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1.
Immunity ; 50(4): 1054-1068.e3, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30926235

ABSTRACT

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2RFP with Zbtb16CreGFP and Bcl11btdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2+Zbtb16+ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16+ ILCPs.


Subject(s)
Gene Expression Regulation/immunology , Hematopoietic Stem Cells/cytology , Immunity, Innate , Inhibitor of Differentiation Protein 2/genetics , Lymphopoiesis/genetics , Adoptive Transfer , Animals , Cell Lineage , GATA3 Transcription Factor/biosynthesis , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/physiology , Genes, Reporter , Hematopoietic Stem Cells/metabolism , Inhibitor of Differentiation Protein 2/biosynthesis , Killer Cells, Natural/cytology , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Models, Immunological , Promyelocytic Leukemia Zinc Finger Protein/biosynthesis , Promyelocytic Leukemia Zinc Finger Protein/genetics , Promyelocytic Leukemia Zinc Finger Protein/physiology , Single-Cell Analysis , T-Lymphocytes, Helper-Inducer/cytology , Transcription, Genetic , Red Fluorescent Protein
2.
Nat Immunol ; 19(12): 1427-1440, 2018 12.
Article in English | MEDLINE | ID: mdl-30374131

ABSTRACT

Multipotent progenitor cells confirm their T cell-lineage identity in the CD4-CD8- double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.


Subject(s)
Lymphopoiesis/immunology , Precursor Cells, T-Lymphoid/immunology , Promyelocytic Leukemia Zinc Finger Protein/biosynthesis , Repressor Proteins/immunology , Tumor Suppressor Proteins/immunology , Animals , Cell Differentiation/immunology , Gene Expression Regulation/immunology , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Precursor Cells, T-Lymphoid/cytology , Promyelocytic Leukemia Zinc Finger Protein/immunology
3.
Mol Med Rep ; 16(3): 3687-3693, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28765931

ABSTRACT

Acute myelocytic leukemia (AML) is the most common type of acute leukemia. Long non­coding RNAs (lncRNAs) serve an important role in regulating gene expression through chromatin modification, transcription and post­transcriptional processing. LncRNA H19 was considered as an independent prognostic marker for patients with tumors. The expression of lncRNA H19 was identified to be significantly upregulated in bone marrow samples from patients with AML­M2. Furthermore, it was demonstrated that the knockdown of lncRNA H19 resulted in increased expression of hsa­microRNA (miR)­19a/b and decreased expression of inhibitor of DNA binding 2 (ID2) in AML cells. The knockdown of lncRNA H19 inhibited the proliferation of AML cells in vitro, which could be partially reversed by ID2 overexpression. Furthermore, the results of the bioinformatic analysis revealed potential hsa­miR­19a/b­3p binding sites in lncRNA H19 and ID2. Altogether, the results of the present study suggest that lncRNA H19 regulates the expression of ID2 through competitive binding to hsa­miR­19a and hsa­miR­19b, which may serve a role in AML cell proliferation.


Subject(s)
Gene Expression Regulation, Leukemic , Inhibitor of Differentiation Protein 2/biosynthesis , Leukemia, Myeloid, Acute/metabolism , MicroRNAs/metabolism , Neoplasm Proteins/biosynthesis , RNA, Long Noncoding/metabolism , RNA, Neoplasm/metabolism , HL-60 Cells , Humans , Inhibitor of Differentiation Protein 2/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , MicroRNAs/genetics , Neoplasm Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Neoplasm/genetics
4.
Cell Death Dis ; 8(2): e2615, 2017 02 16.
Article in English | MEDLINE | ID: mdl-28206987

ABSTRACT

Tumor cells proliferate in cellular environments characterized by a lack of optimal tissue organization resulting oftentimes in compromised cellular metabolism affecting nutrition, respiration, and energetics. The response of tumor cells to adverse environmental conditions is a key feature affecting their pathogenicity. We found that inhibitor of DNA binding 2 (ID2) expression levels significantly correlate with the ability of glioblastoma (GBM)-derived cell lines to survive glucose deprivation. ID2 suppressed mitochondrial oxidative respiration and mitochondrial ATP production by regulating the function of mitochondrial electron transport chain (mETC) complexes, resulting in reduced superoxide and reactive oxygen species (ROS) production from mitochondria. ID2 suppression of ROS production reduced mitochondrial damage and enhanced tumor cell survival during glucose deprivation. Bioinformatics analysis of GBM gene expression data from The Cancer Genome Atlas (TCGA) database revealed that expression of ID2 mRNA is unique among ID gene family members in correlating with the expression of nuclear genes involved in mitochondrial energy metabolism and assembly of mETC. Our data indicate that the expression level of ID2 in GBM cells can predict the sensitivity of GBM-derived tumor cells to decreased glucose levels. Low levels of ID2 expression in human GBM tissues may identify a clinical group in which metabolic targeting of glycolytic pathways can be expected to have the greatest therapeutic efficacy.


Subject(s)
Glioblastoma/metabolism , Inhibitor of Differentiation Protein 2/metabolism , Mitochondria/metabolism , Cell Line, Tumor , Energy Metabolism , Glioblastoma/genetics , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Stress, Physiological , Survival Analysis
5.
Cancer Genomics Proteomics ; 13(6): 493-497, 2016.
Article in English | MEDLINE | ID: mdl-27807072

ABSTRACT

AIM: Inhibitor of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors that generally stimulate cell proliferation and inhibit differentiation. However, the role of ID2 in cancer progression remains ambiguous. Here, we investigated the function of ID2 in ID2-null oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: We introduced an ID2 cDNA construct into ID2-null OSCC cells and compared them with empty-vector-transfected cells in terms of cell proliferation, invasion, and activity and expression of matrix metalloproteinase (MMP). RESULTS: ID2 introduction resulted in enhanced malignant phenotypes. The ID2-expressing cells showed increased N-cadherin, vimentin, and E-cadherin expression and epithelial-mesenchymal transition. In addition, cell invasion drastically increased with increased expression and activity of MMP2. Immunoprecipitation revealed a direct interaction between ID2 and zinc finger transcription factor, snail family transcriptional repressor 1 (SNAIL1). CONCLUSION: ID2 expression triggered a malignant phenotype, especially of invasive properties, through the ID2-SNAIL axis. Thus, ID2 represents a potential therapeutic target for OSCC.


Subject(s)
Carcinoma, Squamous Cell/genetics , Inhibitor of Differentiation Protein 2/genetics , Matrix Metalloproteinase 2/biosynthesis , Mouth Neoplasms/genetics , Snail Family Transcription Factors/genetics , Cadherins/biosynthesis , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Matrix Metalloproteinase 2/genetics , Mouth Neoplasms/pathology , Mouth Neoplasms/therapy , Neoplasm Invasiveness/genetics , Signal Transduction/genetics , Vimentin/biosynthesis
6.
Sci Rep ; 6: 30977, 2016 08 03.
Article in English | MEDLINE | ID: mdl-27485735

ABSTRACT

Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways.


Subject(s)
Cell Differentiation , Interleukin-3/metabolism , Osteoclasts/metabolism , RANK Ligand/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Animals , Gene Expression Regulation , Humans , Inhibitor of Differentiation Protein 1/biosynthesis , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Interleukin-3/genetics , Mice , Mice, Knockout , Osteoclasts/cytology , RANK Ligand/genetics , STAT5 Transcription Factor/genetics
7.
Genes Cells ; 21(8): 915-20, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27364596

ABSTRACT

Inhibitors of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. We previously determined that ID1 was highly expressed in aggressive salivary gland cancer (SGC) cells in culture. Here, we show that ID2 is also expressed in aggressive SGC cells. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. Moreover, ID2 knockdown almost completely suppresses invasion and the expression of matrix metalloproteinase 9. In conclusion, ID2 expression maintains an aggressive phenotype in SGC cells, and ID2 repression triggers a reduction in cell aggressiveness. ID2 therefore represents a potential therapeutic target during SGC progression. ID proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. ID2 therefore represents a potential therapeutic target during SGC progression.


Subject(s)
Cell Proliferation/genetics , Inhibitor of Differentiation Protein 2/genetics , Molecular Targeted Therapy , Salivary Gland Neoplasms/genetics , Cadherins/biosynthesis , Cell Differentiation/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Neoplasm Invasiveness/genetics , Phenotype , Salivary Gland Neoplasms/pathology , Salivary Gland Neoplasms/therapy , Snail Family Transcription Factors/biosynthesis , Vimentin/biosynthesis
8.
J Biol Chem ; 291(20): 10904-15, 2016 May 13.
Article in English | MEDLINE | ID: mdl-27002159

ABSTRACT

Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression.


Subject(s)
Heat-Shock Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Transcriptional Activation , alpha-Macroglobulins/metabolism , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Female , Heat-Shock Proteins/genetics , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Male , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-fos/biosynthesis , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-myc/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , alpha-Macroglobulins/genetics
9.
Sci Rep ; 6: 22966, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26965643

ABSTRACT

Hypoxia, a feature common to most solid tumors, is known to regulate many aspects of tumorigenesis. Recently, it was suggested that hypoxia increased the size of the cancer stem-cell (CSC) subpopulations and promoted the acquisition of a CSC-like phenotype. However, candidate hypoxia-regulated mediators specifically relevant to the stemness-related functions of colorectal CSCs have not been examined in detail. In the present study, we showed that hypoxia specifically promoted the self-renewal potential of CSCs. Through various in vitro studies, we found that hypoxia-induced Wnt/ß-catenin signaling increased the occurrence of CSC-like phenotypes and the level of Id2 expression in colorectal-cancer cells. Importantly, the levels of hypoxia-induced CSC-sphere formation and Id2 expression were successfully attenuated by treatment with a Wnt/ß-catenin-signaling inhibitor. We further demonstrated, for the first time, that the degree of hypoxia-induced CSC-sphere formation (CD44(+) subpopulation) in vitro and of tumor metastasis/dissemination in vivo were markedly suppressed by knocking down Id2 expression. Taken together, these data suggested that Wnt/ß-catenin signaling mediated the hypoxia-induced self-renewal potential of colorectal-cancer CSCs through reactivating Id2 expression.


Subject(s)
Carcinogenesis/genetics , Colorectal Neoplasms/genetics , Hyaluronan Receptors/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Cell Hypoxia/genetics , Cell Line, Tumor , Cell Lineage , Cell Proliferation/genetics , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Inhibitor of Differentiation Protein 2/genetics , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , Wnt Signaling Pathway/genetics , beta Catenin/genetics
10.
Tumour Biol ; 36(6): 4189-96, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25773386

ABSTRACT

With its growth characteristic and chemoresistance, glioblastoma is the most deadly brain tumor. Twenty-five core genes that influence the chemosensitivity of glioblastoma were screened in our previous experiments, and Id2, the inhibitor of DNA binding 2, an oncogene encoding a helix-loop-helix protein, was identified. The elevated expression levels of Id2 have been reported in several malignancies. The aim of this study is to investigate the effects of Id2 expression on the chemosensitivity of glioma cells. In this study, Id2 expression was investigated in a malignant glioma cell line. Then, we silenced the expression of Id2 with the highly specific posttranscriptional suppression of RNA interference (RNAi) in U87 cells. The changes in response to antitumor agents Me-CCNU, VM26, and TMZ were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit. The relationship between Id2 expression and caspase 3 was tested by RT-PCR and Western blot. This study demonstrated that Id2 was significantly upregulated in glioma tissues, and Id2 correlated well with the advancement of glioma grade and a worse prognosis in response to temozolomide treatment. The RNAi-mediated decrease of Id2 expression enhanced chemosensitivity to Me-CCNU, VM26, and TMZ in the U87 cell line. We further discovered that silencing of Id2 expression could promote apoptosis of glioblastoma cells, which could be attributed to the fact that Id2 affects tumor cell chemosensitivity. Downregulation of the Id2 gene by RNAi could increase the chemosensitivity of glioblastoma cells. Id2 could be a good molecular target for glioblastoma gene therapy.


Subject(s)
Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Glioma/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Glioma/drug therapy , Glioma/pathology , Humans , Inhibitor of Differentiation Protein 2/genetics , RNA Interference , Temozolomide
11.
Nat Immunol ; 15(8): 767-76, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24973820

ABSTRACT

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.


Subject(s)
Inflammation/immunology , Inhibitor of Differentiation Protein 2/immunology , Inhibitor of Differentiation Proteins/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Base Sequence , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Movement/immunology , Cell Proliferation , Female , Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation/immunology , Green Fluorescent Proteins/genetics , Inflammation/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/biosynthesis , Inhibitor of Differentiation Proteins/genetics , Interleukin-10/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, T-Cell/immunology , Receptors, CXCR5/biosynthesis , Sequence Analysis, RNA
12.
Br J Haematol ; 166(5): 677-89, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24801987

ABSTRACT

In Waldenström macroglobulinaemia (WM), the mechanism(s) responsible for repression of B-cell differentiation remains unknown. We found that expression of SPIB and ID2 were significantly increased and decreased, respectively, in WM lymphoplasmacytic cells (LPC). Ectopic expression of SPIB in healthy donor CD19(+) cells inhibited plasmacytic differentiation in conjunction with decreased transcription of IRF4 and XBP1 spliced form. In primary WM LPC, knock-down of SPIB induced plasmacytic differentiation in conjunction with increased transcription of PRDM1, XBP1 spliced form, IRF4 and ID2. Knock-down of SPIB also led to decreased BCL2 expression. Given that SPIB is a direct target of POU2AF1 (OBF1) in complex with POU2F2 or POU2F1, we next examined their expression in WM LPC. POU2F2 transcription, as well as POU2F2 and POU2AF1 protein expression was higher in WM LPC. Ectopic expression of POU2F2 in healthy donor CD19(+) cells induced transcription of SPIB and suppressed transcription of PRDM1 and IRF4. Chromatin immunoprecipitation analysis in BCWM.1 WM cells confirmed binding of POU2F2 and POU2AF1 in SPIB and ID2 promoters. These findings establish a molecular hierarchy among POU2F2, SPIB and ID2 during B-cell differentiation, and suggest that aberrant expression of these transcription factors plays an important role in arresting plasmacytic differentiation in WM.


Subject(s)
DNA-Binding Proteins/biosynthesis , Plasma Cells/physiology , Transcription Factors/biosynthesis , Waldenstrom Macroglobulinemia/blood , Waldenstrom Macroglobulinemia/genetics , Aged , B-Lymphocytes/cytology , B-Lymphocytes/physiology , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Female , Gene Knockdown Techniques , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Male , Middle Aged , Octamer Transcription Factor-2/biosynthesis , Octamer Transcription Factor-2/genetics , Plasma Cells/cytology , Trans-Activators/biosynthesis , Trans-Activators/genetics , Transcription Factors/genetics
13.
J Immunol ; 192(5): 2227-36, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24470501

ABSTRACT

Disease outcome is known to be influenced by defined subsets of invariant NKT (iNKT) cells residing in distinct locations within peripheral tissue. However, the factors governing the development of these unique iNKT sublineages during thymic development are unknown. In this study we explored the mechanism by which E protein transcription factors and their negative regulators, the Id proteins, control the development of iNKT sublineages after positive selection. We found that E proteins directly bound the promyelocytic leukemia zinc finger (PLZF) promoter and were required for expression of this lineage-defining transcription factor and for the maturation and expansion of thymic iNKT cells. Moreover, expression of the negative regulators of E proteins, Id2 and Id3, defined distinct iNKT cell sublineages. Id3 was expressed in PLZF(high) NKT2 cells and loss of Id3 allowed for increased thymic iNKT cell expansion and abundance of the PLZF(+) NKT2 sublineage. Id2 was expressed in T-BET(+) NKT1 cells, and both Id proteins were required for the formation of this sublineage. Thus, we provide insight into E and Id protein regulation of iNKT cell proliferation and differentiation to specific sublineages during development in the thymus.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/immunology , Cell Differentiation/physiology , Cell Proliferation , Inhibitor of Differentiation Protein 2/immunology , Inhibitor of Differentiation Proteins/immunology , Natural Killer T-Cells/immunology , Thymus Gland/immunology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Proteins/biosynthesis , Inhibitor of Differentiation Proteins/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/immunology , Mice , Mice, Transgenic , Natural Killer T-Cells/cytology , Promyelocytic Leukemia Zinc Finger Protein , Thymus Gland/cytology , Thymus Gland/metabolism
14.
Histochem Cell Biol ; 141(4): 431-40, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24292846

ABSTRACT

The inhibitors of DNA binding (ID) inhibit basic helix-loop-helix transcription factors and thereby guide cellular differentiation and proliferation. To elucidate the involvement of IDs in hematopoiesis and acute leukemias (AL), we analyzed ID2 and ID3 expression in hematopoiesis and leukemic blasts in bone marrow biopsies (BMB). BMB of healthy stem cell donors (n = 19) and BMB of patients with acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MD; n = 19), de novo AML (n = 20), B-acute lymphoblastic leukemia (B-ALL) (n = 23), T-ALL (n = 19), were immunohistochemically stained for ID2 and ID3 expression. The expression patterns were evaluated and quantified for each hematopoietic lineage and each leukemia subtype. In normal BMB, immature granulopoiesis showed weak ID2 and strong ID3 expression, which was lost during maturation (p < 0.001). Erythropoiesis remained negative for ID2/3 (p < 0.001). ID2/3 expression differed between immature granulopoiesis and leukemic blasts (p < 0.001). Moreover, differential ID2/3 expression was seen between AL subgroups: AML, especially AML-MD, had more ID2- (p < 0.001) and ID3-positive (p < 0.001) blasts than ALL. We show a comprehensive in situ picture of ID2/3 expression in hematopoiesis and AL. Morphologically, ID2/3 proteins seem to be involved in the granulopoietic maturation. Importantly, the distinct ID2/3 expression patterns in AL indicate a specific deregulation of ID2/3 in the various types of AL and may support subtyping of AL.


Subject(s)
Granulocytes/cytology , Granulocytes/metabolism , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Proteins/analysis , Inhibitor of Differentiation Proteins/biosynthesis , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/metabolism , Neoplasm Proteins/analysis , Neoplasm Proteins/biosynthesis , Biopsy , Bone Marrow Cells/chemistry , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Female , Granulocytes/chemistry , Humans , Inhibitor of Differentiation Protein 2/analysis , Leukemia, Myeloid, Acute/diagnosis , Male , Middle Aged , Retrospective Studies
15.
J Neurosci ; 33(48): 18893-905, 2013 Nov 27.
Article in English | MEDLINE | ID: mdl-24285895

ABSTRACT

In utero ethanol exposure from a mother's consumption of alcoholic beverages impacts brain and cognitive development, creating a range of deficits in the child (Levitt, 1998; Lebel et al., 2012). Children diagnosed with fetal alcohol spectrum disorders (FASD) are often born with facial dysmorphology and may exhibit cognitive, behavioral, and motor deficits from ethanol-related neurobiological damage in early development. Prenatal ethanol exposure (PrEE) is the number one cause of preventable mental and intellectual dysfunction globally, therefore the neurobiological underpinnings warrant systematic research. We document novel anatomical and gene expression abnormalities in the neocortex of newborn mice exposed to ethanol in utero. This is the first study to demonstrate large-scale changes in intraneocortical connections and disruption of normal patterns of neocortical gene expression in any prenatal ethanol exposure animal model. Neuroanatomical defects and abnormal neocortical RZRß, Id2, and Cadherin8 expression patterns are observed in PrEE newborns, and abnormal behavior is present in 20-d-old PrEE mice. The vast network of neocortical connections is responsible for high-level sensory and motor processing as well as complex cognitive thought and behavior in humans. Disruptions to this network from PrEE-related changes in gene expression may underlie some of the cognitive-behavioral phenotypes observed in children with FASD.


Subject(s)
Behavior, Animal/drug effects , Cerebral Cortex/metabolism , Fetal Alcohol Spectrum Disorders/physiopathology , Fetal Alcohol Spectrum Disorders/psychology , Gene Expression/drug effects , Prenatal Exposure Delayed Effects/physiopathology , Prenatal Exposure Delayed Effects/psychology , Animals , Cadherins/biosynthesis , Cadherins/genetics , Cell Count , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Ethanol/blood , Female , Fetal Alcohol Spectrum Disorders/genetics , In Vitro Techniques , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Mice , Microscopy, Fluorescence , Nuclear Receptor Subfamily 1, Group F, Member 2/biosynthesis , Nuclear Receptor Subfamily 1, Group F, Member 2/genetics , Osmolar Concentration , Pregnancy , Pregnancy, Animal/drug effects , Prenatal Exposure Delayed Effects/genetics , Weight Gain
16.
J Immunol ; 191(12): 5993-6001, 2013 Dec 15.
Article in English | MEDLINE | ID: mdl-24227775

ABSTRACT

Dendritic cells (DCs) are heterogeneous cell populations represented by different subtypes, each varying in terms of gene expression patterns and specific functions. Recent studies identified transcription factors essential for the development of different DC subtypes, yet molecular mechanisms for the developmental program and functions remain poorly understood. In this study, we developed and characterized a mouse DC progenitor-like cell line, designated DC9, from Irf8(-/-) bone marrow cells as a model for DC development and function. Expression of Irf8 in DC9 cells led to plasmacytoid DCs and CD8α(+) DC-like cells, with a concomitant increase in plasmacytoid DC- and CD8α(+) DC-specific gene transcripts and induction of type I IFNs and IL12p40 following TLR ligand stimulation. Irf8 expression in DC9 cells led to an increase in Id2 and Batf3 transcript levels, transcription factors shown to be important for the development of CD8α(+) DCs. We show that, without Irf8, expression of Id2 and Batf3 was not sufficient for directing classical CD8α(+) DC development. When coexpressed with Irf8, Batf3 and Id2 had a synergistic effect on classical CD8α(+) DC development. We demonstrate that Irf8 is upstream of Batf3 and Id2 in the classical CD8α(+) DC developmental program and define the hierarchical relationship of transcription factors important for classical CD8α(+) DC development.


Subject(s)
Basic-Leucine Zipper Transcription Factors/physiology , Dendritic Cells/cytology , Gene Expression Regulation/immunology , Inhibitor of Differentiation Protein 2/physiology , Interferon Regulatory Factors/physiology , Repressor Proteins/physiology , Animals , Basic-Leucine Zipper Transcription Factors/biosynthesis , Basic-Leucine Zipper Transcription Factors/genetics , CD8 Antigens/analysis , Cell Differentiation/drug effects , Cell Line , Dendrites/ultrastructure , Dendritic Cells/chemistry , Dendritic Cells/classification , Dendritic Cells/ultrastructure , Hematopoietic Stem Cells/cytology , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Interferon Regulatory Factors/biosynthesis , Interferon Regulatory Factors/genetics , Interferon-alpha/biosynthesis , Interferon-alpha/genetics , Interleukin-12 Subunit p40/biosynthesis , Interleukin-12 Subunit p40/genetics , Membrane Proteins/pharmacology , Mice , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Transduction, Genetic
17.
J Immunol ; 191(3): 1210-9, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23825313

ABSTRACT

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2.


Subject(s)
B-Lymphocyte Subsets/immunology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Inhibitor of Differentiation Protein 2/metabolism , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism , Antigens, Nuclear/metabolism , Bone Marrow/metabolism , Cell Differentiation , Cell Proliferation , DNA Nucleotidylexotransferase/metabolism , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Homeodomain Proteins/metabolism , Humans , Infant , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Ki-67 Antigen/biosynthesis , Ku Autoantigen , Lymphocyte Count , Middle Aged , Nuclear Proteins/metabolism , RNA, Messenger/biosynthesis , Signal Transduction/immunology , Up-Regulation , V(D)J Recombination/genetics
18.
Stem Cells ; 31(5): 1010-21, 2013 May.
Article in English | MEDLINE | ID: mdl-23390122

ABSTRACT

Id2 is a helix-loop-helix transcription factor essential for normal development, and its expression is dysregulated in many human neurological conditions. Although it is speculated that elevated Id2 levels contribute to the pathogenesis of these disorders, it is unknown whether dysregulated Id2 expression is sufficient to perturb normal brain development or function. Here, we show that mice with elevated Id2 expression during embryonic stages develop microcephaly, and that females in particular are prone to generalized tonic-clonic seizures. Analyses of Id2 transgenic brains indicate that Id2 activity is highly cell context specific: elevated Id2 expression in naive neural stem cells (NSCs) in early neuroepithelium induces apoptosis and loss of NSCs and intermediate progenitors. Activation of Id2 in maturing neuroepithelium results in less severe phenotypes and is accompanied by elevation of G1 cyclin expression and p53 target gene expression. In contrast, activation of Id2 in committed intermediate progenitors has no significant phenotype. Functional analysis with Id2-overexpressing and Id2-null NSCs shows that Id2 negatively regulates NSC self-renewal in vivo, in contrast to previous cell culture experiments. Deletion of p53 function from Id2-transgenic brains rescues apoptosis and results in increased incidence of brain tumors. Furthermore, Id2 overexpression normalizes the increased self-renewal of p53-null NSCs, suggesting that Id2 activates and modulates the p53 pathway in NSCs. Together, these data suggest that elevated Id2 expression in embryonic brains can cause deregulated NSC self-renewal, differentiation, and survival that manifest in multiple neurological outcomes in mature brains, including microcephaly, seizures, and brain tumors.


Subject(s)
Brain/abnormalities , Brain/cytology , Inhibitor of Differentiation Protein 2/biosynthesis , Neural Stem Cells/metabolism , Animals , Brain/metabolism , Cell Differentiation/physiology , Cells, Cultured , Female , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Stem Cells/cytology
19.
PLoS One ; 7(7): e40269, 2012.
Article in English | MEDLINE | ID: mdl-22848373

ABSTRACT

The transcription factor ID2 is an important repressor of neural differentiation strongly implicated in nervous system cancers. MicroRNAs (miRNAs) are increasingly involved in differentiation control and cancer development. Here we show that two miRNAs upregulated on differentiation of neuroblastoma cells--miR-9 and miR-103--restrain ID2 expression by directly targeting the coding sequence and 3' untranslated region of the ID2 encoding messenger RNA, respectively. Notably, the two miRNAs show an inverse correlation with ID2 during neuroblastoma cell differentiation induced by retinoic acid. Overexpression of miR-9 and miR-103 in neuroblastoma cells reduces proliferation and promotes differentiation, as it was shown to occur upon ID2 inhibition. Conversely, an ID2 mutant that cannot be targeted by either miRNA prevents retinoic acid-induced differentiation more efficient than wild-type ID2. These findings reveal a new regulatory module involving two microRNAs upregulated during neural differentiation that directly target expression of the key differentiation inhibitor ID2, suggesting that its alteration may be involved in neural cancer development.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Inhibitor of Differentiation Protein 2/biosynthesis , MicroRNAs/biosynthesis , Neoplasm Proteins/biosynthesis , Neuroblastoma/metabolism , RNA, Neoplasm/biosynthesis , Tretinoin/pharmacology , Animals , Cattle , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/genetics , Humans , Inhibitor of Differentiation Protein 2/genetics , Macaca mulatta , Mice , MicroRNAs/genetics , Neoplasm Proteins/genetics , Neuroblastoma/genetics , Neuroblastoma/pathology , RNA, Neoplasm/genetics , Rats , Up-Regulation/drug effects , Up-Regulation/genetics
20.
J Immunol ; 189(3): 1400-5, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22745378

ABSTRACT

An effective immune response to Ag challenge is critically dependent on the size of the effector cell population generated from clonal activation of Ag-specific T cells. The transcription network involved in regulating the size of the effector population, particularly for CD4 Th cells, is poorly understood. In this study, we investigate the role of Id2, an inhibitor of E protein transcription factors, in the generation of CD4 effectors. Using a T cell-specific conditional Id2 knockout mouse model, we show that inhibitor of DNA binding (Id)2 is essential for the development of experimental autoimmune encephalomyelitis. Although Ag-specific and IL-17-producing CD4 T cells are produced in these mice, the activated CD4 T cells form a smaller pool of effector cells in the peripheral lymphoid organs, exhibit reduced proliferation and increased cell death, and are largely absent in the CNS. In the absence of Id2, E protein targets, including the proapoptotic protein Bim and SOCS3, are expressed at higher levels among activated CD4 T cells. This study reveals a critical role of Id2 in the control of effector CD4 T cell population size and the development of a Th17-mediated autoimmune disease.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Inhibitor of Differentiation Protein 2/physiology , Transcription, Genetic/immunology , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Gene Knock-In Techniques , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Protein 2/deficiency , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Resting Phase, Cell Cycle/genetics , Resting Phase, Cell Cycle/immunology , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/pathology
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