Complementary Screen Research Articles

Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells.

Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption ofnuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

Babinet's principle and saturation of the resonance frequency of scaled-down complementary metasurfaces

It has been previously demonstrated that the resonant frequency of the Split Ring Resonator (SRR) reaches a saturation value when its size is continuously scaled down, and at the same time, the resonance is attenuated. Now, it is demonstrated that similar saturation and attenuation phenomena happens to its complementary screen, which is commonly called a Complementary SRR (CSRR). It was found that the saturation frequency of the CSRR is near the saturation frequency of the SRR with equal geometrical dimensions. It is possible to control their saturation levels by tuning the geometry of the resonators. This work is relevant for the design of quasi-optical metamaterials based on one or both kinds of structures (i.e., SRRs and CSRRs) at once, especially when they both are required to resonate at the same frequency.

A Broken Mirror Held to History’s Face On the Narrative Use of Computer Screens, Multi Screen Experiences, and a Transmedia Theoretical Console in the Popular Assassin’s Creed Series

This paper presents media theorist Nanna Verhoeff’s concept of the theoretical console, as a popular and overt form of transmedia narrative. The theoretical console is taken to be a transmedia assemblage that draws attention to itself, as comprising diverse and meaningful media objects, that can be connected in a shared narrative. My main examples of this concept here are those popular video games that spatially juxtapose several types of computer screens and computer uses, with a narrative emphasis. With extensive references to theory on screened media and on transmedia narratives, Assassin’s Creed IV: Black Flag is my main example case study. Specifically, this game and its series peers encourage historiographic contemplations, by assembling a theoretical console across several media forms. Other popular video games from that series provide variations of this same transmedia constellation, this “theoretical console”. In its transmedia constellation, with a second screen mobile phone app, and other complementary screen media, the fictionalized history of Assassin’s Creed IV: Black Flag implicates elements from its actual reality, across various forms of engagement. Assassin’s Creed IV: Black Flag presents a high fidelity historical fiction, to be comprehensively enacted. It mirrors the player’s use of differing computer screens diegetically within playable frame story sections. In addition, the complementary affordances of the mobile phone app, and integrated social media websites, all encourage its player to stay involved in this fictional world, even outside immediate play. With this, the game draws many activities into a single transmedial fiction constellation. Moreover, the game diegetically references online repositories for both its fictionalized history, and actual history. This use of computer screens, to form a transmedia constellation in the form of an overt theoretical console, is shown to complement this popular game’s hypermediative narrative of a fictional shadow war secretly driving actual human history, which then meaningfully posits how to theorize history in our everyday lives.

Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

SummaryKaposi’s sarcoma herpesvirus (KSHV) is an oncogenic human virus and the leading cause of mortality in HIV infection. KSHV reactivation from latent- to lytic-stage infection initiates a cascade of viral gene expression. Here we show how these changes remodel the host cell proteome to enable viral replication. By undertaking a systematic and unbiased analysis of changes to the endothelial cell proteome following KSHV reactivation, we quantify >7,000 cellular proteins and 71 viral proteins and provide a temporal profile of protein changes during the course of lytic KSHV infection. Lytic KSHV induces >2-fold downregulation of 291 cellular proteins, including PKR, the key cellular sensor of double-stranded RNA. Despite the multiple episomes per cell, CRISPR-Cas9 efficiently targets KSHV genomes. A complementary KSHV genome-wide CRISPR genetic screen identifies K5 as the viral gene responsible for the downregulation of two KSHV targets, Nectin-2 and CD155, ligands of the NK cell DNAM-1 receptor.

Chemokine (C-X-C Motif) Ligand 4 Is a Restrictor of Respiratory Syncytial Virus Infection and an Indicator of Clinical Severity.

Rationale: Respiratory syncytial virus (RSV) is the leading cause of childhood respiratory infections worldwide; however, no vaccine is available, and treatment options are limited. Identification of host factors pivotal to viral replication may inform the development of novel therapies, prophylaxes, or diagnoses.Objectives: To identify host factors involved in RSV replication and to evaluate their potential for disease management.Methods: A gain-of-function screening was performed on the basis of a genome-wide human complementary DNA library screen for host factors involved in RSV replication. The antiviral mechanism of CXCL4 (chemokine [C-X-C motif] ligand 4) was analyzed. Its clinical role was evaluated via nasopharyngeal aspirates and plasma samples from patients with RSV infection and different disease severities.Measurements and Main Results: Forty-nine host factors restricting RSV replication were identified by gain-of-function screening, with CXCL4 showing the strongest antiviral effect, which was secretion dependent. CXCL4 blocked viral attachment through binding to the RSV main receptor heparan sulfate, instead of through interacting with RSV surface proteins. Intranasal pretreatment with CXCL4 alleviated inflammation in RSV-infected mice, as shown by decreased concentrations of tumor necrosis factor and viral load in BAL fluid samples as well as by viral nucleocapsid protein histological staining in lungs. Compared with non-RSV infections, RSV infections induced elevated CXCL4 concentrations both in plasma and airway samples from mice and pediatric patients. The airway CXCL4 concentration was correlated with viral load and disease severity in patients (P < 0.001).Conclusions: Our results suggest that CXCL4 is an RSV restriction factor that can block viral entry and serve as an indicator of clinical severity in RSV infections.

CRISPR-based identification of factors influencing IL-22 and IL-17 expression in type III innate lymphoid cells

Abstract Cytokine regulation of immune responses is influenced by multiple factors, including the target cell type and its microenvironment. Type III innate lymphoid cells (ILC3s) rapidly produce cytokines, including interleukin (IL)-22 and IL-17, in response to pathogens or injury. Dysregulation of IL-22 or IL-17 contributes to numerous pathologies, including autoimmune diseases and malignancies. Thus, a deeper understanding of the factors that regulate productive expression of IL-17/22 by ILCs or by their type III helper T cell counterparts is warranted. We have used a genome-wide CRISPR interference screen to elucidate genes that drive IL-22/17 expression by ILC3s. We engineered the murine ILC3-like line, MNK3, to inducibly express catalytically dead Cas9 (dCas9) linked to the transcriptional repressor KRAB. Following lentiviral transduction of a sgRNA library, cells were stimulated with IL-23 and IL-1b. Guides from cell populations sorted by levels of IL-17/22 expression were sequenced and analyzed for depletion (target may be necessary for cytokine expression, e.g., il17, jak3, and il23r) and enrichment (target’s suppression may promote cytokine expression). We are currently validating “hits” that are components of several biological pathways, including transcriptional regulation, chromatin modification, notch/wnt and cadherin signaling, and RNA splicing. Results will also be presented for a complementary CRISPR activation screen. These data will contribute new insights into the biological processes necessary for proper expression of type 3 cytokines by innate and adaptive lymphoid cells.

Computational Design of an Allosteric Antibody Switch by Deletion and Rescue of a Complex Structural Constellation.

Therapeutic monoclonal antibodies have transformed medicine, especially with regards to treating cancers and disorders of the immune system. More than 50 antibody-derived drugs have already reached the clinic, the majority of which target cytokines or cell-surface receptors. Unfortunately, many of these targets have pleiotropic functions: they serve multiple different roles, and often not all of these roles are disease-related. This can be problematic because antibodies act throughout the body, and systemic neutralization of such targets can lead to safety concerns. To address this, we have developed a strategy whereby an antibody’s ability to recognize its antigen is modulated by a second layer of control, relying on addition of an exogenous small molecule. In previous studies, we began to explore this idea by introducing a deactivating tryptophan-to-glycine mutation in the domain–domain interface of a single-chain variable fragment (scFv), and then restoring activity by adding back indole to fit the designed cavity. Here, we now describe a novel computational strategy for enumerating larger cavities that can be formed by simultaneously introducing multiple adjacent large-to-small mutations; we then carry out a complementary virtual screen to identify druglike compounds to match each candidate cavity. We first demonstrate the utility of this strategy in a fluorescein-binding single-chain variable fragment (scFv) and experimentally characterize a triple mutant with reduced antigen-binding (Rip-3) that can be rescued using a complementary ligand (Stitch-3). Because our design is built upon conserved residues in the antibody framework, we then show that the same mutation/ligand pair can also be used to modulate antigen-binding in an scFv build from a completely unrelated framework. This set of residues is present in many therapeutic antibodies as well, suggesting that this mutation/ligand pair may serve as a general starting point for introducing ligand-dependence into many clinically relevant antibodies.

Overlapping the electric and magnetic dipole resonances of a silver 2D Babinet-type metasurface: Broadband high reflectance with local field enhancement

Optical properties are numerically studied of a plasmonic planar 2D noble metal nanostructure with the design inspired by Babinet’s principle. We stack a 2D array of nanodisks and a complementary screen—2D periodically perforated film—so that they are separated by a dielectric spacer. Near-field interaction between electric and magnetic dipoles induced in metal metasurfaces results in increased reflectance and suppressed absorption, canceling out initial resonances in the individual nanostructures. The proposed metasurface has polarization- and angle of incidence-independent response in a wide frequency range as compared to the resonance bandwidth. The proposed design is robust to possible structural imperfections.

A spatial proteomic approach to identify members involved in the IFN response to HCMV

During the early stages of viral detection, signalling and translocation by key protein mediators initiate antiviral activity. For example, interferon (IFN) signals through the JAK/STAT pathway, leading to phosphorylation of STAT1/2 and movement in complex with IRF9 into the nucleus. To identify novel proteins involved in the IFN response to viruses, we have employed a variety of unbiased, spatial proteomic screens that detect translocation of proteins between different organelles on a global scale. Using three different spatial screens in IFN-stimulated or control human foreskin fibroblasts, we quantified movement of &gt;8000 proteins between the nucleus, cytoplasm and individual organelles. A novel method enabled simultaneous analysis of &gt;2000 phosphoproteins. Proteins were shortlisted based on significant, reproducible movement. Results were validated by appropriate movement of STAT and IRF9 controls. To focus on proteins involved in the IFN response that are important during HCMV replication, we used a complementary screen of proteins that are rapidly downregulated or degraded by HCMV. The results of these screens will be presented, including a shortlist of novel factors that may have new roles in the IFN response to HCMV.

Hybrid sequencing-based personal full-length transcriptomic analysis implicates proteostatic stress in metastatic ovarian cancer.

Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.

Babinet의 원리와 Generalized Sheet Transition Conditions(GSTCs)를 이용한 메타스크린 해석

Using Babinet’s principle and generalized sheet transition conditions(GSTCs), an analysis method for metascreens is proposed. First, the relation of a complementary screen between a metafilm and metascreen with the same dimensions was confirmed using Babinet’s principle. These properties were verified by comparing the reflection coefficient of the patch and the transmission coefficient of the mesh. The patch and mesh are the basic structures of metafilms and metascreens, respectively. Based on these findings, it is also confirmed that the reflection coefficient of the patch surface calculated by GSTCs has the same value as the transmission coefficient of the mesh surface. It is expected that the proposed analysis methods can be applied to the magnitude, phase, and polarization of electromagnetic waves in the various unit structures of a given metasurface.

Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials

Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.

Variation in Leaf Respiration Rates at Night Correlates with Carbohydrate and Amino Acid Supply.

Plant respiration can theoretically be fueled by and dependent upon an array of central metabolism components; however, which ones are responsible for the quantitative variation found in respiratory rates is unknown. Here, large-scale screens revealed 2-fold variation in nighttime leaf respiration rate (RN) among mature leaves from an Arabidopsis (Arabidopsis thaliana) natural accession collection grown under common favorable conditions. RN variation was mostly maintained in the absence of genetic variation, which emphasized the low heritability of RN and its plasticity toward relatively small environmental differences within the sampling regime. To pursue metabolic explanations for leaf RN variation, parallel metabolite level profiling and assays of total protein and starch were performed. Within an accession, RN correlated strongly with stored carbon substrates, including starch and dicarboxylic acids, as well as sucrose, major amino acids, shikimate, and salicylic acid. Among different accessions, metabolite-RN correlations were maintained with protein, sucrose, and major amino acids but not stored carbon substrates. A complementary screen of the effect of exogenous metabolites and effectors on leaf RN revealed that (1) RN is stimulated by the uncoupler FCCP and high levels of substrates, demonstrating that both adenylate turnover and substrate supply can limit leaf RN, and (2) inorganic nitrogen did not stimulate RN, consistent with limited nighttime nitrogen assimilation. Simultaneous measurements of RN and protein synthesis revealed that these processes were largely uncorrelated in mature leaves. These results indicate that differences in preceding daytime metabolic activities are the major source of variation in mature leaf RN under favorable controlled conditions.

The Plasmonic Pixel: Large Area, Wide Gamut Color Reproduction Using Aluminum Nanostructures.

We demonstrate a new plasmonic pixel (PP) design that produces a full-color optical response over macroscopic dimensions. The pixel design employs arrays of aluminum nanorods "floating" above their Babinet complementary screen, Concepts from conventional cyan magenta yellow key (CMYK) printing techniques and red green blue (RGB) digital displays are integrated with nanophotonic design principles and adapted to the production of PP elements. The fundamental PP color blocks of CMYK are implemented via a composite plasmonic nanoantenna/slot design and then mixed in a digital display analog 3 × 3 array to produce a broad-gamut PP. The PP goes beyond current investigations into plasmonic color production by enabling a broad color gamut and physically large plasmonic color features/devices/images. The use of nanorods also leads to a color response that is polarization tunable. Furthermore, devices are fabricated using aluminum and the fabrication strategy is compatible with inexpensive, rapid-throughput, nanoimprint approaches. Here we quantify, both computationally and experimentally, the performance of the PP. Spectral data from a test palette is obtained and a large area (>1.5 cm lateral dimensions) reproduction of a photograph is generated exemplifying the technqiue.

Analytical models for predicting the effective permittivity of complementary metamaterial structures

We present an analytical formula to calculate the resultant effective permittivity of a metamaterial medium composed of unit cells with complementary structures. Complementary metamaterial can be considered to be the complementary screen of artificial magnetic materials. Based on Babinet's principle and duality, complementary metamaterial provides a negative effective permittivity in the vicinity of its resonance frequency. The developed formula is general and predicts the effective permittivity for a specific topology. The derived model validated by comparison to results obtained using full-wave simulation. © 2013 Wiley Periodicals, Inc. Microwave Opt Technol Lett 55:1565–1569, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/mop.27655

Design and study of the active frequency selective surface based on the complementary screen

Separating the load impedance of the resonant frequency selective surface (FSS) can form the complementary frequency selective surface (CFSS), whose filtering feature is based on the coupling mechanism. The resonance of CFSS can be tuned by controlling the rotation angle of the element on the capacitive surface. Taking tripole element for example, the frequency response of the CFSS is calculated by using the coupled integral equation method. A 250 mm250 mm prototype is tested via free space method. Calculation and test results show that the resonance of the CFSS can be tuned actively as the rotation angle changes. This study provides reference to the active FSS design.

Abstract SY37-01: Cilia and wounding cooperate with Hedgehog signaling to initiate basal cell carcinoma

Abstract Primary cilia are microtubule-based projections found on many human cells. These cilia do not move, but function as cellular antennae to receive and interpret signals. For example, olfactory cilia sense odorants in the environment. Other cilia sense cues from other cells, such as Hedgehog proteins, to coordinate tissue patterning and growth. We have identified mechanisms of ciliary signaling, cilium formation, and the regulation of ciliary composition. Defects in ciliary signaling cause some forms of birth defects, cancer, polycystic kidney disease, and other disorders collectively called ciliopathies. To examine whether cilia can participate in disorders beyond recognized ciliopathies, we assessed the prevalence of cilia in human tumors and the role of cilia in cancer. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog signaling, commonly through acquisition of mutations in Smoothened. We found that human BCCs are frequently ciliated. We tested the role of cilia in BCC by conditionally deleting genes required for ciliogenesis in a Hedgehog pathway-dependent mouse model of BCC. Ciliary ablation strongly inhibited BCC-like tumors induced by misactivation of the Hedgehog pathway at the level of Smoothened, revealing that BCC can be addicted to cilia. Skin tumors, like many other cancers, are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Surprisingly, expression of an activated form of Smoothened by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hedgehog signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hedgehog signal transduction is derepressed, giving rise to superficial BCC-like tumors. Thus, BCCs can originate from follicular stem cells and wounding can promote tumorigenesis by moving oncogene-expressing stem cells out of a suppressive niche. As we found that oncogenic Smoothened requires the cilium to promote tumorigenesis, we investigated how Smoothened moves to the cilium. We found that proteins of the Tectonic family form part of the transition zone, a region at the base of the cilium. Tectonic1 is an extracytosolic glycoprotein that interacts with transmembrane components of the transition zone to connect to a cytosolic transition zone complex comprised of the products of genes associated with two ciliopathies, Joubert and Meckel syndromes. Loss of components of the Tectonic transition zone complex in mice compromise ciliogenesis in some tissues, but in others, it is required for Smoothened and select other membrane proteins to localize to cilia. A complementary small molecule screen is identifying new chemicals that also prevent Smoothened localization to cilia and block oncogenic Hedgehog signaling. Interfering with the cellular machinery that moves Smoothened to cilia may be a complementary means of treating Hedgehog pathway-associated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-01. doi:1538-7445.AM2012-SY37-01

Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1

Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5' of the B cell-specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.