Tet-on Inducible System Research Articles

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.

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.

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.

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.

Abstract 3932: Targeting cyclin K in pancreatic cancer

Abstract This study focuses on investigating the role of Cyclin K in pancreatic cancer growth and chemotherapeutic sensitivity, aiming to provide pre-clinical evidence for Cyclin K-targeted therapy in pancreatic cancer. Unlike other well-known cyclins, Cyclin K is largely understudied in cancers. Data extracted from the TCGA database indicated that Cyclin K was overexpressed in pancreatic ductal adenocarcinoma (PDAC) and associated with reduced overall survival. Consistently, western blotting showed that Cyclin K was abundantly expressed in human and mouse PDAC cell lines. By using a Tet-On inducible system, we established Cyclin K-depleted and Cyclin K-overexpressed cell lines to evaluate the function of Cyclin K in pancreatic cancer. The proliferation assay showed that Cyclin K depletion led to retarded PDAC cell proliferation, whereas Cyclin K overexpression boosted cell growth. We further confirmed that Cyclin K was required for pancreatic cancer growth in vivo. In addition, cell cycle analysis showed that Cyclin K deficiency resulted in G1-S arrest, while Cyclin K overexpression promoted G1-S progression. By using an RT2 cell cycler array, we identified CDC20 as an important target of Cyclin K that mediated Cyclin K-induced PDAC cell proliferation. To investigate the influence of Cyclin K on chemo-sensitivity, we treated the PDAC cell lines with two newly synthesized Cyclin K molecular glue degraders (HQ461 and NCT02). We found that these degraders specifically ablated Cyclin K and its cognate kinase CDK12 in a very efficient manner. Importantly, we demonstrated that Cyclin K abrogation, either by Cyclin K degrader or Cyclin K knockdown, rendered PDAC cells more sensitive to GemTaxol (gemcitabine plus Taxol) treatment and PARP inhibitors (olaparib or niraparib), as indicated by increased cleavage of PARP or caspase 3. Together, our current results suggest: 1. Cyclin K promotes cell proliferation and G1-S transition in pancreatic cancer; 2. CDC20 mediates the function of Cyclin K on pancreatic cancer cell proliferation; 3. Cyclin K depletion synergizes with GemTaxol or PARP inhibitor in pancreatic cancer treatment. Our study revealed for the first time that Cyclin K plays an essential role in pancreatic cancer growth and chemo-sensitivity, and targeting Cyclin K may offer a great therapeutic avenue for pancreatic cancer patients. Citation Format: Yi Xiao, Jixin Dong, Yuanhong Chen. Targeting cyclin K in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3932.

BCR::ABL1 -Dependent Promotion of Hematopoietic Stem Cells (HSC) and Progenitors in an Inducible Embryonic Stem Cell (ESC) Model

Our current understanding of the effect of BCR::ABL1 oncogene in leukemia development largely relies on studies using in vitro and in vivo leukemogenesis models. Among them, in vivo models using retrovirus-mediated BCR-ABL gene transfer in murine embryonic stem cell (ESC)-derived hematopoietic cells suggested that the enforced expression of BCR::ABL1 was sufficient to confer them an in vivo long-term repopulation potential. However, in these models, the unique BCR::ABL1 dependence of the generation of this potential has not been shown. Our study aimed at getting a better understanding of the early events regulated by BCR::ABL1 during the course of ESC differentiation without retroviral-mediated gene transfer. The elucidation of these early events could also be of major interest in therapy as generation of bona fide HSCs from normal ESC or iPSC remains a major challenge. To this end, we engineered a unique Tet-ON inducible system in which BCR::ABL1(p210) expression level can be tightly controlled using doxycycline (Dox). As compared to previously reported ESC-derived models, our system contains a single copy of BCR::ABL1 inserted into the murine Col1a1 locus under the control of Tetracycline operator, is sensitive to Dox, leakproof and reversible. The first striking results was the timing of Dox-inducible BCR-ABL expression during the course of ESC differentiation, impacting both the frequency and emergence of hematopoietic progenitors. Indeed, the expression of BCR::ABL1 at day+5 favors significantly the emergence and maintenance of immature hematopoietic progenitors. Importantly, these cells can self-renew only in the presence of Dox. Several hematopoietic stem-like cell lines were established and two of them (HSCL-A and HSCL-C) have been explored in detail. A major BCR-ABL-dependent proliferative ability was documented in both (Figure 1D). Both expressed Lin-Sca-1+c-Kit+CD150+CD48- and eSLAM, corresponding to phenotypically identified adult mouse HSCs. Importantly, these cells exhibited a normal myeloid and erythroid differentiation capacity in CFC assays, giving rise also to CFU-GEMM. We then evaluated the ability of HSCL to self-renew using long-term culture-initiating cell (LTC-IC) assays. To this purpose, EPCR+CD45+/loCD48-CD150+ (ESLAM) cells were sorted at a single cell level and co-cultured on a supportive AFT024 feeder layers for 4 weeks. LTC-IC-derived progenitors were then assessed using CFC assays. The frequency of ESLAM LTC-ICs was 0.53 from adult bone marrow and 0.52 from HSCL lines demonstrating their self-renewal ability. To better characterize HSCL cell lines, we sorted LSK cell population from HSCL#A, HSCL#C, E14.5 fetal liver and murine adult bone marrow and analyzed their transcriptome signatures by RNA-Seq. Unsupervised principal component analysis with the 1333 genes exhibiting the highest variability revealed that HSCL cell lines clustered together and exhibited distinct molecular signature from fetal and adult LSK. This stratification was validated by clustering with the 50 best variable genes. Among them, we found 17 genes highly expressed in HSCL cell lines associated with HSC quiescence (such as NupR1). GSEA analyses confirmed an enrichment of several cell cycle genes as well as genes involved in unfolded protein response (UPR) and autophagy (p <0.001). Thus, we report here for the first time, a unique BCR-ABL inducible ESC-derived hematopoiesis model which demonstrates the ability of BCR-ABL expression to induce a proliferative signal for extended periods of time in highly primitive hematopoietic cells. This novel tool can be used to dissect CML ontogeny both in vitro and in vivo and perform drug screening at the most primitive stem cell level. It can also be of major interest to uncover molecular events and signaling pathways activated during normal ESC-derived hematopoiesis. Such knowledge will open up new avenues to differentiate human iPSC towards bona fide HSCs for potential clinical applications. Figure 1. Establishment of ES cell-derived hematopoietic cell lines expressing BCR::ABLp210.(A) Representation of the timeline of ES cell differentiation protocol and doxycycline induction. (B) HSCL#A cell morphology. (C) MGG staining of HSCL#A cells. (D) Proliferation curves of HSCL#A and HSCL#C cell lines over 40 days in culture. (E)Kinetics of the effect of doxycycline removal on HSCL#A cells cultured on OP9 cells for 4 consecutive days. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antitumor Effect of Simvastatin in Combination With DNA Methyltransferase Inhibitor on Gastric Cancer via GSDME-Mediated Pyroptosis.

Gasdermin E (GSDME) is one of the executors of pyroptosis, a type of programmed lytic cell death, which can be triggered by caspase-3 activation upon stimulation. Silenced GSDME expression due to promoter hypermethylation is associated with gastric cancer (GC), which is confirmed in the present study by bioinformatics analysis and methylation-specific PCR (MSP) test of GC cell lines and clinical samples. GC cell lines and mouse xenograft models were used to investigate the pyroptosis-inducing effect of the common cholesterol-depleting, drug simvastatin (SIM), allied with upregulating GSDME expression by doxycycline (DOX)- inducible Tet-on system or DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR). Cell viability assessment and xenograft tumour growth demonstrated that the tumour inhibition effects of SIM can be enhanced by elevated GSDME expression. Morphological examinations and assays measuring lactate dehydrogenase (LDH) release and caspase-3/GSDME protein cleavage underlined the stimulation of pyroptosis as an important mechanism. Using short hairpin RNA (shRNA) knockdown of caspase-3 or GSDME, and caspase-3-specific inhibitors, we provided evidence of the requirement of caspase-3/GSDME in the pyroptosis process triggered by SIM. We conclude that reactivating GSDME expression and thereby inducing cancer cell-specific pyroptosis could be a potential therapeutic strategy against GC.

The C-22 sterol desaturase Erg5 is responsible for ergosterol biosynthesis and conidiation in Aspergillus fumigatus

Aspergillus fumigatus is the most prevalent saprophytic fungi and can cause severe invasive aspergillosis in immunocompromised individuals. For infection of A. fumigatus, the small hydrophobic conidia have been shown to play a dominant role. In this study, we found that deletion of erg5, a C-22 sterol desaturase gene which function in the last two steps of ergosterol biosynthesis, was sufficient to block ergosterol biosynthesis and conidiation. The deletion phenotype was further verified by a conditional expression strain of erg5 using the inducible tet-on system. Strikingly, erg5 mutant displays increased susceptibility to antifungal azoles itraconazole. RNA sequencing analysis showed that erg5 deficiency resulted in changes in transcription mainly related to lipid, carbohydrate, and amino acid metabolism. Genes encoding ergosterol biosynthesis-related enzymes were found to be up-regulated in erg5 null mutants. However, genes involved in asexual development, including upstream regulators, melanin biosynthesis enzymes, heterotrimeric G proteins, and MAPK signaling, were down-regulated to various degrees. Furthermore, metabolomic study revealed that erg5 deficiency also resulted in altered lipid and amino acid metabolism, which was consistent with our transcriptomics analysis. Collectively, our study established a link between ergosterol biosynthesis and asexual development at the transcriptomics and metabolomics level in A. fumigatus.

Characterization of a silent azaphilone biosynthesis gene cluster in Aspergillus terreus NIH 2624

Filamentous fungal secondary metabolites are an important source of bioactive components. Genome sequencing ofAspergillus terreusrevealed many silent secondary metabolite biosynthetic gene clusters presumed to be involved in producing secondary metabolites. Activation of silent gene clusters through overexpressing a pathway-specific regulator is an effective avenue for discovering novel fungal secondary metabolites. Replacement of the native promoter of the pathway-specific activator with the inducible Tet-on system to activate thetazpathway led to the discovery of a series of azaphilone secondary metabolites, among which azaterrilone A (1) was purified and identified for the first time. Genetic deletion of core PKS genes and transcriptional analysis further characterized thetazgene cluster to consist of 16 genes with the NR-PKS and the HR-PKS collaborating in a convergent mode. Based on the putative gene functions and the characterized compounds structural information, a biosynthetic pathway of azaterrilone A (1) was proposed.

Narciclasine Display Preferential Pre-Clinical Activity Against KSHV-Associated Primary Effusion Lymphoma By Targeting MYC Via NF-κB

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of PEL. KSHV-encoded viral FLICE-inhibitory protein (vFLIP)-K13 constitutively activates the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. In a K13-isogenic cell line based screening of small molecule libraries, we have identified the natural products narciclasine and its structural analog, 7-deoxynarciclasine (found in daffodils), as having preferential cytotoxicity towards K13-expressing isogenic cell line. Further, the structurally closely related lycorine (another alkaloid found in daffodils) also exhibited similar activity. All the three compounds (Narciclasine, 7-deoxynarciclasine, and lycorine) display preferential cytotoxicity against PEL in a panel of cell lines comprising 6 hematological malignancies. Narciclasine is approximately 10 and 100-fold much effective than its structural analogs, 7-deoxynarciclasine, and lycorine, respectively. Importantly, IC50 of narciclasine in PEL cell lines (⁓10 nM) are well below the physiological concentration that was achieved in murine pharmacokinetic studies (250 nM). Narciclasine arrested cell cycle progression of PEL cells at the G0/G1 phase, with a concomitant decrease in the percentage of cells in S-phase. Further, there is a significant increase in the number of cells in sub-G0 peak, representative of apoptotic cells, which was confirmed by activation of caspase-3/7, AnnexinV staining, and PARP cleavage. Narciclasine blocked the NF-κB promoter activity in PEL cells as observed by a drastic decrease in firefly luciferase activity of PEL cells stably transduced with a lentiviral NF-κB-Luc reporter. IL-6 and IL-10 are known NF-κB targets and growth factors that are constitutively secreted by PEL, striking decrease in their secretory levels were observed upon treatment with narciclasine. Narciclasine preferentially blocked K13-induced NF-κB over the constitutive NF-κB activity in cells, which were engineered to express K13 conditionally (tet-on inducible lentiviral system). IKKγ/NEMO is essential for K13-induced NF-κB. The preference of narciclasine for K13-induced NF-κB is completely abolished upon NEMO knockout by CRISPR-Cas9 system. While probing the effect of narciclasine on the levels of putative NF-κB target genes by qRT-PCR in PEL, we found that there is a significant decrease in mRNA level of MYC, with a concomitant decrease in its protein level. Subsequently, all the direct target genes of MYC (MYB, TYRO3, TERT, PMM2, SLC19A1, and HK2) were significantly down regulated upon treatment with narciclasine. Furthermore, ectopic expression of MYC from a retroviral promoter significantly diminished the anti-proliferative effect of narciclasine and its analogs in PEL. It has been shown that inhibition of MYC is toxic to PEL and compounds that target MYC display selective activity against PEL, which explains the preferential activity of narciclasine and its analogs in PEL. MYC is required for maintenance of KSHV latency, and its down regulation results in the induction of KSHV Replication and Transcription Activator (RTA/ORF50) in PEL. However, narciclasine neither induced the expression of RTA nor the production of infectious KSHV virions in PEL. Most importantly, narciclasine doubled the survival of NSG mice bearing orthotopic - Cell Line Xenograft (CLX) and Patient Derived Xenograft (PDX) models of PEL. Finally, the IC50 of narciclasine in PBMCs from normal healthy donors is approximately 4-fold higher than the PEL cells directly isolated from the mice bearing the PDX model of the disease and PEL cell lines, indicating that there is a significant therapeutic window for narciclasine, which can be exploited for the treatment of KSHV-associated malignancies. DisclosuresChaudhary:University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript.

Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a promising biomarker for the early prediction of acute kidney injury (AKI). The production of recombinant NGAL is considered to be necessary for the development of a detection method. This study intended to express the recombinant NGAL protein in 293T cell under the Tet-On inducible system and human serum albumin signal sequence (HSA-SS). The transfection efficiency and protein modulation were assessed by detecting the expression of the enhanced green fluorescent protein (EGFP) and secreted NGAL protein. Both proteins were detected only in the presence of a doxycycline (Dox) inducer. Cell toxicity was not found under any conditions. Moreover, a higher level of soluble NGAL protein in the supernatant secreted by HSA-SS compared with a native signal peptide (Nat-SS) was observed. In summary, this work successfully optimized the conditions for induction of NGAL expression. This system will provide as an efficient strategy to produce other recombinant proteins secreted from a mammalian cell.

Reg4 regulates pancreatic regeneration following pancreatitis via modulating the Notch signaling.

Pancreatic regeneration after acute pancreatitis is critical in the normal restoration of pancreatic exocrine function, the inhibition of which can cause severe complications including pancreatic exocrine insufficiency. However, the regulators of pancreatic regeneration and the underlying mechanisms remain uncovered. Here, using the inducible Tet-on system, we found that regenerating family member 4 (Reg4) knockdown significantly impaired pancreatic regeneration after pancreatitis. Both acinar-to-ductal metaplasia and the resolution of pancreatitis during regeneration were affected by Reg4 knockdown. Further investigations confirmed that Reg4 exerted its function through regulating Notch activation both in vitro and in vivo. Our study revealed Reg4 as a new regulator and potential therapeutic target for pancreatic regeneration.

Abrogation of mesenchyme-specific TGF-β signaling results in lung malformation with prenatal pulmonary cysts in mice.

The TGF-β signaling pathway plays a pivotal role in controlling organogenesis during fetal development. Although the role of TGF-β signaling in promoting lung alveolar epithelial growth has been determined, mesenchymal TGF-β signaling in regulating lung development has not been studied in vivo due to a lack of genetic tools for specifically manipulating gene expression in lung mesenchymal cells. Therefore, the integral roles of TGF-β signaling in regulating lung development and congenital lung diseases are not completely understood. Using a Tbx4 lung enhancer-driven Tet-On inducible Cre transgenic mouse system, we have developed a mouse model in which lung mesenchyme-specific deletion of TGF-β receptor 2 gene (Tgfbr2) is achieved. Reduced airway branching accompanied by defective airway smooth muscle growth and later peripheral cystic lesions occurred when lung mesenchymal Tgfbr2 was deleted from embryonic day 13.5 to 15.5, resulting in postnatal death due to respiratory insufficiency. Although cell proliferation in both lung epithelium and mesenchyme was reduced, epithelial differentiation was not significantly affected. Tgfbr2 downstream Smad-independent ERK1/2 may mediate these mesenchymal effects of TGF-β signaling through the GSK3β-β-catenin-Wnt canonical pathway in fetal mouse lung. Our study suggests that Tgfbr2-mediated TGF-β signaling in prenatal lung mesenchyme is essential for lung development and maturation, and defective TGF-β signaling in lung mesenchyme may be related to abnormal airway branching morphogenesis and congenital airway cystic lesions.

Levels of sgRNA as a Major Factor Affecting CRISPRi Knockdown Efficiency in K562 Cells

To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. Single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries in four cell clones with various KRAB-dCas9 expression levels. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.

ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation

BackgroundBone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear.MethodsMesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo.ResultsWe found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse.ConclusionsThese results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease.

Genetic manipulation of ureteric bud tip progenitors in the mammalian kidney through an Adamts18 enhancer driven tet-on inducible system

The ureteric epithelial progenitor (UEP) population within the embryonic kidney generates the arborized epithelial network of the kidney’s collecting system and plays a critical role in the expansion and induction of the surrounding nephron progenitor pool. Adamts18 shows UEP- restricted expression in the kidney and progenitor tip-restricted expression in several other organs undergoing branching epithelial growth. Adamts18 is encoded by 23 exons. Genetic removal of genomic sequence spanning exons 1 to 3 led to a specific loss of Adamts18 expression in UEPs, suggesting this region may encode a UEP-specific enhancer. Intron 2 (3 ​kb) was shown to have enhancer activity driving expression of the doxycycline inducible tet-on transcriptional regulator (rtTA) in an Adamts18en-rtTA transgenic mouse strain. Crossing Adamts18en-rtTA mice to a doxycycline dependent GFP reporter mouse enabled the live imaging of embryonic kidney explants. This facilitated the analysis of ureteric epithelial branching events at the cellular level. Ablation of UEPs at the initiation of ureteric bud outgrowth through the doxycycline-mediated induction of Diphtheria Toxin A (DTA) generated a range of phenotypes from complete kidneys agenesis, to duplex kidneys with double ureters. The latter outcome points to the potential of regulative processes to restore UEPs. In contrast, overexpression of YAP prior to ureteric bud outgrowth led to a complete failure of kidney development. Elevating YAP levels at later stages retarded branching growth. A similar phenotype was observed with the overexpression of MYC within the branch-tip localized UEP population. These experiments showcase the utility of the Adamts18en-rtTA transgenic model to the investigation of cellular and molecular events specific to branch tip progenitors within the mammalian kidney complementing existing CRE-dependent genetic tools. Further, the illustrative examples point to areas where new insight may be gained into the regulation of UEP programs.

Heparanase promotes glioma progression via enhancing CD24 expression.

Heparanase is an endo-β-d-glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans. Compelling evidence tie heparanase levels with all steps of tumor formation including tumor initiation, growth, metastasis and chemo-resistance, likely involving augmentation of signaling pathways and gene transcription. In order to reveal the molecular mechanism(s) underlying the protumorigenic properties of heparanase, we established an inducible (Tet-on) system in U87 human glioma cells and applied gene array methodology in order to identify genes associated with heparanase induction. We found that CD24, a mucin-like cell adhesion protein, is consistently upregulated by heparanase and by heparanase splice variant devoid of enzymatic activity, whereas heparanase gene silencing was associated with decreased CD24 expression. This finding was further substantiated by a similar pattern of heparanase and CD24 immunostaining in glioma patients (Pearson's correlation; R = 0.66, p = 0.00001). Noteworthy, overexpression of CD24 stimulated glioma cell migration, invasion, colony formation in soft agar and tumor growth in mice suggesting that CD24 functions promote tumor growth. Likewise, anti-CD24 neutralizing monoclonal antibody attenuated glioma tumor growth, and a similar inhibition was observed in mice treated with a neutralizing mAb directed against L1 cell adhesion molecule (L1CAM), a ligand for CD24. Importantly, significant shorter patient survival was found in heparanase-high/CD24-high tumors vs. heparanase-high/CD24-low tumors for both high-grade and low-grade glioma (p = 0.02). Our results thus uncover a novel heparanase-CD24-L1CAM axis that plays a significant role in glioma tumorigenesis.

The Late Endosomal Pathway Regulates the Ciliary Targeting of Tetraspanin Protein Peripherin 2.

Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.

Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish.

ABSTRACTCardiac muscle troponin T (Tnnt2) mediates muscle contraction in response to calcium ion dynamics, and Tnnt2 mutations are associated with multiple types of cardiomyopathy. Here, we employed a zebrafish model to investigate the genetic replenishment strategies of using conditional and inducible promoters to rescue the deficiencies in the heart. tnnt2a mutations were induced in zebrafish via the CRISPR/Cas9 technique, and the mutants displayed heart arrest and dilated cardiomyopathy-like phenotypes. We first utilized the classic myocardial promoter of the myl7 and TetOn inducible system to restore tnnt2a expression in myocardial tissue in tnnt2a mutant zebrafish. However, this attempt failed to recover normal heart function and circulation, although heart pumping was partially restored. Further analyses via both RNA-seq and immunofluorescence indicated that Tnnt2a, which was also expressed in a novel group of myl7-negative smooth muscle cells on the outflow tract (OFT), was indispensably responsible for the normal mechanical dynamics of OFT. Lastly, tnnt2 expression induced by OFT cells in addition to the myocardial cells successfully rescued heart function and circulation in tnnt2a mutant zebrafish. Together, our results reveal the significance of OFT expression of Tnnt2 for cardiac function and demonstrate zebrafish larva as a powerful and convenient in vivo platform for studying cardiomyopathy and the relevant therapeutic strategies.

STAT3-Inducible Mouse ESCs: A Model to Study the Role of STAT3 in ESC Maintenance and Lineage Differentiation.

Studies have demonstrated that STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. However, there is still lack of direct evidence on STAT3 functions in ESCs and embryogenesis because constitutive STAT3 knockout (KO) mouse is embryonic lethal at E6.5–E7.5, prior to potential functional role in early development can be assessed. Therefore, in this study, two inducible STAT3 ESC lines were established, including the STAT3 knockout (InSTAT3 KO) and pSTAT3 overexpressed (InSTAT3 CA) using Tet-on inducible system in which STAT3 expression can be strictly controlled by doxycycline (Dox) stimulation. Through genotyping, deletion of STAT3 alleles was detected in InSTAT3 KO ESCs following 24 hours Dox stimulation. Western blot also showed that pSTAT3 and STAT3 protein levels were significantly reduced in InSTAT3 KO ESCs while dominantly elevated in InSTAT3 CA ECSs upon Dox stimulation. Likewise, it was found that STAT3-null ESCs would affect the differentiation of ESCs into mesoderm and cardiac lineage. Taken together, the findings of this study indicated that InSTAT3 KO and InSTAT3 CA ESCs could provide a new tool to clarify the direct targets of STAT3 and its role in ESC maintenance, which will facilitate the elaboration of the mechanisms whereby STAT3 maintains ESC pluripotency and regulates ESC differentiation during mammalian embryogenesis.