmid and is a natural colonizer of the murine gut. Using this model, we validated that MP1 is competent for colicin production and then directly interrogated the importance of colicin production and immunity for MP1 survival in the murine gut. We showed that colicin production is dispensable for sustained colonization in the unperturbed gut. A strain lacking colicin production or immunity shows minimal fitness defects and can resist displacement by colicin producers. This report extends our understanding of the role that colicin production may play for during gut colonization and suggests that colicin production is not essential for a commensal to persist in its physiologic niche in the absence of exogenous challenges.
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http://dx.doi.org/10.3390/microorganisms8050664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284606PMC
May 2020

Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N-Demethylation.

Angew Chem Int Ed Engl 2020 07 11;59(28):11312-11315. Epub 2020 May 11.

Department of Medicine, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104, USA.

TET family enzymes are known for oxidation of the 5-methyl substituent on 5-methylcytosine (5mC) in DNA. 5mC oxidation generates the stable base 5-hydroxymethylcytosine (5hmC), starting an indirect, multi-step process that ends with reversion of 5mC to unmodified cytosine. While probing the nucleobase determinants of 5mC recognition, we discovered that TET enzymes are also proficient as direct N-demethylases of cytosine bases. We find that N-demethylase activity can be readily observed on substrates lacking a 5-methyl group and, remarkably, TET enzymes can be similarly proficient in either oxidation of 5mC or demethylation of N4-methyl substituents. Our results indicate that TET enzymes can act as both direct and indirect demethylases, highlight the active-site plasticity of these Fe /α-ketoglutarate-dependent dioxygenases, and suggest activity on unexplored substrates that could reveal new TET biology.
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http://dx.doi.org/10.1002/anie.202002751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332413PMC
July 2020

The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe.

ACS Chem Biol 2020 05 5;15(5):1127-1133. Epub 2020 Feb 5.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.
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http://dx.doi.org/10.1021/acschembio.9b00886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230020PMC
May 2020

A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Nature 2019 05 1;569(7757):581-585. Epub 2019 May 1.

State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.
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http://dx.doi.org/10.1038/s41586-019-1160-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628258PMC
May 2019

Improving the Fluorescent Probe Acridonylalanine Through a Combination of Theory and Experiment.

J Phys Org Chem 2018 Aug 22;31(8). Epub 2018 Feb 22.

Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA.

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior FRET acceptor to Acd in a peptide cleavage assay, and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives which can be used in peptides and proteins.
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http://dx.doi.org/10.1002/poc.3813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454874PMC
August 2018

Advancement of the 5-Amino-1-(Carbamoylmethyl)-1H-1,2,3-Triazole-4-Carboxamide Scaffold to Disarm the Bacterial SOS Response.

Front Microbiol 2018 18;9:2961. Epub 2018 Dec 18.

Fox Chase Chemical Diversity Center, Inc., Doylestown, PA, United States.

Many antibiotics, either directly or indirectly, cause DNA damage thereby activating the bacterial DNA damage (SOS) response. SOS activation results in expression of genes involved in DNA repair and mutagenesis, and the regulation of the SOS response relies on two key proteins, LexA and RecA. Genetic studies have indicated that inactivating the regulatory proteins of this response sensitizes bacteria to antibiotics and slows the appearance of resistance. However, advancement of small molecule inhibitors of the SOS response has lagged, despite their clear promise in addressing the threat of antibiotic resistance. Previously, we had addressed this deficit by performing a high throughput screen of ∼1.8 million compounds that monitored for inhibition of RecA-mediated auto-proteolysis of LexA, the reaction that initiates the SOS response. In this report, the refinement of the 5-amino-1-(carbamoylmethyl)-1H-1,2,3-triazole-4-carboxamide scaffold identified in the screen is detailed. After development of a modular synthesis, a survey of key activity determinants led to the identification of an analog with improved potency and increased breadth, targeting auto-proteolysis of LexA from both and . Comparison of the structure of this compound to those of others in the series suggests structural features that may be required for activity and cross-species breadth. In addition, the feasibility of small molecule modulation of the SOS response was demonstrated by the suppression of the appearance of resistance. These structure activity relationships thus represent an important step toward producing Drugs that Inhibit SOS Activation to Repress Mechanisms Enabling Resistance (DISARMERs).
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http://dx.doi.org/10.3389/fmicb.2018.02961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305444PMC
December 2018

Exploiting Substrate Promiscuity To Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes.

J Am Chem Soc 2018 12 11;140(50):17329-17332. Epub 2018 Dec 11.

Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme's crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.
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http://dx.doi.org/10.1021/jacs.8b04722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470038PMC
December 2018

The SOS Response Mediates Sustained Colonization of the Mammalian Gut.

Infect Immun 2019 02 24;87(2). Epub 2019 Jan 24.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Bacteria have a remarkable ability to survive, persist, and ultimately adapt to environmental challenges. A ubiquitous environmental hazard is DNA damage, and most bacteria have evolved a network of genes to combat genotoxic stress. This network is known as the SOS response and aids in bacterial survival by regulating genes involved in DNA repair and damage tolerance. Recently, the SOS response has been shown to play an important role in bacterial pathogenesis, and yet the role of the SOS response in nonpathogenic organisms and in physiological settings remains underexplored. Using a commensal strain, MP1, we showed that the SOS response plays a vital role during colonization of the murine gut. In an unperturbed environment, the SOS-off mutant is impaired for stable colonization relative to a wild-type strain, suggesting the presence of genotoxic stress in the mouse gut. We evaluated the possible origins of genotoxic stress in the mouse gut by examining factors associated with the host versus the competing commensal organisms. In a dextran sulfate sodium (DSS) colitis model, the SOS-off colonization defect persisted but was not exacerbated. In contrast, in a germ-free model, the SOS-off mutant colonized with efficiency equal to that seen with the wild-type strain, suggesting that competing commensal organisms might be a significant source of genotoxic stress. This report extends our understanding of the importance of a functional SOS response for bacterial fitness in the context of a complex physiological environment and highlights the SOS response as a possible mechanism that contributes to ongoing genomic changes, including potential antibiotic resistance, in the microbiome of healthy hosts.
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http://dx.doi.org/10.1128/IAI.00711-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346138PMC
February 2019

Selectivity and Promiscuity in TET-Mediated Oxidation of 5-Methylcytosine in DNA and RNA.

Biochemistry 2019 02 14;58(5):411-421. Epub 2018 Nov 14.

Department of Chemistry , University of North Texas , Denton , Texas 76201 , United States.

Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.
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http://dx.doi.org/10.1021/acs.biochem.8b00912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363868PMC
February 2019

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development.

Elife 2018 10 16;7. Epub 2018 Oct 16.

Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United States.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1's function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.
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http://dx.doi.org/10.7554/eLife.34870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214653PMC
October 2018

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Nat Biotechnol 2018 Oct 8. Epub 2018 Oct 8.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.
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http://dx.doi.org/10.1038/nbt.4204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453757PMC
October 2018

Systematic Evaluation of Soluble Protein Expression Using a Fluorescent Unnatural Amino Acid Reveals No Reliable Predictors of Tolerability.

ACS Chem Biol 2018 10 20;13(10):2855-2861. Epub 2018 Sep 20.

Department of Medicine, Department of Biochemistry and Biophysics , University of Pennsylvania , Philadelphia , Pennsylvania 19104 , United States.

Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.
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http://dx.doi.org/10.1021/acschembio.8b00696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195468PMC
October 2018

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PLoS Genet 2018 06 1;14(6):e1007405. Epub 2018 Jun 1.

Department of Medicine, Division of Infectious Diseases, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America.

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.
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http://dx.doi.org/10.1371/journal.pgen.1007405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999292PMC
June 2018

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Nature 2018 06 30;558(7709):307-312. Epub 2018 May 30.

Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.
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http://dx.doi.org/10.1038/s41586-018-0178-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320248PMC
June 2018

Harnessing natural DNA modifying activities for editing of the genome and epigenome.

Curr Opin Chem Biol 2018 08 13;45:10-17. Epub 2018 Feb 13.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

The introduction of site-specific DNA modifications to the genome or epigenome presents great opportunities for manipulating biological systems. Such changes are now possible through the combination of DNA-modifying enzymes with targeting modules, including dCas9, that can localize the enzymes to specific sites. In this review, we take a DNA modifying enzyme-centric view of recent advances. We highlight the variety of natural DNA-modifying enzymes-including DNA methyltransferases, oxygenases, deaminases, and glycosylases-that can be used for targeted editing and discuss how insights into the structure and function of these enzymes has further expanded editing potential by introducing enzyme variants with altered activities or by improving spatiotemporal control of modifications.
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http://dx.doi.org/10.1016/j.cbpa.2018.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076857PMC
August 2018

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

ACS Infect Dis 2018 03 8;4(3):349-359. Epub 2018 Jan 8.

Department of Medicine, Department of Biochemistry and Biophysics , University of Pennsylvania , 3610 Hamilton Walk , Philadelphia , Pennsylvania 19104 , United States.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.
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http://dx.doi.org/10.1021/acsinfecdis.7b00122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893282PMC
March 2018

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Nano Lett 2017 11 5;17(11):7110-7116. Epub 2017 Oct 5.

Department of Biomedical Engineering, Virginia Tech-Wake Forest University, School of Biomedical Engineering and Sciences, Wake Forest School of Medicine , Winston-Salem, North Carolina 27101, United States.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.
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http://dx.doi.org/10.1021/acs.nanolett.7b03911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704975PMC
November 2017

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage.

ACS Synth Biol 2017 11 1;6(11):2067-2076. Epub 2017 Sep 1.

Department of Medicine, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.
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http://dx.doi.org/10.1021/acssynbio.7b00108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696648PMC
November 2017

APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA.

Nucleic Acids Res 2017 Jul;45(13):7655-7665

Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
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http://dx.doi.org/10.1093/nar/gkx345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570014PMC
July 2017

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.

Org Biomol Chem 2017 May;15(17):3603-3610

Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.
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http://dx.doi.org/10.1039/c7ob00582bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507695PMC
May 2017