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1.
Cells ; 9(1)2020 01 15.
Article in English | MEDLINE | ID: mdl-31952221

ABSTRACT

BACKGROUND: The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. METHODS: Generation of an in vitroETV6/RUNX1 knock out model using the CRISPR/Cas9 gene editing system. Functional characterization by RNA sequencing, proliferation assays, apoptosis and pharmacologic studies, and generation of edited-cell xenograft model. RESULTS: The expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukemic cells. The loss of fusion gene expression led to the deregulation of biological processes affecting survival such as apoptosis resistance and cell proliferation capacity. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. CONCLUSIONS: ETV6/RUNX1 fusion protein seems to play an important role in the maintenance of the leukemic phenotype and could thus become a potential therapeutic target.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Models, Biological , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Repressor Proteins/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Cell Proliferation , Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , Gene Editing , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/deficiency , Repressor Proteins/genetics , Tumor Cells, Cultured , ETS Translocation Variant 6 Protein
2.
Am J Respir Cell Mol Biol ; 62(3): 373-381, 2020 03.
Article in English | MEDLINE | ID: mdl-31596609

ABSTRACT

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.


Subject(s)
Epithelial Cells/drug effects , Gene Targeting/methods , Interleukin-13/physiology , Mucociliary Clearance/physiology , Proto-Oncogene Proteins c-ets/physiology , Ribonucleoproteins/genetics , Bronchi/cytology , CRISPR-Cas Systems , Cells, Cultured , Down-Regulation , Epithelial Cells/metabolism , Gene Expression Regulation , Goblet Cells/metabolism , Humans , Metaplasia , Mucin 5AC/biosynthesis , Mucin 5AC/genetics , Primary Cell Culture , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , RNA, Guide, Kinetoplastida/genetics , Ribonucleoproteins/administration & dosage , Transcriptome
3.
J Clin Invest ; 129(10): 4433-4450, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31524632

ABSTRACT

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1ß, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1ß alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1ß induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1ß are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1ß-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.


Subject(s)
Cystic Fibrosis/metabolism , Interleukin-1beta/metabolism , Mucus/metabolism , Animals , Case-Control Studies , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Female , Gene Expression Regulation , Humans , Interleukin-1alpha/genetics , Interleukin-1alpha/metabolism , Interleukin-1beta/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucin 5AC/genetics , Mucin 5AC/metabolism , Mucin-5B/genetics , Mucin-5B/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-1 Type I/deficiency , Receptors, Interleukin-1 Type I/genetics , Receptors, Interleukin-1 Type I/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Signal Transduction
4.
Biomed Pharmacother ; 111: 76-85, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30576937

ABSTRACT

Gastric cancer is one of the most common malignant tumors worldwide and has the second highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Here, we evaluated PDEF expression in 30 cases of human gastric carcinoma and the corresponding peritumoral tissues, using immunohistochemistry and immunofluorescence. Significantly higher levels of PDEF were detected in tumors compared to peritumoral tissues. We then investigated PDEF expression in the gastric cancer cell lines SGC and AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. Cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells were evaluated using CCK-8, flow cytometry, scratch wound, and transwell assays, respectively. The results illustrated that PDEF-knockout inhibited AGS cell proliferation, migration, and invasion. Taken together, the results imply that PDEF plays important roles in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer.


Subject(s)
CRISPR-Cas Systems/physiology , Cell Movement/physiology , Gene Knockdown Techniques/methods , Neoplasm Invasiveness/genetics , Proto-Oncogene Proteins c-ets/genetics , Stomach Neoplasms/genetics , Base Sequence , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Line, Tumor , Gastric Mucosa/metabolism , Gastric Mucosa/pathology , Humans , Neoplasm Invasiveness/pathology , Proto-Oncogene Proteins c-ets/deficiency , Random Allocation , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology
5.
Nat Commun ; 8(1): 1426, 2017 11 10.
Article in English | MEDLINE | ID: mdl-29127283

ABSTRACT

Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Proto-Oncogene Proteins c-ets/immunology , Proto-Oncogene Proteins/immunology , Trans-Activators/immunology , Animals , B-Lymphocytes/cytology , CD40 Antigens/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Female , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/metabolism , Immunity, Humoral/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Plasma Cells/cytology , Plasma Cells/immunology , Plasma Cells/metabolism , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Signal Transduction , Toll-Like Receptors/metabolism , Trans-Activators/deficiency , Trans-Activators/genetics
6.
PLoS One ; 9(9): e107048, 2014.
Article in English | MEDLINE | ID: mdl-25203538

ABSTRACT

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.


Subject(s)
Arteries/abnormalities , Joint Instability/pathology , Neovascularization, Pathologic/pathology , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Retina/pathology , Retinal Neovascularization/pathology , Retinal Vessels/pathology , Skin Diseases, Genetic/pathology , Vascular Malformations/pathology , Angiopoietins/genetics , Angiopoietins/metabolism , Animals , Arteries/metabolism , Arteries/pathology , Disease Models, Animal , Female , Joint Instability/genetics , Joint Instability/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Receptors, TIE/genetics , Receptors, TIE/metabolism , Retina/metabolism , Retinal Neovascularization/genetics , Retinal Neovascularization/metabolism , Retinal Vessels/metabolism , Serum Response Factor/genetics , Serum Response Factor/metabolism , Signal Transduction/physiology , Skin Diseases, Genetic/genetics , Skin Diseases, Genetic/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factors/genetics , Vascular Endothelial Growth Factors/metabolism , Vascular Malformations/genetics , Vascular Malformations/metabolism
7.
Clin Lymphoma Myeloma Leuk ; 14(5): e151-5, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25022600
8.
Am J Pathol ; 183(1): 35-48, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23665202

ABSTRACT

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1ß, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.


Subject(s)
Conjunctiva/pathology , Disease Models, Animal , Dry Eye Syndromes/pathology , Goblet Cells/pathology , Proto-Oncogene Proteins c-ets/deficiency , Animals , Biomarkers/metabolism , Cell Differentiation , Conjunctiva/metabolism , Down-Regulation , Dry Eye Syndromes/genetics , Dry Eye Syndromes/metabolism , Female , Genetic Markers , Goblet Cells/metabolism , Humans , Immunohistochemistry , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Phenotype , Proto-Oncogene Proteins c-ets/metabolism , RNA/analysis , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sjogren's Syndrome/metabolism , Up-Regulation
9.
Mucosal Immunol ; 6(4): 838-46, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23212199

ABSTRACT

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell-specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B-deficient mice showed reduced expression of most, but not all, other M-cell-specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B-deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B-deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.


Subject(s)
Peyer's Patches/immunology , Peyer's Patches/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Signal Transduction , Animals , Antigens, Surface/genetics , Antigens, Surface/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Differentiation , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression Regulation , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Mice , Mice, Knockout , Microvilli/metabolism , Organ Specificity/genetics , Peyer's Patches/cytology , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Uromodulin/genetics , Uromodulin/metabolism
10.
PLoS One ; 6(10): e26348, 2011.
Article in English | MEDLINE | ID: mdl-22028862

ABSTRACT

BACKGROUND: ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. FINDINGS: Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the "cell activation", "immune response", "apoptosis", "signal transduction" and "development and differentiation" categories, whereas in the E/R KD-DOWN set only the "PI3K/AKT/mTOR signaling" and "hematopoietic stem cells" categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories "stem cell properties", "B-cell differentiation", "immune response", "cell adhesion" and "DNA damage" with RT-qPCR. CONCLUSION: Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.


Subject(s)
Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , Gene Fusion/genetics , Gene Silencing , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/deficiency , Repressor Proteins/genetics , Cell Line, Tumor , Child , Gene Knockdown Techniques , Humans , Oligonucleotide Array Sequence Analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Reproducibility of Results , Transcriptome/genetics , ETS Translocation Variant 6 Protein
11.
Gastroenterology ; 137(4): 1333-45.e1-3, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19549527

ABSTRACT

BACKGROUND & AIMS: Stem cells within the intestinal epithelium generate daughter cells that undergo lineage commitment and maturation through the combined action of the Wnt and Notch signaling cascades. Both pathways, in turn, regulate transcription factor networks that further define differentiation toward either enterocytes or 1 of 3 secretory cell lineages (Paneth, goblet, or enteroendocrine cells). In this study, we investigated the role of the Wnt-responsive, Ets-domain transcription factor Spdef in the differentiation of goblet and Paneth cells. METHODS: The in vivo function of Spdef was examined by disrupting the Spdef gene in mice (Spdef(-/-) mice) and analyzing the intestinal phenotype using a range of histologic techniques and DNA microarray profiling. RESULTS: In accordance with expression data, we found that loss of Spdef severely impaired the maturation of goblet and Paneth cells and, conversely, led to an accumulation of immature secretory progenitors. Spdef appears to positively and negatively regulate a specific subset of goblet and Paneth cell genes, including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2. CONCLUSIONS: Spdef acts downstream of Math1 to promote terminal differentiation of a secretory progenitor pool into Paneth and goblet cells.


Subject(s)
Cell Differentiation , Colon/metabolism , Goblet Cells/metabolism , Intestine, Small/metabolism , Paneth Cells/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Stem Cells/metabolism , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Lineage , Colon/ultrastructure , Gene Expression Profiling/methods , Gene Expression Regulation , Genotype , Goblet Cells/ultrastructure , Intestine, Small/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Paneth Cells/ultrastructure , Phenotype , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Stem Cells/ultrastructure , Transcription, Genetic
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