1. Label-free blood screening instruments (Masters work). Whole blood analysis is a critical and familiar part of clinical workflows. Widely used clinical blood cytometers are generally expensive and bulky, require proprietary reagents, and provide limited quantitative information on cell morphology. When these automated cytometers do find abnormalities, manual examination of a stained blood smear using light microscopy is required for further diagnosis. This process is costly, slow, and not quantitative. In my early graduate career, working Prof. Gabriel Popescu, I set out to address these issues by utilizing Quantitative Phase Imaging (QPI). In QPI the phase shift (optical delay) of light passing through a sample is measured with sub-nanometer accuracy using interferometry providing high resolution topographic and tomographic data (Mir et al, Prog. Optics, 2012). I started by building a new QPI instrument utilizing commercial CD-ROM technology (Mir et al., Opt. Express., 2009) with the goal of developing a low-cost platform for an automated point-of-care blood screening tool. A comparison of results on patient samples with those from a clinical cytometer shows excellent agreement (Mir et al., J. Biomed. Opt., 2010 & Biomed. Opt. Exp., 2011) for clinical parameters on red blood cells. Additionally, my approach provides quantitative single cell morphological data, uses 2 orders of magnitude lower input sample, requires no reagents other than whole blood, and significantly lowers instrument cost. Collectively, these studies established that QPI based technologies are more sensitive than currently used clinical whole blood analyzers for common parameters used to characterize erythrocytes and also provide additional information that could lead to earlier diagnosis of certain diseases.
a) M. Mir, B. Bhaduri, R. Wang, R. Zhu, G. Popescu, Chapter 3 – Quantitative Phase Imaging, Progress in Optics, Ed: W. Emil, Elsevier, Volume 57, pp. 133-217 (2012)
b) M. Mir, Z. Wang, K. Tangella and G. Popescu, Diffraction Phase Cytometry: Blood on a CD-ROM, Optics Express, 17 (4), 2579-2585 (2009)
c) M. Mir, H. Ding, Z. Wang, J. Reedy, K. Tangella and G. Popescu, Blood Screening using Diffraction Phase Cytometry, Journal of Biomedical Optics, 15 (2), 027016 (2010)
d) M. Mir, K. Tangella and G. Popescu, Blood Testing at the Single Cell Level using Quantitative Phase and Amplitude Microscopy, Biomedical Optics Express, 2 (12), 3259-3266. (2011)
2. Non-perturbative, quantitative, and label-free live imaging spanning broad spatio-temporal scales (PhD work). A major challenge in the live imaging of cells and tissues is the need to non-destructively capture data at a broad range of temporal and spatial scales. High temporal and spatial resolution data is required to measure dynamics and morphological changes at the sub-cellular level, at sub-micron and millisecond scales, while at the same time placing this data in the context of the population (e.g. whole tissue) at scales of millimeters and days. In Prof. Gabriel Popescu’s lab we developed new quantitative phase imaging (QPI) hardware and analytical tools which provide quantitative information on morphology, mass density, and dynamics while spanning this broad range of spatial-temporal scales. Critically, these new microscopes use low-power white-light illumination and are sensitive to the endogenous optical properties of the sample, circumventing challenges associated with fluorescence microscopy such as photobleaching and phototoxicity (at the expense of molecular specificity). One of our instruments, known as the Spatial Light Interference Microscope (Wang et al, Opt. Express, 2011), for which I developed control hardware and software, has also been successfully commercialized (phioptics.com). I used these new tools to 1) Characterize single cell mass growth in a cell-cycle dependent manner, overturning widely used models of constant exponential growth (Mir et al, PNAS, 2011); 2) Measure the effects of estrogen and estrogen agonists on the growth and dynamics of estrogen sensitive breast cancer cells, showing that estrogen blockers lead to slower growing cells with lower cell masses (Mir et al, PLoS One, 2014); 3) Quantify neuronal network formation in human stem cell derived neurons and showed correlations between single cell mass growth, transport within the forming network, and the emergence of self-organization of the developing network (Mir et al, Sci. Rep., 2014). These studies have had a major impact on the rapid growth of the field of Quantitative Phase Imaging and its adoption by life scientists which is best illustrated by the increasing presence of dedicated QPI instruments in core facilities and laboratories around the world.
a) Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. Gillette, and G. Popescu, Spatial Light Interference Microscopy, Optics Express, 19 (2), 1016-1026 (2011)
b) M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding and G. Popescu*, Optical Measurement of Cell Cycle Dependent Growth, Proceedings of the National Academies of Sciences (PNAS), 108 (32), 13124-13129 (2011)
c) M. Mir, A. Bergamaschi, B.S. Katzenellenbogen and G. Popescu, Highly sensitive quantitative imaging for single cancer cell growth kinetics and drug response, PLoS ONE, 9 (2), e89000 (2014)
d) M. Mir, T. Kim, A. Majumder, M. Xiang, R. Wang, S. C. Liu, M. U. Gillette, S. Stice and G. Popescu, Label-Free Characterization of Emerging Human Neuronal Networks” Scientific Reports, 4, 4434 (2014)
3. Computational Imaging (PhD and Postdoc work). Live-cell microscopy involves harsh tradeoffs between spatial resolution, temporal resolution, and sensitivity. In all optical microscopes spatial resolution is limited by the diffraction limit of light. Additionally, in the case of fluorescence microscopy, photo-bleaching and photo-toxicity present significant barriers for high-speed 3D imaging. Over the past two decades the field of compressed sensing has emerged to take advantage of an intrinsic property of natural images known as sparsity. Sparsity can be exploited to reconstruct high resolution images from lower resolution data (time or space) provided this data is acquired in an appropriate manner. Such computational imaging approaches, integrate hardware design and computational algorithms to boost spatial and temporal imaging resolution. During my graduate work, I developed a 3D deconvolution method (with Dr. D. Babacan) that exploits the sparsity of quantitative phase images to provide super-resolution 3D tomograms of living cells in a completely label-free manner (Mir et al., PLoS ONE, 2012). Using this approach I was able to resolve and characterize coiled and helical sub-cellular structures in E. coli which were previously only visible using fluorescence based super-resolution methods. We then further refined this approach by including a near-complete physical model of our optical system in a technique known as White-Light Diffraction Tomography (Kim et al., Nat. Photonics, 2014) which provides quantitative 3D tomograms of live cells with sub-cellular resolution. During my post-doctoral work, I led a project (senior author) to develop a generalizable compressed sensing scheme which provides a 5-10 fold reduction of light exposure and acquisition time in 3D fluorescence microscopy (Woringer et al., Opt. Express 2017). Collectively these works have demonstrated novel computational imaging approaches to improve the temporal and spatial resolution of light-microscopes and decrease photo-bleaching and photo-toxicity in 3D fluorescence imaging.
a) M. Mir, D. Babacan, M. Bednarz, I. Golding and G. Popescu, Three Dimensional Deconvolution Spatial Light Interference Tomography for studying Subcellular Structure in E. coli , PLoS ONE, 7 (6), e38916 (2012)
b) T. Kim, R. Zhou, M. Mir, S. D. Babacan, P. S. Carney, L. L. Goddard, and G. Popescu, White-light diffraction tomography of unlabelled live cells, Nature Photonics, (2014)
c) M. Woringer, X. Darzacq, C. Zimmer, and M. Mir, Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme, Optics Express, 25(12), 13668-13683, (2017)
4. Single molecule tracking and high-resolution 4D imaging in live embryos (Postdoc work). The application of single-molecule tracking to study transcription factor dynamics has shed significant insight on their search and DNA-binding kinetics over the past decade in cell culture models. At the start of my postdoc the technology to perform such measurements in live embryos did not exist. I overcame this problem by building a customized lattice light-sheet microscope to acquire the first data on single-molecule kinetics of proteins within the nuclei of living animal embryos (demonstrated in Mice, C. elegans, and Drosophila, Mir et al., Methods Mol Biol., 2018). In addition to enabling single molecule tracking in embryos, lattice light-sheet microscopy also provides high resolution 4D imaging over large fields-of-views and extended periods of times. These unique imaging capabilities provided opportunities for several collaborative efforts centered on questions of nuclear organization and transcription regulation (these collaborative works are in addition to my primary focus on studying transcription factor binding in Drosophila embryos as described below). Notably, these efforts include 1) Revealing the role of phase separation in the formation of heterochromatin domains in collaboration with Gary Karpen’s group (Strom et al, Nature 2017), 2) Characterizing the role of intrinsically disordered regions in replication initiation factor loading in collaboration with Michael Botchan’s group (Parker et al., eLife 2019), 3) Developing capabilities to label non-repetitive genomic loci with Cas9 in collaboration with Ahmet Yildiz’s group (Qin et al, 2017) and 4) Using single molecule-imaging in C. elegans embryos to study the regulation of RNA polymerase II recruitment to chromatin during X-chromosome dosage compensation in collaboration with Barbara Meyer’s group (in preparation). These collaborative efforts have provided unique biophysical insights on nuclear organization, chromatin biology, and transcription regulation and have helped mature the technologies of single molecule imaging in live embryos and software to analyze large high-dimensional imaging datasets.
a) M. Mir, A. Reimer, M. Stadler, A. Tangara, A. S. Hansen, D. Hockemeyer, M. B. Eisen, H. Garcia, X. Darzacq*, Chapter 32 – Single Molecule Imaging in Live Embryos using Lattice Light-Sheet Microscopy, Methods in Molecular Biology, Nanoscale Imaging: Methods and Protocols, Volume 1814, Ed: Y. L. Lyubchenko, Springer, pp 541-559 (2018)
b) A. R. Strom, A. V. Emelyanov, M. Mir, D. V. Fyodorov, X. Darzacq, and G. Karpen, Phase separation drives heterochromatin domain formation, Nature, doi:10.1038/nature22989 (2017)
c) M. W. Parker, M. Bell, M. Mir, J. A. Kao, X. Darzacq, M. R. Botchan, J. M. Berger, A new class of disordered elements controls DNA replication through initiator self-assembly, eLife 8:e48562, (2019) Download PDF
d) P. Qin, M. Parlak, C. Kuscu, J. Bandaria, M. Mir, K. Szlachta, R. Singh, X. Darzacq, A. Yildiz, and M. Adli, Live cell imaging of low- and non- repetitive chromosome loci using CRISPR-Cas9, Nature Communications, 8, 14725, (2017)
5. High concentration multi-factor hubs accentuate transcription factor binding and gene activation (Postdoc work). During my postdoc I became fascinated with linking transcription regulation at the molecular scale to cell-fate patterning during embryonic development. Central to this question is the biophysical problem of how transcription factors find their specific genomic targets in the crowded nuclear milieu. I utilized single molecule imaging to study the anteroposterior morphogen gradient formed by the transcription factor Bicoid in Drosophila melanogaster embryos. Bicoid has long been a model for classical cooperative binding, in which protein-protein interactions stabilize its interaction with target sites. However, when I analyzed Bicoid dynamics in nuclei along its anterior to posterior gradient, I saw no evidence for the concentration-dependent binding off-rates this model predicts. My data instead revealed that Bicoid binding occurs in high-local concentration hubs, which are dependent on the presence of a maternally deposited pioneer factor, Zelda. These multi-factor hubs increase the frequency of Bicoid binding events and thus result in higher Bicoid time-averaged occupancy without directly stabilizing its interactions with DNA (Mir et al, Genes Dev. 2017). Remarkably, these hubs themselves are highly dynamic, forming and melting on the order of seconds. By combining single-molecule tracking, high-speed 3D imaging, and live-visualization of nascent transcription I showed that Zelda-Bicoid hubs drive enrichment of transcription factors at their target loci through transient but preferential interactions (Mir et al, eLife, 2018) which then result in bursts of transcriptional activity (unpublished). Recently I have shown that hub formation is dependent on interactions between the disordered protein domains of hub components and independent of DNA binding (unpublished). These discoveries have come at a time when there is great interest in the role of disordered proteins in driving nuclear organization, particularly in the context of liquid-liquid phase separation. However, my work illustrates that disordered protein interactions can lead to dynamic compartmentalization distinct from phase separation, emphasizing that we have barely begun to understand the myriad biophysical forces that are at play (Mir et al, Development, 2019; McSwiggen et al., Genes Dev 2019).
a) M. Mir, A. Reimer, J. E. Haines, X.Y. Li, M. Stadler, H. Garcia, M. B. Eisen, X. Darzacq, Dense Bicoid hubs accentuate binding along the morphogen gradient, Genes Dev., 31, 1784-1794 (2017)
b) M. Mir, M. R. Stadler, S.A. Ortiz, X. Darzacq, M. B. Eisen, Dynamic Multifactor hubs transiently interact with sites of active transcription in Drosophila embryos, eLife, 7:e40497, (2018)
c) M. Mir, W. Bickmore, E. E. M. Furlong, G. J. Narlikar, Chromatin topology, condensates, and gene regulation: shifting paradigms or just a phase? Development, 146,dev182766 (2019)
d) D. T. McSwiggen, M. Mir, X. Darzacq, R. Tjian, Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences, Genes Dev, (2019)
Gene expression is regulated by a complex choreography of highly dynamic events, including the binding of transcription factors to non-coding regulatory regions of the genome, regulation of chromatin topology, and the assembly of large macromolecular complexes, all of which occur in the crowded nuclear environment. Our understanding of these dynamic processes has largely been driven by approaches that provide population averaged and static snapshots which have delivered remarkable insights, but are inherently ill suited for elucidating processes that vary greatly in space and time. Comprehending the mechanisms that regulate gene expression, and the role of nuclear organization in this regulation, requires technological and theoretical approaches that bridge spatial scales from molecular to organismal and temporal scales from milliseconds to days. We develop and utilize high-resolution microscopy methods which allows us to probe this vast range of spatial and temporal scales within living embryos. For example, we acquire high-speed volumetric data to quantify chromatin dynamics, multi-color datasets to study the interaction and distribution of protein domains associated with gene activation or repression as cell fates are determined in young embryos, and use single-molecule localization techniques to quantify the kinetics of individual transcription factors as they whizz around the nucleoplasm searching for and binding to their genomic targets. The goal of our lab is to use these advanced imaging technologies in combination with biophysical modelling, genomics, and gene editing to comprehend and manipulate the interplay between nuclear organization, transcription regulation, and gene expression patterns during cell-fate determination.
The Perelman School of Medicine at the University of Pennsylvania
Department of Cell and Developmental Biology
9-101 Smilow Center for Translational Research
3400 Civic Center Blvd
Philadelphia, PA 19104-6059
Deciphering Mechanisms of Genome Mis-folding In Cancer
Our lab deploys data-rich experimental techniques to elucidate the role of genome mis-folding in controlling oncogenic gene expression programs. Specifically, we are interested in moving beyond the status quo to understand how oncogenic subversion of lineage-determining transcription factors set topology of cancer genome. To tackle this question, we combine genomics and super-resolution imaging and focus on investigating molecular mechanisms of genome mis-folding in breast and blood cancers. These mechanistic studies aim to identify precise epigenetic vulnerabilities of cancer cells and guide treatments disrupting cancer cells’ transcriptional addiction.
Oncogenic Notch Promotes Long-Range Regulatory Interactions Within Hyperconnected 3D Cliques. Petrovic J*, Zhou Y*, Fasolino M, Goldman N, Schwartz GW, Mumbach MR, Nguyen SC, Rome KS, Sela Y, Zapataro Z, Blacklow SC, Kruhlak MJ, Shi J, Aster JC, Joyce EF, Little SC, Vahedi G, Pear WS, Faryabi RB Molecular Cell. 2019;73(6):1174-90 e12
Determining Epigenetic Mechanisms Of Resistance To Targeted Therapies
Targeting oncogenic drivers of cancers commonly leads to drug resistance. Mechanisms of acquiring resistance to oncology drugs mostly remain unknown, partly due to the limitations of population-based assays in elucidating heterogeneity of drug-naive and complexity of drug-induced tumor evolution. Using single-cell genomics and imaging, we study how heterogeneity and plasticity of transcriptional dependencies confer resistance to targeted therapeutics such as Notch inhibitors.
TooManyCells Identifies And Visualizes Relationships Of Single-cell Clades. Schwartz GW, Zhou Y, Petrovic J, Fasolino M, Xu L, Shaffer SM, Pear WS, Vahedi G, Faryabi RB Nature Methods, 2020; 17: 405-413
Innovating Computational Methods To Enable Cancer Discovery
Our lab innovates statistical and machine learning approaches to accelerate discovery of novel therapeutics and biomarkers by elucidating complexity and heterogeneity of tumors. Recently, we have developed a computational ecosystem for mapping molecular and spatial heterogeneity in tumors. As part of the Center for Personalized Diagnostics, we also mine cancer patient genotypic/phenotypic data to improve patient health. patient health.
Classes of ITD Predict Outcomes in AML Patients Treated With FLT3 Inhibitors. Schwartz GW, Manning B, Zhou Y, Velu P, Bigdeli A, Astles R, Lehman AW, Morrissette JJD, Perl AE, Li M, Carroll M, Faryabi RB Clinical Cancer Research. 2019;25(2):573-83
Cancer is typically considered a genetic disease. However, recent progress in our understanding of epigenetic aberrations in cancer has challenged this view. Overarching goal of our lab is to understand epigenetic mechanisms of transcriptional addiction in cancer and exploit this information to advance cancer therapeutics.
To pursue this objective, we use cutting-edge chromatin conformation capture, high-content imaging, single-cell epigenomics, functional genomics, and combine these technologies with our expertise in computational sciences to systematically explore: i) how epigenetic control of gene expression is disrupted in cancer, ii) why transcriptional addiction can develop, and iii) how heterogeneity and plasticity of transcriptional dependencies enable drug resistance.
Resolving the enigmatic mechanisms of DNA demethylation. Despite the wealth of studies on the importance of 5-methylcytosine (mC) in mammalian genomes, the mechanism by which DNA can be demethylated has remained elusive. While high profile studies had suggested that deamination of mC or related analogs could be involved in DNA demethylation, the biochemical feasibility of this reaction had not been established. Further, with the discovery of TET family enzymes that can oxidize mC, new avenues for demethylation have been recently proposed. We were the first to show that enzymatic deamination of oxidized analogs of mC is a disfavored route for demethylation and also the first to show that TDG results in specific depletion of 5-formylcytosine from genomes. We have also recently demonstrated that TET2 shows catalytic processivity, providing a mechanism for the generation of highly oxidized mC species despite the relative dearth of their precursors. We have also found key active site determinants that control step-wise oxidation and fund TET enzymes that stall at hmC and provide a means to dissociate the different activities of hmC, fC and caC. We continue to probe the mechanism of TET family enzymes, aiming to unravel the many permutations of modifications with five cytosine states (C, mC, hmC, fC, cac), on two opposite strand (CpG pairs) that can be generated by three different TET enzymes (TET1, TET2, TET3).
Nabel CS, Jia H, Ye Y, Shen L, Goldschmidt HL, Stivers JT, Zhang Y, Kohli RM (2012) AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation, Nature Chem Biol 8:751-8. (PMC3427411)
Kohli RM, Zhang Y (2013) TET enzymes, TDG and the Dynamics of DNA Demethylation, Nature 502: 472-9. [Peer-reviewed Review] (PMC4046508)
Crawford DJ, Liu MY, Nabel CS, Cao XJ, Garcia BA, Kohli RM. (2016) Tet2 catalyzes stepwise 5-methylcytosine oxidation by an iterative and de novo mechanism. J Am Chem Soc 138:730-3.
Liu MY, Torabifard H, Crawford DJ, DeNizio JE, Cao XJ, Garcia BA, Cisneros GA, Kohli RM (2017) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine, Nature Chem Biol, 13:181-187.
Explaining how targeted mutagenesis drives antibody maturation and genomic modification. Our lab has made great strides in understanding how targeted and purposeful mutation is used to improve our immune defenses. The enzyme Activation Induced Deaminase (AID) is the key driver of antibody maturation, catalyzing the targeted deamination of cytosine to generate uracil within the immunoglobulin locus. This targeted mutation is the initiating step in somatic hypermutation and class switch recombination. We have helped to decipher how targeting takes place at the molecular level, demonstrating how particular hotspots in the genome are targeted, and how the enzyme can discriminate DNA from RNA. These cytosine deaminase enzymes have been proposed to play a role in DNA demethylation. Furthermore, they offer new and unexploited tools to understand cytosine modification states in the genome.
Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT (2009) A portable hotspot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase, J Biol Chem 284: 22898-22904 (PMC2755697)
Nabel CS, Lee JW ,Wang LC Kohli RM (2013) Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase, Proc Natl Acad Sci USA 110: 14225–14230 (PMC3761612)
Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase, Nucleic Acids Res 42: 9964-75 (PMC4150791)
Schutsky EK, Nabel CS, Davis AKF, DeNizio JE, Kohli RM (2017) APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA, Nucleic Acids Res. doi: 10.1093/nar/gkx345 (PMID 28472485).
In mammalian cells, DNA modifications are centered to the largest extent around cytosine bases, which are targeted by three different DNA modifying processes: methylation, oxidation and deamination. Research in the Kohli laboratory focused on the biochemistry and chemical biology of the enzymes that make cytosine such a dynamic base in the genome.
Cytosine methylation by DNA Methyltransferases (DNMTs) generates 5-methylcytosine (5mC), an epigenetic modification associated with silencing, while TET family enzymes can catalyze step-wise oxidation of 5mC to generate three new oxidized 5mC bases (ox-mCs) – 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These bases that are critical intermediates in the cycle of DNA demethylation and can also potentially serve as independent epigenetic marks. Deamination of either cytosine or modified cytosine bases by AID/APOBEC family enzymes yields targeted transition mutations in the genome. ‘Purposeful’ mutation by AID/APOBECs is used to garble foreign genomes, is exploited by the immune system to mature antibody responses, and has been posited to play roles in DNA demethylation. Such activity also carries risks and, accordingly, the deamination signatures of AID/APOBECs have been prominently left on cancer genomes.
In the Kohli laboratory, we utilize a broad array of approaches, which include: 1) biochemical characterization of enzyme mechanisms, 2) chemical synthesis of enzyme probes, and 3) biological assays spanning epigenetics and immunology to study DNA modifying enzymes.
Professor and Program Leader, Gene Expression and Regulation Program
Director, Center for Chemical Biology and Translational Medicine
Vice Chair for Research, Pathology & Laboratory Medicine
Director, Experimental Pathology Division
Work in my lab addresses the dynamic interactions between viruses and host cells when their genomes are in conflict. My lab pioneered the study of cellular damage sensing machinery as an intrinsic defense to virus infection. We have studied the DNA damage responses with a range of human DNA viruses and identified distinct ways that they manipulate signaling networks and DNA repair processes. Studying DNA damage as part of the cellular response to infection has opened up a new area in the biology of virus-host interactions. It has also provided a platform for interrogating cellular pathways involved in recognition and processing of DNA damage. This work revealed that the MRN complex is the mammalian sensor of DNA breaks and viral genomes, and that it is required for efficient activation of ATM/ATR damage signaling.
Stracker, TH, Carson, CT and Weitzman, MD. (2002) Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex. Nature, 418, 348-352.
Carson, CT, Schwartz, RA, Stracker, TH, Lilley, CE, Lee, DV and Weitzman, MD (2003). The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J, 22,6610-6620.
Lilley, CE, Carson, CT, Muotri, AR, Gage, FH and Weitzman, MD (2005). DNA repair proteins affect the HSV-1 lifecycle. Proc Natl Acad Sci USA, 102, 5844-5849.
Lilley, CE, Chaurushiya, MS, Boutell, C, Everett, RD, and Weitzman, MD (2011). The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0. PLoS Pathog 7:e1002084. PMC3116817
Chaurushiya, MS, Lilley, CE, Aslanian, A, Meisenhelder, J, Scott, DC, Landry, S, Ticau, S, Boutell, C, Yates, JR, Schulman, BA, Hunter, T and Weitzman, MD (2012). Viral E3 ubiquitin-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain. Mol Cell 46, 79-90. PMC3648639
I have had a long-standing interest in viral and host proteins that bind DNA and chromatin. As a postdoc I used biochemical approaches to identify a recognition sequence for the Rep protein of AAV within the site-specific integration site on chromosome 19 (AAVS1). I demonstrated that Rep proteins can mediate interaction between cellular and viral DNA to promote targeted integration. We have recently employed proteomic approaches to identify proteins associated with viral DNA genomes during infection, as well as the modifications that occur to chromatin on the host genome. We have analyzed histone post-translational modifications during virus infection and shown how these are altered by viruses. We recently discovered that the histone-like protein VII encoded by Adenovirus for packaging of its genome, can also affect the composition of cellular chromatin by retaining danger signals to overcome immune signaling. We are now interested in looking at how viruses impact genome and nuclear architecture and the effects this has on gene expression.
Weitzman, MD, Kyöstiö, SRM, Kotin, RM and Owens, RA (1994). Rep proteins of adeno-associated virus (AAV) mediate a complex formation between AAV DNA and the AAV integration site on human chromosome 19. Proc Natl Acad Sci USA, 91, 5808-5812.
Kulej, K, Avgousti, DC, Weitzman, MD and Garcia, BA (2015). Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics. Methods 90, 8-20.
Avgousti, DC, Herrmann, C, Sekulic, N, Kulej, K, Petrescu, J, Molden, RC, Pancholi, NJ, Reyes, ED, Seeholzer, SH, Black, BE, Garcia, BA and Weitzman, MD (2016). A core viral protein binds host nucleosomes to sequester immune danger signals. Nature 535, 173-177. PMC4950998
Kulej, K, Avgousti, DC, Sidoli, S, Herrman C, Della Fera, AN, Kim ET, Garcia, BA and Weitzman, MD (2017). Time-resolved global and chromatin proteomics during Herpes Simplex Virus Type 1 (HSV-1) infection. Mol Cell Proteomics 16, S92-S107.
Reyes, RD, Kulej, K, Akhtar, LN, Avgousti, DC, Pancholi, NJ, Kim, ET, Bricker, D, Koniski, S, Seeholzer, SH, Isaacs, SN, Garcia, BA, and Weitzman, MD. Identifying host factors associated with DNA replicated during virus infection. (in press).
My lab is interested in cellular responses that restrict virus replication. APOBEC3 proteins belong to a family of cytidine deaminases that provide a line of defense against retroviruses and endogenous mobile retroelements. We were the first to show that human APOBEC3A (A3A) is a catalytically active cytidine deaminase, with a preference for ssDNA. We demonstrated that A3A is a potent inhibitor of endogenous retroelements such as LINE1, and also blocks replication of single-stranded parvoviruses such as AAV and MVM. We have also shown how the SAMHD1 protein limits replication of the DNA virus HSV-1. We discovered ways that cellular DNA repair proteins can act as species-specific barriers through their interaction with viral proteins.
Chen, H, Lilley, CE, Yu, Q, Lee, DV, Chou, J, Narvaiza, I, Landau, NR and Weitzman, MD (2006). APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr Bio 16, 480-485.
Narvaiza, I, Linfesty, DC, Greener, BN, Hakata, Y, Pintel, DJ, Logue, E, Landau, NR, and Weitzman, MD (2009). Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase. PLoS Pathog 5, e1000439. PMC2678267
Richardson, SR, Narvaiza, I, Planegger, RA, Weitzman, MD and Moran, JV (2014). APOBEC3A deaminates transiently exposed single-strand DNA that arises during LINE-1 retrotransposition. eLife 3, e02008. PMC4003774
Kim, ET, White, TE, Brandariz-Nunez, A, Diaz-Griffero, F, and Weitzman, MD (2013). SAMHD1 restricts herpes simplex virus type 1 (HSV-1) in macrophages by limiting DNA replication. J Virol 87, 12949-12956. PMC3838123
Lou, DI, Kim, ET, Shan, S, Meyerson, NR, Pancholi, NJ, Mohni, KM, Enard, D, Petrov, DA, Weller, SK, Weitzman, MD*, and Sawyer, SL (2016). An intrinsically disordered region of the DNA repair protein Nbs1 is a species-specific barrier to Herpes Simplex Virus 1 in primates. Cell Host & Microbe 20, 178-188. (*Co-corresponding author)
Proteins that mutate viral genetic material must also be carefully regulated to prevent deleterious effects on the host genome. While studying antiviral functions for A3A we discovered that the enzyme can also act on the cellular genome, inducing DNA breaks and cell cycle arrest. We suggested therefore that APOBEC proteins cause genomic instability and contribute to malignancy, and we are now studying how they are regulated to prevent inappropriate mutations. This body of work demonstrates how studying virus-host interactions can lead to insights into fundamental processes that impact cellular genomic integrity. We have recently found A3A upregulated in a subset of human leukemias and demonstrated how this provides vulnerability for targeted cancer therapies.
Landry, S, Narvaiza, I, Linfesty, DC and Weitzman, MD (2011). APOBEC3A can activate the DNA damage response and cause cell cycle arrest. EMBO Reports 12, 444-450. PMC3090015
Narvaiza, I, Landry, S, and Weitzman, MD (2012). APOBEC3 proteins and genome stability: The high cost of a good defense? Invited Extraview in Cell Cycle 11, 33-38.
Green, AM, Landry, S, Budagyan, K, Avgousti, D, Shalhout, S, Bhagwat, AS and Weitzman, MD (2016). APOBEC3A damages the cellular genome during DNA replication. Cell Cycle 15, 998-1008. PMC4889253
Green, AM, Budagyan, K, Hayer, KE, Reed, MA, Savani, MR, Wertheim, GB and Weitzman, MD (2017). Cytosine deaminase APOBEC3A sensitizes leukemia cells to inhibition of the DNA replication checkpoint. Cancer Research (in press)
Our lab aims to understand host responses to virus infection, and the cellular environment encountered and manipulated by viruses. We study multiple viruses in an integrated experimental approach that combines biochemistry, molecular biology, genetics and cell biology. We have chosen viral models that provide tractable systems to investigate the dynamic interplay between viral genetic material and host defense strategies. We have used proteomic approaches to probe the dynamic interactions that take place on viral and cellular genomes during infection, and have uncovered ways that viruses manipulate histones and chromatin as they take control of cellular processes. The pathways illuminated are key to fighting diseases of viral infection, provide insights into fundamental processes that maintain genome instability, and have implications for the development of efficient viral vectors for gene therapy.