Julia Belk

All Publications

• Clonal hematopoiesis is associated with protection from Alzheimer's disease. Nature medicine Bouzid, H., Belk, J. A., Jan, M., Qi, Y., Sarnowski, C., Wirth, S., Ma, L., Chrostek, M. R., Ahmad, H., Nachun, D., Yao, W., Beiser, A., Bick, A. G., Bis, J. C., Fornage, M., Longstreth, W. T., Lopez, O. L., Natarajan, P., Psaty, B. M., Satizabal, C. L., Weinstock, J., Larson, E. B., Crane, P. K., Keene, C. D., Seshadri, S., Satpathy, A. T., Montine, T. J., Jaiswal, S. 2023

  Abstract

  Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant expansion of mutated hematopoietic stem cells. As CHIP-associated mutations are known to alter the development and function of myeloid cells, we hypothesized that CHIP may also be associated with the risk of Alzheimer's disease (AD), a disease in which brain-resident myeloid cells are thought to have a major role. To perform association tests between CHIP and AD dementia, we analyzed blood DNA sequencing data from 1,362 individuals with AD and 4,368 individuals without AD. Individuals with CHIP had a lower risk of AD dementia (meta-analysis odds ratio (OR) = 0.64, P = 3.8 × 10-5), and Mendelian randomization analyses supported a potential causal association. We observed that the same mutations found in blood were also detected in microglia-enriched fraction of the brain in seven of eight CHIP carriers. Single-nucleus chromatin accessibility profiling of brain-derived nuclei in six CHIP carriers revealed that the mutated cells comprised a large proportion of the microglial pool in the samples examined. While additional studies are required to validate the mechanistic findings, these results suggest that CHIP may have a role in attenuating the risk of AD.

  View details for DOI 10.1038/s41591-023-02397-2

  View details for PubMedID 37322115

  View details for PubMedCentralID 4306669

• A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages. Immunity Brioschi, S., Belk, J. A., Peng, V., Molgora, M., Rodrigues, P. F., Nguyen, K. M., Wang, S., Du, S., Wang, W. L., Grajales-Reyes, G. E., Ponce, J. M., Yuede, C. M., Li, Q., Baer, J. M., DeNardo, D. G., Gilfillan, S., Cella, M., Satpathy, A. T., Colonna, M. 2023

  Abstract

  Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.

  View details for DOI 10.1016/j.immuni.2023.01.028

  View details for PubMedID 36791722

• Enhanced T cell effector activity by targeting the Mediator kinase module. Science (New York, N.Y.) Freitas, K. A., Belk, J. A., Sotillo, E., Quinn, P. J., Ramello, M. C., Malipatlolla, M., Daniel, B., Sandor, K., Klysz, D., Bjelajac, J., Xu, P., Burdsall, K. A., Tieu, V., Duong, V. T., Donovan, M. G., Weber, E. W., Chang, H. Y., Majzner, R. G., Espinosa, J. M., Satpathy, A. T., Mackall, C. L. 2022; 378 (6620): eabn5647

  Abstract

  T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

  View details for DOI 10.1126/science.abn5647

  View details for PubMedID 36356142

• Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer cell Belk, J. A., Yao, W., Ly, N., Freitas, K. A., Chen, Y. T., Shi, Q., Valencia, A. M., Shifrut, E., Kale, N., Yost, K. E., Duffy, C. V., Daniel, B., Hwee, M. A., Miao, Z., Ashworth, A., Mackall, C. L., Marson, A., Carnevale, J., Vardhana, S. A., Satpathy, A. T. 2022

  Abstract

  T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

  View details for DOI 10.1016/j.ccell.2022.06.001

  View details for PubMedID 35750052

• Epigenetic regulation of T cell exhaustion. Nature immunology Belk, J. A., Daniel, B., Satpathy, A. T. 2022

  Abstract

  Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.

  View details for DOI 10.1038/s41590-022-01224-z

  View details for PubMedID 35624210

• Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions. Cell Flynn, R. A., Belk, J. A., Qi, Y., Yasumoto, Y., Wei, J., Alfajaro, M. M., Shi, Q., Mumbach, M. R., Limaye, A., DeWeirdt, P. C., Schmitz, C. O., Parker, K. R., Woo, E., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. R., Wilen, C. B., Satpathy, A. T. 2021

  Abstract

  SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

  View details for DOI 10.1016/j.cell.2021.03.012

  View details for PubMedID 33743211

• Coordinated inheritance of extrachromosomal DNAs in cancer cells. Nature Hung, K. L., Jones, M. G., Wong, I. T., Curtis, E. J., Lange, J. T., He, B. J., Luebeck, J., Schmargon, R., Scanu, E., Bruckner, L., Yan, X., Li, R., Gnanasekar, A., Chamorro Gonzalez, R., Belk, J. A., Liu, Z., Melillo, B., Bafna, V., Dorr, J. R., Werner, B., Huang, W., Cravatt, B. F., Henssen, A. G., Mischel, P. S., Chang, H. Y. 2024; 635 (8037): 201-209

  Abstract

  The chromosomal theory of inheritance dictates that genes on the same chromosome segregate together while genes on different chromosomes assort independently1. Extrachromosomal DNAs (ecDNAs) are common in cancer and drive oncogene amplification, dysregulated gene expression and intratumoural heterogeneity through random segregation during cell division2,3. Distinct ecDNA sequences, termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells4. How multiple ecDNA species within a tumour cell are assorted and maintained across somatic cell generations is unclear. Here we show that cooperative ecDNA species are 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 before any selection. Intermolecular proximity and active transcription at the start of mitosis facilitate the coordinated segregation of ecDNA species, and transcription inhibition reduces co-segregation. Computational modelling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. 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 10.1038/s41586-024-07861-8

  View details for PubMedID 39506152

• Deciphering regulation of FOXP3 expression in human conventional T cells. bioRxiv : the preprint server for biology Umhoefer, J. M., Arce, M. M., Dajani, R., Belk, J. A., Mowery, C. T., Nguyen, V., Gowen, B. G., Simeonov, D. R., Curie, G. L., Corn, J. E., Chang, H. Y., Marson, A. 2024

  Abstract

  FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

  View details for DOI 10.1101/2024.08.30.610436

  View details for PubMedID 39282425

  View details for PubMedCentralID PMC11398386

• Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair. Cancer discovery Rose, J. C., Belk, J. A., Wong, I. T., Luebeck, J., Horn, H. T., Daniel, B., Jones, M. G., Yost, K. E., Hung, K. L., Kolahi, K. S., Curtis, E. J., Kuo, C. J., Bafna, V., Mischel, P. S., Chang, H. Y. 2024

  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 ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 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 can 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 10.1158/2159-8290.CD-23-1117

  View details for PubMedID 39109936

• Clonal inactivation of TERT impairs stem cell competition. Nature Hasegawa, K., Zhao, Y., Garbuzov, A., Corces, M. R., Neuhöfer, P., Gillespie, V. M., Cheung, P., Belk, J. A., Huang, Y. H., Wei, Y., Chen, L., Chang, H. Y., Artandi, S. E. 2024

  Abstract

  Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.

  View details for DOI 10.1038/s41586-024-07700-w

  View details for PubMedID 39020172

  View details for PubMedCentralID 7377944

• Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation. Immunity Buquicchio, F. A., Fonseca, R., Yan, P. K., Wang, F., Evrard, M., Obers, A., Gutierrez, J. C., Raposo, C. J., Belk, J. A., Daniel, B., Zareie, P., Yost, K. E., Qi, Y., Yin, Y., Nico, K. F., Tierney, F. M., Howitt, M. R., Lareau, C. A., Satpathy, A. T., Mackay, L. K. 2024

  Abstract

  The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

  View details for DOI 10.1016/j.immuni.2024.06.014

  View details for PubMedID 39043184

• Bidirectional epigenetic editing reveals hierarchies in gene regulation. Nature biotechnology Pacalin, N. M., Steinhart, Z., Shi, Q., Belk, J. A., Dorovskyi, D., Kraft, K., Parker, K. R., Shy, B. R., Marson, A., Chang, H. Y. 2024

  Abstract

  CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

  View details for DOI 10.1038/s41587-024-02213-3

  View details for PubMedID 38760566

• Xist ribonucleoproteins promote female sex-biased autoimmunity. Cell Dou, D. R., Zhao, Y., Belk, J. A., Zhao, Y., Casey, K. M., Chen, D. C., Li, R., Yu, B., Srinivasan, S., Abe, B. T., Kraft, K., Hellström, C., Sjöberg, R., Chang, S., Feng, A., Goldman, D. W., Shah, A. A., Petri, M., Chung, L. S., Fiorentino, D. F., Lundberg, E. K., Wutz, A., Utz, P. J., Chang, H. Y. 2024; 187 (3): 733-749.e16

  Abstract

  Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.

  View details for DOI 10.1016/j.cell.2023.12.037

  View details for PubMedID 38306984

• Chromatin Accessibility Landscapes of CD4+T cells in Monozygotic Discordant Twin pairs for Asthma. Zhou, X., Sindher, S., Chinthrajah, R., Belk, J., Chang, H., Nadeau, K. MOSBY-ELSEVIER. 2024: AB379

  View details for Web of Science ID 001267526000875

• Xist Ribonucleoproteins Promote Female Sex-biased Autoimmunity Dou, D., Zhao, Y., Belk, J., Zhao, Y., Casey, K., Chen, D., Li, R., Yu, B., Srinivasan, S., Abe, B., Kraft, K., Hellstroem, C., Sjoeberg, R., Chang, S., Feng, A., Goldman, D., Shah, A., Petri, M., Chung, L., Fiorentino, D., Lundberg, E., Wutz, A., Utz, P., Chang, H. WILEY. 2023: 25-26

  View details for Web of Science ID 001190014300018

• 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

• Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis. Nature Weinstock, J. S., Gopakumar, J., Burugula, B. B., Uddin, M. M., Jahn, N., Belk, J. A., Bouzid, H., Daniel, B., Miao, Z., Ly, N., Mack, T. M., Luna, S. E., Prothro, K. P., Mitchell, S. R., Laurie, C. A., Broome, J. G., Taylor, K. D., Guo, X., Sinner, M. F., von Falkenhausen, A. S., Kääb, S., Shuldiner, A. R., O'Connell, J. R., Lewis, J. P., Boerwinkle, E., Barnes, K. C., Chami, N., Kenny, E. E., Loos, R. J., Fornage, M., Hou, L., Lloyd-Jones, D. M., Redline, S., Cade, B. E., Psaty, B. M., Bis, J. C., Brody, J. A., Silverman, E. K., Yun, J. H., Qiao, D., Palmer, N. D., Freedman, B. I., Bowden, D. W., Cho, M. H., DeMeo, D. L., Vasan, R. S., Yanek, L. R., Becker, L. C., Kardia, S. L., Peyser, P. A., He, J., Rienstra, M., Van der Harst, P., Kaplan, R., Heckbert, S. R., Smith, N. L., Wiggins, K. L., Arnett, D. K., Irvin, M. R., Tiwari, H., Cutler, M. J., Knight, S., Muhlestein, J. B., Correa, A., Raffield, L. M., Gao, Y., de Andrade, M., Rotter, J. I., Rich, S. S., Tracy, R. P., Konkle, B. A., Johnsen, J. M., Wheeler, M. M., Smith, J. G., Melander, O., Nilsson, P. M., Custer, B. S., Duggirala, R., Curran, J. E., Blangero, J., McGarvey, S., Williams, L. K., Xiao, S., Yang, M., Gu, C. C., Chen, Y. I., Lee, W. J., Marcus, G. M., Kane, J. P., Pullinger, C. R., Shoemaker, M. B., Darbar, D., Roden, D. M., Albert, C., Kooperberg, C., Zhou, Y., Manson, J. E., Desai, P., Johnson, A. D., Mathias, R. A., Blackwell, T. W., Abecasis, G. R., Smith, A. V., Kang, H. M., Satpathy, A. T., Natarajan, P., Kitzman, J. O., Whitsel, E. A., Reiner, A. P., Bick, A. G., Jaiswal, S. 2023

  Abstract

  Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

  View details for DOI 10.1038/s41586-023-05806-1

  View details for PubMedID 37046083

  View details for PubMedCentralID 4624443

• Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation. Genes & development Liu, T. T., Ou, F., Belk, J. A., Bagadia, P., Anderson, D. A., Durai, V., Yao, W., Satpathy, A. T., Murphy, T. L., Murphy, K. M. 2023

  Abstract

  Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.

  View details for DOI 10.1101/gad.350339.122

  View details for PubMedID 36990511

• TCR-BERT: learning the grammar of T-cell receptors for flexible antigen-binding analyses Wu, K., Yost, K. E., Daniel, B., Belk, J. A., Xia, Y., Egawa, T., Satpathy, A., Chang, H. Y., Zou, J., Knowles, D. A., Mostafavi, S. JMLR-JOURNAL MACHINE LEARNING RESEARCH. 2023

  View details for Web of Science ID 001221072500010

• Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state. Molecular cell Daniel, B., Belk, J. A., Meier, S. L., Chen, A. Y., Sandor, K., Czimmerer, Z., Varga, Z., Bene, K., Buquicchio, F. A., Qi, Y., Kitano, H., Wheeler, J. R., Foster, D. S., Januszyk, M., Longaker, M. T., Chang, H. Y., Satpathy, A. T. 2022

  Abstract

  Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings invivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

  View details for DOI 10.1016/j.molcel.2022.11.017

  View details for PubMedID 36521490

• Engineered cell entry links receptor biology with single-cell genomics. Cell Yu, B., Shi, Q., Belk, J. A., Yost, K. E., Parker, K. R., Li, R., Liu, B. B., Huang, H., Lingwood, D., Greenleaf, W. J., Davis, M. M., Satpathy, A. T., Chang, H. Y. 2022

  Abstract

  Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between Tcell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual Tcells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory Tcell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

  View details for DOI 10.1016/j.cell.2022.11.016

  View details for PubMedID 36516854

• Divergent clonal differentiation trajectories of T cell exhaustion. Nature immunology Daniel, B., Yost, K. E., Hsiung, S., Sandor, K., Xia, Y., Qi, Y., Hiam-Galvez, K. J., Black, M., J Raposo, C., Shi, Q., Meier, S. L., Belk, J. A., Giles, J. R., Wherry, E. J., Chang, H. Y., Egawa, T., Satpathy, A. T. 2022

  Abstract

  Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

  View details for DOI 10.1038/s41590-022-01337-5

  View details for PubMedID 36289450

• RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature Carnevale, J., Shifrut, E., Kale, N., Nyberg, W. A., Blaeschke, F., Chen, Y. Y., Li, Z., Bapat, S. P., Diolaiti, M. E., O'Leary, P., Vedova, S., Belk, J., Daniel, B., Roth, T. L., Bachl, S., Anido, A. A., Prinzing, B., Ibanez-Vega, J., Lange, S., Haydar, D., Luetke-Eversloh, M., Born-Bony, M., Hegde, B., Kogan, S., Feuchtinger, T., Okada, H., Satpathy, A. T., Shannon, K., Gottschalk, S., Eyquem, J., Krenciute, G., Ashworth, A., Marson, A. 2022

  Abstract

  The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulationandcan increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in thebone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

  View details for DOI 10.1038/s41586-022-05126-w

  View details for PubMedID 36002574

• Author Correction: Engineering circular RNA for enhanced protein production. Nature biotechnology Chen, R., Wang, S. K., Belk, J. A., Amaya, L., Li, Z., Cardenas, A., Abe, B. T., Chen, C., Wender, P. A., Chang, H. Y. 2022

  View details for DOI 10.1038/s41587-022-01472-2

  View details for PubMedID 35978134

• Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection. Nature genetics Biering, S. B., Sarnik, S. A., Wang, E., Zengel, J. R., Leist, S. R., Schafer, A., Sathyan, V., Hawkins, P., Okuda, K., Tau, C., Jangid, A. R., Duffy, C. V., Wei, J., Gilmore, R. C., Alfajaro, M. M., Strine, M. S., Nguyenla, X., Van Dis, E., Catamura, C., Yamashiro, L. H., Belk, J. A., Begeman, A., Stark, J. C., Shon, D. J., Fox, D. M., Ezzatpour, S., Huang, E., Olegario, N., Rustagi, A., Volmer, A. S., Livraghi-Butrico, A., Wehri, E., Behringer, R. R., Cheon, D., Schaletzky, J., Aguilar, H. C., Puschnik, A. S., Button, B., Pinsky, B. A., Blish, C. A., Baric, R. S., O'Neal, W. K., Bertozzi, C. R., Wilen, C. B., Boucher, R. C., Carette, J. E., Stanley, S. A., Harris, E., Konermann, S., Hsu, P. D. 2022

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

  View details for DOI 10.1038/s41588-022-01131-x

  View details for PubMedID 35879412

• Engineering circular RNA for enhanced protein production. Nature biotechnology Chen, R., Wang, S. K., Belk, J. A., Amaya, L., Li, Z., Cardenas, A., Abe, B. T., Chen, C., Wender, P. A., Chang, H. Y. 2022

  Abstract

  Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.

  View details for DOI 10.1038/s41587-022-01393-0

  View details for PubMedID 35851375

• Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature cancer Mabe, N. W., Huang, M., Dalton, G. N., Alexe, G., Schaefer, D. A., Geraghty, A. C., Robichaud, A. L., Conway, A. S., Khalid, D., Mader, M. M., Belk, J. A., Ross, K. N., Sheffer, M., Linde, M. H., Ly, N., Yao, W., Rotiroti, M. C., Smith, B. A., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C. S., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. 2022

  Abstract

  Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

  View details for DOI 10.1038/s43018-022-00405-x

  View details for PubMedID 35817829

• Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells Freitas, K. A., Belk, J. A., Sotillo, E., Daniel, B., Sandor, K., Klysz, D., Duong, V. T., Xu, P., Malipatlolla, M., Weber, E. W., Majzner, R. G., Chang, H. Y., Satpathy, A. T., Mackall, C. AMER ASSOC CANCER RESEARCH. 2022

  View details for Web of Science ID 000892509507415

• 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

• Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer. Cancer cell Kersten, K., Hu, K. H., Combes, A. J., Samad, B., Harwin, T., Ray, A., Rao, A. A., Cai, E., Marchuk, K., Artichoker, J., Courau, T., Shi, Q., Belk, J., Satpathy, A. T., Krummel, M. F. 2022

  Abstract

  T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

  View details for DOI 10.1016/j.ccell.2022.05.004

  View details for PubMedID 35623342

• Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell Reticker-Flynn, N. E., Zhang, W., Belk, J. A., Basto, P. A., Escalante, N. K., Pilarowski, G. O., Bejnood, A., Martins, M. M., Kenkel, J. A., Linde, I. L., Bagchi, S., Yuan, R., Chang, S., Spitzer, M. H., Carmi, Y., Cheng, J., Tolentino, L. L., Choi, O., Wu, N., Kong, C. S., Gentles, A. J., Sunwoo, J. B., Satpathy, A. T., Plevritis, S. K., Engleman, E. G. 2022

  Abstract

  For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

  View details for DOI 10.1016/j.cell.2022.04.019

  View details for PubMedID 35525247

• Loquacious modulates flaviviral RNA replication in mosquito cells. PLoS pathogens Shivaprasad, S., Weng, K. F., Ooi, Y. S., Belk, J., Carette, J. E., Flynn, R., Sarnow, P. 2022; 18 (4): e1010163

  Abstract

  Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.

  View details for DOI 10.1371/journal.ppat.1010163

  View details for PubMedID 35482886

• Leveraging nonstructural data to predict structures and affinities of protein-ligand complexes. Proceedings of the National Academy of Sciences of the United States of America Paggi, J. M., Belk, J. A., Hollingsworth, S. A., Villanueva, N., Powers, A. S., Clark, M. J., Chemparathy, A. G., Tynan, J. E., Lau, T. K., Sunahara, R. K., Dror, R. O. 1800; 118 (51)

  Abstract

  Over the past five decades, tremendous effort has been devoted to computational methods for predicting properties of ligands-i.e., molecules that bind macromolecular targets. Such methods, which are critical to rational drug design, fall into two categories: physics-based methods, which directly model ligand interactions with the target given the target's three-dimensional (3D) structure, and ligand-based methods, which predict ligand properties given experimental measurements for similar ligands. Here, we present a rigorous statistical framework to combine these two sources of information. We develop a method to predict a ligand's pose-the 3D structure of the ligand bound to its target-that leverages a widely available source of information: a list of other ligands that are known to bind the same target but for which no 3D structure is available. This combination of physics-based and ligand-based modeling improves pose prediction accuracy across all major families of drug targets. Using the same framework, we develop a method for virtual screening of drug candidates, which outperforms standard physics-based and ligand-based virtual screening methods. Our results suggest broad opportunities to improve prediction of various ligand properties by combining diverse sources of information through customized machine-learning approaches.

  View details for DOI 10.1073/pnas.2112621118

  View details for PubMedID 34921117

• 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

• Clonal Hematopoiesis is Associated with Reduced Risk of Alzheimer's Disease Bouzid, H., Belk, J., Jan, M., Qi, Y., Sarnowski, C., Wirth, S., Ma, L., Chrostek, M., Ahmad, H., Nachun, D., Yao, W., Beiser, A., Bick, A. G., Bis, J., Fornage, M., Longstreth, W. T., Lopez, O., Nataranjan, P., Psaty, B., Satizabal, C., Weinstock, J., Larson, E., Crane, P., Keene, C., Seshadri, S., Satpathy, A. T., Montine, T., Jaiswal, S. AMER SOC HEMATOLOGY. 2021

  View details for DOI 10.1182/blood-2021-151064

  View details for Web of Science ID 000736398800006

• A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance. The Journal of experimental medicine Warshauer, J. T., Belk, J. A., Chan, A. Y., Wang, J., Gupta, A. R., Shi, Q., Skartsis, N., Peng, Y., Phipps, J. D., Acenas, D., Smith, J. A., Tamaki, S. J., Tang, Q., Gardner, J. M., Satpathy, A. T., Anderson, M. S. 2021; 218 (8)

  Abstract

  Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

  View details for DOI 10.1084/jem.20210759

  View details for PubMedID 34115115

• Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis. Immunity Huang, X., Ferris, S. T., Kim, S., Choudhary, M. N., Belk, J. A., Fan, C., Qi, Y., Sudan, R., Xia, Y., Desai, P., Chen, J., Ly, N., Shi, Q., Bagadia, P., Liu, T., Guilliams, M., Egawa, T., Colonna, M., Diamond, M. S., Murphy, T. L., Satpathy, A. T., Wang, T., Murphy, K. M. 2021

  Abstract

  The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Delta-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Delta-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Delta-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at+164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

  View details for DOI 10.1016/j.immuni.2021.04.015

  View details for PubMedID 34004142

• The Role of Aire in the selection of Regulatory T cells in Type 1 Diabetes Bridge, J., Balolong, J., Belk, J., Dong, S., Cramer, N., Mowery, C., Crawford, F., Ye, J., Satpathy, A., Kappler, J. W., Bluestone, J. A., Anderson, M. S. AMER ASSOC IMMUNOLOGISTS. 2021

  View details for Web of Science ID 000713665800398

• Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science (New York, N.Y.) Weber, E. W., Parker, K. R., Sotillo, E., Lynn, R. C., Anbunathan, H., Lattin, J., Good, Z., Belk, J. A., Daniel, B., Klysz, D., Malipatlolla, M., Xu, P., Bashti, M., Heitzeneder, S., Labanieh, L., Vandris, P., Majzner, R. G., Qi, Y., Sandor, K., Chen, L., Prabhu, S., Gentles, A. J., Wandless, T. J., Satpathy, A. T., Chang, H. Y., Mackall, C. L. 2021; 372 (6537)

  Abstract

  T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.

  View details for DOI 10.1126/science.aba1786

  View details for PubMedID 33795428

• Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses. Immunity Wong, R., Belk, J. A., Govero, J., Uhrlaub, J. L., Reinartz, D., Zhao, H., Errico, J. M., D'Souza, L., Ripperger, T. J., Nikolich-Zugich, J., Shlomchik, M. J., Satpathy, A. T., Fremont, D. H., Diamond, M. S., Bhattacharya, D. 2020

  Abstract

  Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

  View details for DOI 10.1016/j.immuni.2020.09.001

  View details for PubMedID 33010224

• Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection. bioRxiv : the preprint server for biology Flynn, R. A., Belk, J. A., Qi, Y. n., Yasumoto, Y. n., Schmitz, C. O., Mumbach, M. R., Limaye, A. n., Wei, J. n., Alfajaro, M. M., Parker, K. R., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. n., Wilen, C. B., Satpathy, A. T. 2020

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.· ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.

  View details for DOI 10.1101/2020.10.06.327445

  View details for PubMedID 33052334

  View details for PubMedCentralID PMC7553159