King L Hung

All Publications

• CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing. bioRxiv : the preprint server for biology Zhu, K., Jones, M. G., Luebeck, J., Bu, X., Yi, H., Hung, K. L., Wong, I. T., Zhang, S., Mischel, P. S., Chang, H. Y., Bafna, V. 2024

  Abstract

  Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in approximately 15% of early stage cancers and 30% of late-stage cancers. EcDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy-number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, where the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graph have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together of complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose CoRAL (Complete Reconstruction of Amplifications with Long reads), for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 9 datasets from previously-characterized cell-lines as compared to previous short-read-based tools. As long-read usage becomes wide-spread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors. Availability: https://github.com/AmpliconSuite/CoRAL.

  View details for DOI 10.1101/2024.02.15.580594

  View details for PubMedID 38405779

• Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma. Nature genetics Chapman, O. S., Luebeck, J., Sridhar, S., Wong, I. T., Dixit, D., Wang, S., Prasad, G., Rajkumar, U., Pagadala, M. S., Larson, J. D., He, B. J., Hung, K. L., Lange, J. T., Dehkordi, S. R., Chandran, S., Adam, M., Morgan, L., Wani, S., Tiwari, A., Guccione, C., Lin, Y., Dutta, A., Lo, Y. Y., Juarez, E., Robinson, J. T., Korshunov, A., Michaels, J. A., Cho, Y. J., Malicki, D. M., Coufal, N. G., Levy, M. L., Hobbs, C., Scheuermann, R. H., Crawford, J. R., Pomeroy, S. L., Rich, J. N., Zhang, X., Chang, H. Y., Dixon, J. R., Bagchi, A., Deshpande, A. J., Carter, H., Fraenkel, E., Mischel, P. S., Wechsler-Reya, R. J., Bafna, V., Mesirov, J. P., Chavez, L. 2023

  Abstract

  Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

  View details for DOI 10.1038/s41588-023-01551-3

  View details for PubMedID 37945900

  View details for PubMedCentralID 5334176

• Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair. bioRxiv : the preprint server for biology Rose, J. C., Wong, I. T., Daniel, B., Jones, M. G., Yost, K. E., Hung, K. L., Curtis, E. J., Mischel, P. S., Chang, H. Y. 2023

  Abstract

  Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

  View details for DOI 10.1101/2023.10.22.563489

  View details for PubMedID 37961138

• Coordinated inheritance of extrachromosomal DNA species in human cancer cells. bioRxiv : the preprint server for biology Hung, K. L., Jones, M. G., Wong, I. T., Lange, J. T., Luebeck, J., Scanu, E., He, B. J., Brückner, L., Li, R., González, R. C., Schmargon, R., Dörr, J. R., Belk, J. A., Bafna, V., Werner, B., Huang, W., Henssen, A. G., Mischel, P. S., Chang, H. Y. 2023

  Abstract

  The chromosomal theory of inheritance has dominated human genetics, including cancer genetics. Genes on the same chromosome segregate together while genes on different chromosomes assort independently, providing a fundamental tenet of Mendelian inheritance. Extrachromosomal DNA (ecDNA) is a frequent event in cancer that drives oncogene amplification, dysregulated gene expression and intratumoral heterogeneity, including through random segregation during cell division. Distinct ecDNA sequences, herein termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells. However, how multiple ecDNA species within a tumor cell are assorted and maintained across somatic cell generations to drive cancer cell evolution is not known. Here we show that cooperative ecDNA species can be coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. EcDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy number gains in multiple ecDNA species prior to any selection. Computational modeling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Finally, we show that coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

  View details for DOI 10.1101/2023.07.18.549597

  View details for PubMedID 37503111

  View details for PubMedCentralID PMC10371175

• Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells. Nature genetics Chamorro González, R., Conrad, T., Stöber, M. C., Xu, R., Giurgiu, M., Rodriguez-Fos, E., Kasack, K., Brückner, L., van Leen, E., Helmsauer, K., Dorado Garcia, H., Stefanova, M. E., Hung, K. L., Bei, Y., Schmelz, K., Lodrini, M., Mundlos, S., Chang, H. Y., Deubzer, H. E., Sauer, S., Eggert, A., Schulte, J. H., Schwarz, R. F., Haase, K., Koche, R. P., Henssen, A. G. 2023

  Abstract

  Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

  View details for DOI 10.1038/s41588-023-01386-y

  View details for PubMedID 37142849

  View details for PubMedCentralID 5334176

• The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nature communications Gonzalez-Sandoval, A., Pekrun, K., Tsuji, S., Zhang, F., Hung, K. L., Chang, H. Y., Kay, M. A. 2023; 14 (1): 2448

  Abstract

  Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

  View details for DOI 10.1038/s41467-023-38106-3

  View details for PubMedID 37117181

  View details for PubMedCentralID 4829541

• Profiling oncogenic extra-chromosomal DNA in cancer NATURE GENETICS Hung, K. L., Chang, H. Y. 2022: 1591-1592

  View details for DOI 10.1038/s41588-022-01193-x

  View details for Web of Science ID 000877757400001

  View details for PubMedID 36319855

• Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH. Nature genetics Hung, K. L., Luebeck, J., Dehkordi, S. R., Colon, C. I., Li, R., Wong, I. T., Coruh, C., Dharanipragada, P., Lomeli, S. H., Weiser, N. E., Moriceau, G., Zhang, X., Bailey, C., Houlahan, K. E., Yang, W., Gonzalez, R. C., Swanton, C., Curtis, C., Jamal-Hanjani, M., Henssen, A. G., Law, J. A., Greenleaf, W. J., Lo, R. S., Mischel, P. S., Bafna, V., Chang, H. Y. 2022

  Abstract

  Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

  View details for DOI 10.1038/s41588-022-01190-0

  View details for PubMedID 36253572

• Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo. Nature communications Cheng, R. Y., Hung, K. L., Zhang, T., Stoffers, C. M., Ott, A. R., Suchland, E. R., Camp, N. D., Khan, I. F., Singh, S., Yang, Y., Rawlings, D. J., James, R. G. 2022; 13 (1): 6110

  Abstract

  Due to their unique longevity and capacity to secrete high levels of protein, plasma B cells have the potential to be used as a cell therapy for protein replacement. Here, we show that ex vivo engineered human plasma cells exhibit single-cell RNA profiles, scanning electron micrograph ultrastructural features, and in vivo homing capacity of long-lived plasma cells. After transferring human plasma cells to immunodeficient mice in the presence of the human cytokines BAFF and IL-6, we observe increases in retention of plasma cells in the bone marrow, with engraftment exceeding a year. The most profound in vivo effects of human IL-6 are observed within 20 days of transfer and could be explained by decreased apoptosis in newly differentiated plasma cells. Collectively, these results show that ex vivo engineered and differentiated human plasma cells have the potential for long-lived in vivo protein secretion, which can be modeled in small animals.

  View details for DOI 10.1038/s41467-022-33787-8

  View details for PubMedID 36245034

• The evolutionary dynamics of extrachromosomal DNA in human cancers. Nature genetics Lange, J. T., Rose, J. C., Chen, C. Y., Pichugin, Y., Xie, L., Tang, J., Hung, K. L., Yost, K. E., Shi, Q., Erb, M. L., Rajkumar, U., Wu, S., Taschner-Mandl, S., Bernkopf, M., Swanton, C., Liu, Z., Huang, W., Chang, H. Y., Bafna, V., Henssen, A. G., Werner, B., Mischel, P. S. 2022

  Abstract

  Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.

  View details for DOI 10.1038/s41588-022-01177-x

  View details for PubMedID 36123406

• Gene regulation on extrachromosomal DNA. Nature structural & molecular biology Hung, K. L., Mischel, P. S., Chang, H. Y. 2022

  Abstract

  Oncogene amplification on extrachromosomal DNA (ecDNA) is prevalent in human cancer and is associated with poor outcomes. Clonal, megabase-sized circular ecDNAs in cancer are distinct from nonclonal, small sub-kilobase-sized DNAs that may arise during normal tissue homeostasis. ecDNAs enable profound changes in gene regulation beyond copy-number gains. An emerging principle of ecDNA regulation is the formation of ecDNA hubs: micrometer-sized nuclear structures of numerous copies of ecDNAs tethered by proteins in spatial proximity. ecDNA hubs enable cooperative and intermolecular sharing of DNA regulatory elements for potent and combinatorial gene activation. The 3D context of ecDNA shapes its gene expression potential, selection for clonal heterogeneity among ecDNAs, distribution through cell division, and reintegration into chromosomes. Technologies for studying gene regulation and structure of ecDNA are starting to answer long-held questions on the distinct rules that govern cancer genes beyond chromosomes.

  View details for DOI 10.1038/s41594-022-00806-7

  View details for PubMedID 35948767

• Dissecting the role of Stag2 in lung adenocarcinoma Ashkin, E. L., Cai, H., Tang, Y. J., Li, C., Chew, S., Hung, K., Belk, J., Karmakar, S., Hebert, J., Yousefi, M., Swanton, C., Petrov, D. A., Winslow, M. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509506122

• Oncogene Convergence in Extrachromosomal DNA Hubs. Cancer discovery Weiser, N. E., Hung, K. L., Chang, H. Y. 2022: OF1-OF4

  Abstract

  Extrachromosomal DNA circles (ecDNA) are a common mechanism for oncogene amplification and are associated with worse clinical outcomes compared with other types of oncogene amplification. Several recent discoveries of ecDNA hubs-local congregations of ecDNAs in the nucleus-highlight unique features of ecDNA biology that may contribute to higher oncogene expression and rapid tumor evolution.

  View details for DOI 10.1158/2159-8290.CD-22-0076

  View details for PubMedID 35398879

• The Primate Selective Transduction of rAAV-LK03 Vectors is Related to Variation in Histone and Histone Post-Translational Modifications on the Viral Genome in the Host Nucleus Gonzalez-Sandoval, A., Tsuji, S., Hung, K. L., Zhang, F., Kay, M. A. CELL PRESS. 2022: 408

  View details for Web of Science ID 000794043702045

• ecDNA hubs drive cooperative intermolecular oncogene expression. Nature Hung, K. L., Yost, K. E., Xie, L., Shi, Q., Helmsauer, K., Luebeck, J., Schopflin, R., Lange, J. T., Chamorro Gonzalez, R., Weiser, N. E., Chen, C., Valieva, M. E., Wong, I. T., Wu, S., Dehkordi, S. R., Duffy, C. V., Kraft, K., Tang, J., Belk, J. A., Rose, J. C., Corces, M. R., Granja, J. M., Li, R., Rajkumar, U., Friedlein, J., Bagchi, A., Satpathy, A. T., Tjian, R., Mundlos, S., Bafna, V., Henssen, A. G., Mischel, P. S., Liu, Z., Chang, H. Y. 2021

  Abstract

  Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

  View details for DOI 10.1038/s41586-021-04116-8

  View details for PubMedID 34819668

• Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell Nunez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J. Y., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., Weissman, J. S. 2021

  Abstract

  A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

  View details for DOI 10.1016/j.cell.2021.03.025

  View details for PubMedID 33838111

• A functional taxonomy of tumor suppression in oncogenic KRAS-driven lung cancer. Cancer discovery Cai, H. n., Chew, S. K., Li, C. n., Tsai, M. K., Andrejka, L. n., Murray, C. W., Hughes, N. W., Shuldiner, E. G., Ashkin, E. L., Tang, R. n., Hung, K. L., Chen, L. C., Lee, S. Y., Yousefi, M. n., Lin, W. Y., Kunder, C. A., Cong, L. n., McFarland, C. D., Petrov, D. A., Swanton, C. n., Winslow, M. M. 2021

  Abstract

  Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multi-step process, however the importance and specific roles of many of these genes during tumor initiation, growth and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of forty-eight known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, while the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression.

  View details for DOI 10.1158/2159-8290.CD-20-1325

  View details for PubMedID 33608386

• Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells MOLECULAR THERAPY Hung, K. L., Meitlis, I., Hale, M., Chen, C., Singh, S., Jackson, S. W., Miao, C. H., Khan, I. F., Rawlings, D. J., James, R. G. 2018; 26 (2): 456–67

  Abstract

  The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

  View details for PubMedID 29273498

  View details for PubMedCentralID PMC5835153

• Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm DEVELOPMENT Bouldin, C. M., Manning, A. J., Peng, Y., Farr, G. H., Hung, K. L., Dong, A., Kimelman, D. 2015; 142 (14): 2499-+

  Abstract

  Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitors have a limited rate of proliferation and their maintenance is crucial for completion of the anterior-posterior axis. How they leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that zebrafish Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative we are able to show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together, our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high-Wnt environment.

  View details for PubMedID 26062939

  View details for PubMedCentralID PMC4510867