Honors & Awards
Postdoctoral Fellowship, Stanford University Dean's Postdoctoral Fellowship (2018)
Postdoctoral Fellowship, American Heart Association (2018-2020)
Pilot Grant Award, Stanford Translational Research and Applied Medicine (2020-2022)
LRP Award, National Institutes of Health (2021-2023)
Helen Blau, Postdoctoral Research Mentor
Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.
Stem cell reports
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.
View details for DOI 10.1016/j.stemcr.2021.04.018
View details for PubMedID 34019816
Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome.
2020; 15 (12): e0243905
Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.
View details for DOI 10.1371/journal.pone.0243905
View details for PubMedID 33351840
Reprogramming cell fate with artificial transcription factors.
2018; 592 (6): 888–900
Transcription factors (TFs) reprogram cell states by exerting control over gene regulatory networks and the epigenetic landscape of a cell. Artificial transcription factors (ATFs) are designer regulatory proteins comprised of modular units that can be customized to overcome challenges faced by natural TFs in establishing and maintaining desired cell states. Decades of research on DNA-binding proteins and synthetic molecules has provided a molecular toolkit for ATF design and the construction of genome-scale libraries of ATFs capable of phenotypic manipulation and reprogramming of cell states. Here, we compare the unique strengths and limitations of different ATF platforms, highlight the advantages of cooperative assembly, and present the potential of ATF libraries in revealing gene regulatory networks that govern cell fate choices.
View details for DOI 10.1002/1873-3468.12993
View details for PubMedID 29389011
View details for PubMedCentralID PMC5869137
Synthetic transcription elongation factors license transcription across repressive chromatin.
Science (New York, N.Y.)
2017; 358 (6370): 1617–22
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.
View details for DOI 10.1126/science.aan6414
View details for PubMedID 29192133
View details for PubMedCentralID PMC6037176
Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin).
Journal of visualized experiments : JoVE
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.
View details for DOI 10.3791/53510
View details for PubMedID 26863565
View details for PubMedCentralID PMC4781686
Reprogramming cell fate with a genome-scale library of artificial transcription factors.
Proceedings of the National Academy of Sciences of the United States of America
2016; 113 (51): E8257–E8266
Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.
View details for DOI 10.1073/pnas.1611142114
View details for PubMedID 27930301
View details for PubMedCentralID PMC5187731
Mapping polyamide-DNA interactions in human cells reveals a new design strategy for effective targeting of genomic sites.
Angewandte Chemie (International ed. in English)
2014; 53 (38): 10124–28
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.
View details for DOI 10.1002/anie.201405497
View details for PubMedID 25066383
View details for PubMedCentralID PMC4160732
Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers.
The Biochemical journal
2014; 462 (3): 397–413
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.
View details for DOI 10.1042/BJ20140400
View details for PubMedID 25145439
View details for PubMedCentralID PMC4205157
Mitigation of peroxynitrite-mediated nitric oxide (NO) toxicity as a mechanism of induced adaptive NO resistance in the CNS
JOURNAL OF NEUROCHEMISTRY
2009; 109 (1): 74-84
During CNS injury and diseases, nitric oxide (NO) is released at a high flux rate leading to formation of peroxynitrite (ONOO(*)) and other reactive nitrogenous species, which nitrate tyrosines of proteins to form 3-nitrotyrosine (3NY), leading to cell death. Previously, we have found that motor neurons exposed to low levels of NO become resistant to subsequent cytotoxic NO challenge; an effect dubbed induced adaptive resistance (IAR). Here, we report IAR mitigates, not only cell death, but 3NY formation in response to cytotoxic NO. Addition of an NO scavenger before NO challenge duplicates IAR, implicating reactive nitrogenous species in cell death. Addition of uric acid (a peroxynitrite scavenger) before cytotoxic NO challenge, duplicates IAR, implicating peroxynitrite, with subsequent 3NY formation, in cell death, and abrogation of this pathway as a mechanism of IAR. IAR is dependent on the heme-metabolizing enzyme, heme oxygenase-1 (HO1), as indicated by the elimination of IAR by a specific HO1 inhibitor, and by the finding that neurons isolated from HO1 null mice have increased NO sensitivity with concomitant increased 3NY formation. This data indicate that IAR is an HO1-dependent mechanism that prevents peroxynitrite-mediated NO toxicity in motor neurons, thereby elucidating therapeutic targets for the mitigation of CNS disease and injury.
View details for DOI 10.1111/j.1471-4159.2009.05884.x
View details for Web of Science ID 000264022700007
View details for PubMedID 19183270
View details for PubMedCentralID PMC2692600
Differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species: implications for multiple sclerosis
JOURNAL OF NEUROCHEMISTRY
2009; 109 (1): 93-104
Depending on its concentration, nitric oxide (NO) has beneficial or toxic effects. In pathological conditions, NO reacts with superoxide to form peroxynitrite, which nitrates proteins forming nitrotyrosine residues (3NY), leading to loss of protein function, perturbation of signal transduction, and cell death. 3NY immunoreactivity is present in many CNS diseases, particularly multiple sclerosis. Here, using the high flux NO donor, spermine-NONOate, we report that oligodendrocytes are resistant to NO, while motor neurons are NO sensitive. Motor neuron sensitivity correlates with the NO-dependent formation of 3NY, which is significantly more pronounced in motor neurons when compared with oligodendrocytes, suggesting peroxynitrite as the toxic molecule. The heme-metabolizing enzyme, heme-oxygenase-1 (HO1), is necessary for oligodendrocyte NO resistance, as demonstrated by loss of resistance after HO1 inhibition. Resistance is reinstated by peroxynitrite scavenging with uric acid further implicating peroxynitrite as responsible for NO sensitivity. Most importantly, differential sensitivity to NO is also present in cultures of primary oligodendrocytes and motor neurons. Finally, motor neurons cocultured with oligodendrocytes, or oligodendrocyte-conditioned media, become resistant to NO toxicity. Preliminary studies suggest oligodendrocytes release a soluble factor that protects motor neurons. Our findings challenge the current paradigm that oligodendrocytes are the exclusive target of multiple sclerosis pathology.
View details for DOI 10.1111/j.1471-4159.2009.05891.x
View details for Web of Science ID 000264022700009
View details for PubMedID 19226373
View details for PubMedCentralID PMC2756289