Resident physician scientist with interests in protein engineering, chemical biology, nucleic acids, epigenetic modifications, assay development, clinical diagnostics, personalized medicine, and laboratory developed tests
- Clinical Pathology
BS, University of Wisconsin-Madison, Honors in Chemistry (2015)
PhD, Perelman School of Medicine at the University of Pennsylvania, Biochemistry and Molecular Biophysics (2021)
MD, Perelman School of Medicine at the University of Pennsylvania (2023)
Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects.
Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multimodal single-cell data integration, enable accurate identification of neuronal subtypes and uncover context-specific regulatory effects on cell-type-specific genes by TET enzymes.
View details for DOI 10.1038/s41587-023-01909-2
View details for PubMedID 37640946
View details for PubMedCentralID 6197482
Revealing Drivers for Carboxy-S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase.
ACS chemical biology
Methylation of DNA plays a key role in diverse biological processes spanning from bacteria to mammals. DNA methyltransferases (MTases) typically employ S-adenosyl-l-methionine (SAM) as a critical cosubstrate and the relevant methyl donor for modification of the C5 position of cytosine. Recently, work on the CpG-specific bacterial MTase, M.MpeI, has shown that a single N374K point mutation can confer the enzyme with the neomorphic ability to use the sparse, naturally occurring metabolite carboxy-S-adenosyl-l-methionine (CxSAM) in order to generate the unnatural DNA modification, 5-carboxymethylcytosine (5cxmC). Here, we aimed to investigate the mechanistic basis for this DNA carboxymethyltransferase (CxMTase) activity by employing a combination of computational modeling and in vitro characterization. Modeling of substrate interactions with the enzyme variant allowed us to identify a favorable salt bridge between CxSAM and N374K that helps to rationalize selectivity of the CxMTase. Unexpectedly, we also discovered a potential role for a key active site E45 residue that makes a bidentate interaction with the ribosyl sugar of CxSAM, located on the opposite face of the CxMTase active site. Prompted by these modeling results, we further explored the space-opening E45D mutation and found that the E45D/N374K double mutant in fact inverts selectivity, preferring CxSAM over SAM in biochemical assays. These findings provide new insight into CxMTase active site architecture and may offer broader utility given the numerous opportunities offered by using SAM analogs for selective molecular labeling in concert with nucleic acid or even protein-modifying MTases.
View details for DOI 10.1021/acschembio.3c00184
View details for PubMedID 37379458
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution.
Nature chemical biology
5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.
View details for DOI 10.1038/s41589-023-01318-1
View details for PubMedID 37322153
View details for PubMedCentralID 6197482
The Base-Editing Enzyme APOBEC3A Catalyzes Cytosine Deamination in RNA with Low Proficiency and High Selectivity
ACS CHEMICAL BIOLOGY
2022; 17 (3): 629-636
Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, mutagenesis by A3A has also been readily detected in both cellular DNA and RNA, activities that have been implicated in cancer. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G), which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.
View details for DOI 10.1021/acschembio.1c00919
View details for Web of Science ID 000778547200016
View details for PubMedID 35262324
View details for PubMedCentralID PMC9949940
Mutant IDH Inhibits IFN gamma-TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma
2022; 12 (3): 812-835
Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy.Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.
View details for DOI 10.1158/2159-8290.CD-21-1077
View details for Web of Science ID 000767261500001
View details for PubMedID 34848557
View details for PubMedCentralID PMC8904298
Enzymatic approaches for profiling cytosine methylation and hydroxymethylation
2022; 57: 101314
In mammals, modifications to cytosine bases, particularly in cytosine-guanine (CpG) dinucleotide contexts, play a major role in shaping the epigenome. The canonical epigenetic mark is 5-methylcytosine (5mC), but oxidized versions of 5mC, including 5-hydroxymethylcytosine (5hmC), are now known to be important players in epigenomic dynamics. Understanding the functional role of these modifications in gene regulation, normal development, and pathological conditions requires the ability to localize these modifications in genomic DNA. The classical approach for sequencing cytosine modifications has involved differential deamination via the chemical sodium bisulfite; however, bisulfite is destructive, limiting its utility in important biological or clinical settings where detection of low frequency populations is critical. Additionally, bisulfite fails to resolve 5mC from 5hmC.To summarize how enzymatic rather than chemical approaches can be leveraged to localize and resolve different cytosine modifications in a non-destructive manner.Nature offers a suite of enzymes with biological roles in cytosine modification in organisms spanning from bacteriophages to mammals. These enzymatic activities include methylation by DNA methyltransferases, oxidation of 5mC by TET family enzymes, hypermodification of 5hmC by glucosyltransferases, and the generation of transition mutations from cytosine to uracil by DNA deaminases. Here, we describe how insights into the natural reactivities of these DNA-modifying enzymes can be leveraged to convert them into powerful biotechnological tools. Application of these enzymes in sequencing can be accomplished by relying on their natural activity, exploiting their ability to discriminate between cytosine modification states, reacting them with functionalized substrate analogs to introduce chemical handles, or engineering the DNA-modifying enzymes to take on new reactivities. We describe how these enzymatic reactions have been combined and permuted to localize DNA modifications with high specificity and without the destructive limitations posed by chemical methods for epigenetic sequencing.
View details for DOI 10.1016/j.molmet.2021.101314
View details for Web of Science ID 000788014200006
View details for PubMedID 34375743
View details for PubMedCentralID PMC8829811
Controllable genome editing with split-engineered base editors
NATURE CHEMICAL BIOLOGY
2021; 17 (12): 1262-1270
DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.
View details for DOI 10.1038/s41589-021-00880-w
View details for Web of Science ID 000708337400001
View details for PubMedID 34663942
View details for PubMedCentralID PMC8981362
Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency
2021; 81 (4): 859-869.e8
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.
View details for DOI 10.1016/j.molcel.2020.11.045
View details for Web of Science ID 000631251700003
View details for PubMedID 33352108
View details for PubMedCentralID PMC7897302
Discovery of an Unnatural DNA Modification Derived from a Natural Secondary Metabolite
CELL CHEMICAL BIOLOGY
2021; 28 (1): 97-+
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.
View details for DOI 10.1016/j.chembiol.2020.09.006
View details for Web of Science ID 000610622900011
View details for PubMedID 33053370
View details for PubMedCentralID PMC7855694
Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq)
2021; 2198: 349-367
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.
View details for DOI 10.1007/978-1-0716-0876-0_27
View details for Web of Science ID 000707160600028
View details for PubMedID 32822044
Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N-Demethylation
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2020; 59 (28): 11312-11315
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 FeII /α-ketoglutarate-dependent dioxygenases, and suggest activity on unexplored substrates that could reveal new TET biology.
View details for DOI 10.1002/anie.202002751
View details for Web of Science ID 000531327300001
View details for PubMedID 32271979
View details for PubMedCentralID PMC7332413
Recognition of Class II MHC Peptide Ligands That Contain beta-Amino Acids
JOURNAL OF IMMUNOLOGY
2019; 203 (6): 1619–28
Proteins are composed of α-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from β-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II-binding peptides derived from OVA, in which at least one α-amino acid residue was replaced with a homologous β-amino acid residue. The latter contain an extra methylene unit in the peptide backbone but retain the original side chain. We show that several of these α/β-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One α/β-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs.
View details for DOI 10.4049/jimmunol.1900536
View details for Web of Science ID 000484842100021
View details for PubMedID 31391235
View details for PubMedCentralID PMC6736755
Consequences of Periodic alpha-to-beta(3) Residue Replacement for Immunological Recognition of Peptide Epitopes
ACS CHEMICAL BIOLOGY
2015; 10 (3): 844-854
Oligomers that contain both α- and β-amino acid residues, or "α/β-peptides", have emerged as promising mimics of signal-bearing polypeptides that can inhibit or augment natural protein-protein interactions. α/β-Peptides that contain a sufficient proportion of β residues evenly distributed along the sequence can be highly resistant to enzymatic degradation, which is favorable with regard to in vivo applications. Little is known, however, about recognition of α/β-peptides by the immune system. Prior studies have focused almost entirely on examples that contain a single β residue; such α/β-peptides frequently retain the immunological profile of the analogous α-peptide. We have conducted α-peptide vs α/β-peptide comparisons involving higher β residue content, focusing on molecules with αααβ and ααβαααβ backbone repeat patterns. Among analogues of an 18-mer derived from the Bim BH3 domain and an 8-mer derived from secreted phospholipase-2 (sPLA2), we find that recognition by antibodies raised against the prototype α-peptide is suppressed by periodic α → β replacements. Complementary studies reveal that antibodies raised against Bim BH3- or sPLA2-derived α/β-peptides fail to recognize prototype α-peptides displaying identical side chain repertoires. Because polypeptides containing d-α-amino acid residues are of growing interest for biomedical applications, we included the enantiomer of the sPLA2-derived α-peptide in these studies; this d-peptide is fully competent as a hapten, but the resulting antibodies do not cross react with the enantiomeric peptide. Among analogues of the 9-mer CD8(+) T-cell viral epitope GP33, we observe that periodic α → β replacements suppress participation in the MHC I + peptide + T-cell receptor ternary complexes that activate cytotoxic T-lymphocytes, due in part to disruption of MHC binding.
View details for DOI 10.1021/cb500888q
View details for Web of Science ID 000351558700023
View details for PubMedID 25559929
View details for PubMedCentralID PMC4372116