Tet-on System Research Articles

Generation and application of CES1-knockout Tet-Off-regulated CYP3A4 and UGT1A1-expressing Caco-2 cells

Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1-knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs.

CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION

Epithelial-to-mesenchymal transition (EMT) is a crucial process that occurs during normal development, such as embryogenesis and organ formation. If it is inappropriately activated, it can lead to pathological processes like metastasis. Although EMT is well studied at the morphological and transcriptome level, cytoskeleton changes during this process are less understood. In our work we aimed to describe the morphological changes that occurred in MCF-7, A-549 and HaCaT cells after EMT, analyze microtubule dynamics, spatial distribution and its contribution to cell motility, identify changes in actin filament organization and study focal adhesion turnover in the cells after EMT. We hypothesized that the dynamics of microtubules in cells undergoing EMT might change. Cells undergoing EMT were expected to have more dynamic microtubules. Also, cells undergoing EMT are expected to adhere more efficiently to diverse substrates, and therefore spread and move more easily. Focal contacts in cells undergoing EMT were expected to be more pronounced and dynamic than in cells that not undergoing EMT. The research methods used in the study included inducing EMT in modified MCF-7 cells through an inserted inducible Tet-on system and stimulation of EMT in A-549 and HaCaT cells using TGF-β. The cells were observed using bright field microscopy, and immunofluorescence analysis was conducted to visualize microtubules and actin filaments. Transfection with EB-3–RFP protein was done to describe and measure microtubule dynamics, while transduction with Talin-RFP and transient transfection with Ptag-RFP-vinculin was done to visualize focal adhesions. Time-lapse fluorescent microscopy was used to record films, and the Fiji Image J program was used to analyze the data. All statistical analysis was performed using GraphPad Prism (Dotmatics, USA), and a nonparametric Mann-Whitney U test or parametric t-test with Welch correction. The actin filament measurements were completed using Matlab scripts. The major research findings: In all three-cell models after EMT, cell morphology changed. Cells increased in size. MCF-7 and HaCaT became spread out, while A-549 became elongated. Some of the cells lost cell-cell contacts. All three cell models after EMT had alterations in microtubule organization and dynamics. MCF-7 and HaCaT cells showed more frequently individual MTs at cell edges, while A-549 had less covered nucleus by MTs after EMT. Microtubule dynamics (growth velocity) increased, and the length of microtubule growth tracks became longer. The average angle of MT growth trajectories to cell radius decreased. Actin fibers rearranged into more pronounced stress fibers after EMT. Alterations in the focal adhesion behaviors were different in three models used. These results indicate that cytoskeletal changes during EMT mainly include increased microtubule dynamics.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

The Tet-on system for controllable gene expression in the rock-inhabiting black fungus Knufia petricola

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K.petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K.petricola cell or for using K. petricola as host for the expression of heterologous genes.

An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri.

Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.

A new hiPSC-based viral expression system to study virus host-cell interaction in hiPSC-derived cardiomyocytes

Abstract Coxsackievirus B3 (CVB3) belongs to the Picornaviridae family and is a member of the enterovirus genus. CVB3 is the most frequently detected pathogen in viral myocarditis, which leads to acute heart infections in 20-40% of cases and to dilated cardiomyopathy (DCM) in cases of prolonged viral persistence. Complex arrhythmias and sudden deaths due to CVB infections have also been reported, particularly in newborns and children. Observed arrhythmias include bradycardia, AV block and junctional ectopic tachycardia, suggesting that in addition to the functioning myocardium, the pacemaker cells and cardiac conduction system may also be impaired by CVB3 infection. The symptomatic phenotype is highly variable and depends on unclear host factors. It ranges from inconspicuous inflammation and rapid healing to acute congestive heart failure or viral persistence with progressive chronic disease. Despite the increasing and profound knowledge about viral myocarditis, it is still a challenge to identify further host factors that contribute to acute and chronic myocarditis, as science lacks sensitive and controllable disease models. The presented project shows the generation of a controlled, inducible virus-expressing hiPSC line that provides a solution for the beforementioned problems. The new human induced pluripotent stem cell line (hiPSC) i can be used to carry out virologic experiments in a controlled manner and without risk to the user. For this purpose, the modified and non-infectious genome of the enterovirus Coxsackievirus B3 (CVB3) is stably integrated into the genome of a wild-type hiPSC line. The CVB3 genome to be stably integrated was modified to prevent the formation of functional capsids by a DVP0 mutation. In addition, the 3'UTR and 5'UTR regions of the CVB3 genome were removed, which prevents uncontrolled self-replication of viral RNA. As a result, work with this CVB3DVP0-UTR genome is classified as S1 compliant. The expression of the CVB3DP0-UTR virus genome can be controlled by adding doxycycline to the cell culture medium by a TeT-On System. The transfected cells are then cultivated as clones and tested for virus expression. The dose-dependent CVB3DVP0 expression is verified using qPCR and FACS after 1 day of doxycycline application in the cell culture medium. Clones isolated and positively tested for CVB3 expression are used to carry out initial experiments on the interaction of the expressed viral proteins with the cell. Particular focus is placed on the induction of cell death, remodelling of the cytoskeleton and increase of reactive oxygen species (ROS) in mitochondria after CVB3DVP0-UTR induction in order to validate the cell system for application. Differentiation of the CVB3-expressing hiPSC-line into cardiomyocytes with subsequent virus induction results in the elevation of mitochondrial ROS, degradation of the cytoskeleton and arrhythmic behaviour of the cells.

Abstract 2187: Influences of high-fat diet on tumor biology in a mouse model of colorectal cancer

Abstract Purpose: To test the influence of high-fat diet on tumor biology in the inducible Kras, Apc and p53 (iKAP) model (Boutin, et al Genes & Development 2017, 31:370). Methods: At weening, both male and female mice were fed either a high-fat (60%) or low-fat (10%) diet for 5-6 weeks prior to tumor induction. Mutant Kras expression was controlled using a tet-on system. Expression of mutant Kras was activated by the addition of doxycycline hyclate in the drinking water. Tumor growth was monitored by bi-weekly endoscopy and calculated as percent luminal occlusion. Tumor biology was assessed with proteomics and phospho-proteomics with comparisons by sex and diet. Results: In the absence of mutant Kras expression (no doxycycline in drinking water), tumors were larger in females on high-fat diet compared to low-fat diet. Tumor sizes were not significantly different in males or in the presence of mutant Kras expression. Proteomics analyses of tumor tissue identified significantly different patterns by diet in female mice, including altered levels of KIF4, DAB2iP, JNK cascade and JUN kinase activity (Table). Conclusion: Based on our preliminary data, high-fat diet may alter tumor kinase activities, even in a genetically engineered mouse model. Replication and validation studies are ongoing. Table of significantly upregulated biologic processes in female mice on high-fat diet compared to lo XXXXXXXXXXXXXXXX XXXXXXXXXXXXXXX Citation Format: Anindita Mahanty, Cara Wallingford, Revan Hammontree, Muturi Njoka, Zachary Clark, Michaella Rekowski, Michael Washburn, Jennifer S. Davis. Influences of high-fat diet on tumor biology in a mouse model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2187.

Abstract 1444: Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer characterized by very poor survival outcomes. Genomic sequencing of human PDAC tissues and cell lines have identified four molecular subtypes: quasimesenchymal (QM), pancreatic progenitor, immunogenic, and aberrantly differentiated endocrine exocrine (ADEX). QM-PDAC has the worse prognosis, but no in vivo models exist for this molecular subtype. Our preliminary analysis using publicly available data demonstrated that the epithelial-to-mesenchymal transition (EMT) transcription factor TWIST1 is upregulated in QM-PDAC. We hypothesized that the TWIST1-dependent plasticity program was a key regulator of QM-PDAC subtype development. We created a tetracycline-inducible tissue specific Twist1 PDAC genetically engineered mouse model (GEMM). Tumor development in the mouse pancreatic ductal epithelium was directed using the Pdx1 promoter-Cre (P) allele leading to stable native expression of the LSL-KrasG12D oncogene (R) and LSL-rtTA-IRES-GFP (G) alleles. The non-Twist1 GEMM (PGR) was crossed with the Twist1-tetO-Luc (T) mouse to create the PGRT model which allows inducible expression of Twist1 and luciferase in pancreatic epithelium under doxycycline treatment (Tet-ON system). Tumor progression, lineage tracing and metastatic invasion are followed by fluorescence (GFP) and bioluminescence (BLI) imaging. The characterization of this novel PGRT mouse model showed decreased overall survival compared to control non-Twist1 containing mice (~35 weeks PGRT (n=17) vs ~60 weeks PGR (n=10)). Histological and immunohistochemistry analyses showed the PGRT pancreatic tumors resemble the QM-PDAC subtype and vimentin overexpression correlates with Twist1-dependent induction of EMT. We attempted to confirm the QM-PDAC profile via cellular markers: GATA6-, HNF1A-, KRT81+, KRT17+, KRT20+, P63+ and ∆P63+. The PGR and standard KPC (Kras+/LSL-G12D; Trp53+LSL-R172H; Pdx1-Cre) PDAC model were also profiled. We observed that 75% of the PGRT tumors displayed a QM-PDAC profile, contrasting with the absence of such profile in both PGR and KPC tumors. Examining the tumors from PGR and KPC, 100% of the PGR and 72.27% KPC tumors exhibited a non-squamous PDAC profile while an intermediate squamous profile was observed in 25% PGRT tumors and 27.27% KPC tumors. Furthermore, Twist1 induction resulted in intra-abdominal and lung metastases. We observed that 87.5% of the PGRT mice were positive for metastases while we observed no positivity for PGR mice and 63.64% positivity in KPC mice. In conclusion, these data suggest that Twist1 overexpression cooperates with KrasG12D for PDAC tumor development mimicking the human QM-PDAC subtype to promote progression towards metastasis and early lethality. Potential uses of this novel model include testing treatment strategies to ultimately improve patient outcomes with QM-PDAC. Citation Format: Muhammad Ajmal Khan, Jinhee Chang, Triet Nguyen, Danielle N. Council, Dipanwita Dutta Chowdhury, Amol Shetty, Yang Song, Aaron Chan, Christine Lam, Priyanshi Patel, Lucas Tran, Jiayu Chen, Katriana Nugent, Matthew Ballew, Ghali Lemtiri-Chlieh, Francesca A. Carrieri, Reem Malek, Hailun Wang, Noelle R. Thielman, Keyu Li, Lei Zheng, Kathleen Gabrielson, Phuoc T. Tran, Audrey Lafargue. Modeling Twist1 overexpression in a pancreatic ductal adenocarcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1444.

Abstract B019: Deciphering the role of homologous recombination deficiency mutations in the response to combined immune checkpoint and PARP inhibitors

Abstract Background: Over 2 million women develop breast cancer globally every year despite immense advances in disease biology, diagnostic strategies and novel treatment options. The deeper knowledge of disease biology and genomic landscape has fostered a broader mechanistic approach to patients and their therapeutic choices. Many preclinical and clinical studies suggest a benefit of combining PARP inhibitors (PARPi) and immune checkpoint inhibitors (ICPi) in patients with homologous recombination deficiency (HRD) intact and mutated cancers. However, similar to the varied benefits of either drug class based on tissue and mutational context, the benefits of such combination are strongly variable based on tumor type, tissue and mutation context. Methods: To study how select HRD mutations respond to a combination of a PARPi inhibitor with an ICPi, we used CRISPR engineering to functionally impair or repress BRCA1, BRCA2, ATM, CHEK2, PALB2 in different mouse breast cancer cell lines. EMT6 and 4T1 cells were engineered to expressed deactivated cas9 nuclease (dcas9) fused to the repressor domain Kruppel-associated- box (KRAB) and mcherry florescent protein. The dcas9-KRAB-mcherry breast cancer cells were further engineered to express BFP-tagged guide RNAs targeting selected genes under a doxycycline inducible tet-on system. After establishing functional loss/repression of the targeted HRD mutations, cells were then implanted in the mammary fat pad of balb/cJ mice and treated with PARPi and ICPi to determine the ICPi, PARPi and their combination induced changes in inflammatory signals and their association with therapy response. Results: Induced suppression of BRCA1 and PALB2 in these breast cancer cells showed exquisite sensitivity to PARPi (IC50 0.01 uM and 0.098 uM) and diminishing sensitivity to PARPi in BRCA2 and ATM (IC50 0.16 uM and IC50 0.28 uM). In contrast, inducible suppression of CHEK2 showed resistance to PARPi versus parental EMT6 cells (IC50 13.9 uM versus 5.3 uM). Evaluation of changes in expression levels of PDL1 and select chemokines including CCL2, CCL5, CXCL9, CXCL10 and CXCL11 suggest a differential response to PARPi depending on specific HRD mutations. HRD mutations and tissue-context will be further characterized in combination therapy of ICPi and PARPi. Conclusion: Using inducible suppression of HRD function in an immune competent mouse breast cancer model helps to understand the differential role of individual HRD mutations (BRCA1, BRCA2, ATM, CHEK2, PALB2) in altering inflammatory signals in response to PARPi and ICI. This data may guide the clinical development of such combination in breast cancer and other cancers. Citation Format: Sibapriya Chaudhuri, Scott Thomas, Luika Timmerman, Pamela Munster. Deciphering the role of homologous recombination deficiency mutations in the response to combined immune checkpoint and PARP inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B019.

Promoters and Synthetic Promoters in Trichoderma reesei.

Trichoderma reesei holds immense promise for large-scale protein production, rendering it an excellent subject for deeper exploration using genetic engineering methods to achieve a comprehensive grasp of its cellular physiology. Understanding the genetic factors governing its intrinsic regulatory network is crucial, as lacking this knowledge could impede the expression of target genes. Prior and ongoing studies have concentrated on advancing new expression systems grounded in synthetic biology principles. These methodologies involve utilizing established potent promoters or engineered variations. Genomic and transcriptomic analyses have played a pivotal role in identifying robust promoters and expression systems, including light-responsive, copper-inducible, L-methionine-inducible, and Tet-On systems, among others. This chapter seeks to highlight various research endeavors focusing on tunable and constitutive promoters, the impact of different promoters on both native and foreign protein expression, the discovery of fresh promoters, and strategies conducive to future research aimed at refining and enhancing protein expression in T. reesei. Characterizing new promoters and adopting innovative expression systems hold the potential to significantly expand the molecular toolkit accessible for genetically engineering T. reesei strains. For instance, modifying potent inducible promoters such as Pcbh1 by replacing transcriptional repressors (cre1, ace1) with activators (xyr1, ace2, ace3, hap2/3/5) and integrating synthetic expression systems can result in increased production of crucial enzymes such as endoglucanases (EGLs), β-glucosidases (BGLs), and cellobiohydrolases (CBHs). Similarly, robust constitutive promoters such as Pcdna1 can be converted into synthetic hybrid promoters by incorporating activation elements from potent inducible promoters, facilitating cellulase induction and expression even under repressive conditions. Nevertheless, further efforts are necessary to uncover innovative promoters and devise novel expression strategies to enhance the production of desired proteins on an industrial scale.

Oxidative and ER stress by elevated insulin biosynthesis and palmitic acid in insulin-producing cells.

The early phase of type 2 diabetes mellitus (T2DM) is characterised by insulin resistance, which can initially be compensated by elevated insulin secretion. However, as postulated by the workload hypothesis, over time harming insulin requirements contribute to β-cell dysfunction and death. The mechanisms behind this transition are complex and not fully understood but involve factors such as endoplasmic reticulum (ER) stress raised by gluco/lipotoxicity. To investigate the effect of excessive insulin folding on ER luminal H2O2 generation, ER stress and viability, insulin was expressed glucose-independently by a doxycycline-regulated Tet-On system in insulin-producing RINm5F cells. Additionally, the effect of palmitic acid (PA) as a subsidiary T2DM-associated factor was examined in this model system. Elevated insulin expression increased ER luminal H2O2 concentration quantified by the fluorescent sensor protein TriPer and reduced viability, but did not activate apoptosis. However, when combined with PA, insulin expression resulted in a significant increase in ER stress and apoptosis. Expression of ER-localised catalase verified the specificity of the applied H2O2 detection method without attenuating ER stress, caspase activation or viability loss. These findings suggest that hyperinsulinism alone can cause increased ER luminal H2O2 generation, mild ER stress and reduced viability, while hyperinsulinism in combination with PA accelerates these processes and triggers apoptosis. The inability of ER catalase to counteract these effects suggests that further damaging factors besides H2O2 are involved in cell dysfunction. Finally, reducing the high insulin demand in the initial phase of T2DM may be crucial in preventing further β-cell damage caused by gluco/lipotoxicity.

In Vivo Optical Interrogation of Neuronal Responses to Genetic, Cell Type-Specific Silencing.

We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.

The Cellular Impact, Immediate Targets and Mechanism of Action of NSD2 in t(4;14) Multiple Myeloma

INTRODUCTION: NSD2 is a potential therapeutic target in the 15% of cases of multiple myeloma that carry a t(4;14) translocation. As a consequence of the translocation, strong Ig enhancers are placed in proximity to NSD2 and FGFR3. FGFR3 is deleted at a subsequent time point in 25% of cases, while all cases retain NSD2 expression consistent with it likely being the key oncogenic driver. NSD2 is an H3K36 dimethyl transferase, a histone modification mark associated with active transcription, but the molecular targets and the mechanism by which NSD2 regulates its targets is unknown. The cellular function of NSD2 is also largely unknown, but previous shRNA knockdown studies have suggested it is involved in cell proliferation. The major limitation to understanding the role of NSD2 is the lack of an appropriate experimental system with which to investigate its molecular and cellular function. To address this deficit and to identify the primary direct targets of NSD2 as a means to elucidate its mechanism of action we have generated NSD2 dTAG and NSD2 KO cells together with NSD2 reconstitution systems. METHODS: We have utilized CRISPR gene editing to knock-in a dTAG in NSD2 in the t(4;14) KMS-11 cell line. This system enables the inducible and rapid degradation of NSD2 following the addition of a small-molecule degrader which is otherwise inert. We have also generated clonal NSD2 KO KMS-11 cells that allow us to observe and study the stable phenotypes of NSD2 KO. Because H3K36me2 deposited by NSD2 leads to induction of DNMT3A-mediated DNA methylation and suppression of PRC2-mediated H3K27me3, we have generated DNMT3A KO on top of NSD2dTAG/KO and have used two different PRC2 inhibitors to dissect the dependency of NSD2 function on such secondary modifications. To systematically dissect the dependency on NSD2 isoforms and its methyltransferase activity, we have reconstituted NSD2 in both NSD2 dTAG and NSD2 KO KMS-11 cells, including WT NSD2I WT, NSD2II WT, NSD2 Y1179A (LOF), NSD2 E1099K (GOF). To maximally match the timing of NSD2 perturbation, NSD2 reconstitution was generated in NSD2 dTAG and NSD2 KO cells in a Tet-ON and Tet-OFF expression system, respectively. Further we have generated KMS-11 cells overexpressing Dox-inducible H3.3K36M to investigate the consequences of abrogation of H3K36me2 by this dominant negative mutant of H3.3. We have done SLAM-seq to assess the transcriptional targets of NSD2 and will extend this with ATAC-seq and CUT&Tag to identify the molecular targets of NSD2. RESULTS: We show that NSD2 is rapidly degraded within 1 h when a degrader is added and subsequently a significant reduction of H3K36me2 is seen by 24 hours although it takes more than 3 days to see its deep depletion. We observed a reciprocal increase of H3K27me3 as previously noted. Depletion of H3K36me2 and increase in H3K27me3 was also seen in the NSD2 KO KMS-11 cells. Paradoxically we didn't see an induction of apoptosis in response to the degradation of NSD2 or its KO. In sharp contrast to the previously published shRNA results only a mild inhibition of cell proliferation was observed in NSD2 KO cells and over an extended period of 3 weeks in the dTAG system. We didn't observe an effect of H3.3 K36M overexpression on cell proliferation although it abolished H3K36me2 very efficiently. These experiments demonstrate that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth, which is likely independent of its H3K36me2 methyltransferase activity. SLAMseq results show that NSD2 degradation in the dTAG system causes transcriptional changes of a specific set of newly synthesized mRNAs. ATAC-seq and CUT&Tag experiments are being run and will be superimposed on the SLAM-seq data to identify immediate targets of NSD2. Subsequently, use of PRC2 inhibitors and DNMT3A KO cells in the dTAG cells and the NSD2 reconstitution experiments will uncover the mechanisms of how NSD2 controls the transcription of its targets. CONCLUSION: We show that NSD2 is not essential for viability of KMS-11 cells but is required for optimal cell growth and that NSD2 controls the transcription of a specific set of genes and does not exert a global impact on the transcriptome. We will further define the immediate targets of NSD2 by leveraging our SLAM-seq, ATAC-seq, and CUT&Tag data in NSD2 dTAG/KO and NSD2-reconstituted cells and by blocking the crosstalk with other epigenetic marks (i.e. H3K27me3 and DNAme).

PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

Uniform transgene activation in Tet-On systems depends on sustained rtTA expression

Application of the tetracycline-inducible gene expression system (Tet-On) in human induced pluripotent stem cells (hiPSCs) has become a fundamental transgenic tool owing to its regulatable gene expression. One of the major hurdles in hiPSC application is non-uniform transgene activation. Here, we report that the supplementation of reverse tetracycline transactivator (rtTA) in polyclonal hiPSCs populations can achieve the uniform transgene activation of Tet-On. Furthermore, the choice of antibiotic selection markers connected by an internal ribosomal entry site (IRES) can influence the expression of upstream transgenes. In particular, expression of the rtTA is more uniform in cell populations when linked to puromycin as compared to neomycin, obviating the need for sub-cloning or supplementation of rtTA. Finally, to expand the range of applications, we adopted our findings to tetracycline-inducible MyoD vector (Tet-MyoD). Our Tet-MyoD promises efficient, robust, and reproducible directed myogenic differentiation of hiPSCs.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells

Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. Its cytolytic activity is sequestered by the intramolecular autoinhibitory mechanism. Upon caspase-1/11 mediated cleavage of GSDMD, the N-terminal pore-forming domain (GD-NT) is released to mediate pyroptosis. However, it remains unclear how GD-NT is regulated once it is generated. In the current study, we developed a TetOn system in which GD-NT was selectively induced in tumor cells to explore how the cytolytic activity of GD-NT is regulated. We found that the cytolytic activity of GD-NT was negatively regulated by the AMP-activated protein kinase (AMPK) and AMPK activation rendered tumor cells resistant to GD-NT-mediated pyroptosis. Mechanistically, AMPK phosphorylated GD-NT at the serine 46 (pS46-GD), which altered GD-NT oligomerization and subsequently eliminated its pore-forming ability. In our in vivo tumor model, AMPK-mediated phosphorylation abolished GD-NT-induced anti-tumor activity and resulted in an aggressive tumor growth. Thus, our data demonstrate the critical role of AMPK in negatively regulating the cytolytic activity of GD-NT. Our data also highlight an unexpected link between GSDMD-mediated pyroptosis and the AMPK signaling pathway in certain tumor cells.

A conditional gene expression system in Porphyromonas gingivalis for study of the secretion mechanisms of lipoproteins and T9SS cargo proteins.

The Gram-negative anaerobe, Porphyromonas gingivalis, is known to be a pathogen associated with chronic periodontitis. P. gingivalis possesses virulence factors such as fimbriae and gingipain proteinases. Fimbrial proteins are secreted to the cell surface as lipoproteins. In contrast, gingipain proteinases are secreted into the bacterial cell surface via the type IX secretion system (T9SS). The transport mechanisms of lipoproteins and T9SS cargo proteins are entirely different and remain unknown. Therefore, using the Tet-on system developed for the genus Bacteroides, we newly created a conditional gene expression system in P. gingivalis. We succeeded in establishing conditional expression of nanoluciferase and its derivatives for lipoprotein export, of FimA for a representative of lipoprotein export, and of T9SS cargo proteins such as Hbp35 and PorA for representatives of type 9 protein export. Using this system, we showed that the lipoprotein export signal, which has recently been found in other species in the phylum Bacteroidota, is also functional in FimA, and that a proton motive force inhibitor can affect type 9 protein export. Collectively, our conditional protein expression method is useful for screening inhibitors of virulence factors, and may be used to investigate the role of proteins essential to bacterial survival in vivo.

BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells

Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.

#3553 TUBULE CELL-DERIVED ANGIOPOIETIN-2 EXACERBATES INFLAMMATION AND IS DETRIMENTAL TO RENAL FIBROSIS

Abstract Background and Aims Our previous studies have shown that angiogenic growth factors are dysregulated in fibrotic kidneys. In murine models of progressive renal disease, angiopoietin-2 (Angpt2) was upregulated in injured tubule epithelial cells. We hypothesize that overexpression of Angpt2 in tubule epithelial cells would exacerbate renal fibrosis and inflammation. Method The unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) are used as the murine model of chronic kidney disease. We use the tetracycline/doxycycline-controlled Tet-on system to induce tubule cell-specific overexpression of Angpt2 before the induction of renal fibrosis. Transgenic mice carrying both Pax8-rtTA and pTetos-hAngpt2 genes (double transgenic mice) are used for Angpt2 overexpression. Littermates that inherit single or no transgene serve as experimental controls. Doxycycline is administered to adult (8 weeks) mice to overexpress Angpt2 in tubule epithelial cells. Results Doxycycline administration induce overexpression of human Anpgt2 in the tubule epithelial cells of both sham and fibrotic kidneys. Compared to littermate controls after the UUO and UIRI surgery, the expression of fibrosis-related genes, Col1a1, Col3a1, and Acta2 are higher in the kidneys of double transgenic mice. Deposition of picrosirius red-stained collagen fibrils is significantly higher in double transgenic mice. Inflammatory cell infiltration is also significantly higher in double transgenic mice. Conclusion Overall, these findings suggest that increased Angpt2 in tubule epithelial cells deteriorate renal fibrosis through detrimental inflammation. Further studies are necessary to explore the participating cellular components and pathogenic mechanisms.