Deltex1 Research Articles

Overexpression of DTX1 inhibits D-GalN/TNF-α-induced pyroptosis and inflammation in hepatocytes by regulating NLRP3 ubiquitination.

Acute liver injury (ALI) is characterized by massive hepatocyte death and has high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the pathophysiology of ALI and is involved in the inflammatory response mediated by NOD-like receptor protein 3 (NLRP3) inflammasome activation. Deltex 1 (DTX1) is a single transmembrane protein with ubiquitin E3 ligase activity and is closely involved in cell growth, differentiation, and apoptosis, as well as intracellular signal transduction. However, little is known about the influence of DTX1 on ALI. This study aimed to investigate the role of DTX1 in pyroptosis and inflammation induced by D-galactosamine (D-GalN) and tumor necrosis factoralpha (TNF-α) in human hepatocytes (LO2 cells) in vitro. Cell pyroptosis was measured by flow cytometry. The levels of DTX1, pyroptosis-associated proteins, and inflammatory cytokines were detected by quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Immunofluorescence staining, co-immunoprecipitation, ubiquitination, and luciferase reporter and chromatin immunoprecipitation assays were performed to detect the regulation between DTX1 and NLRP3 or hepatocyte nuclear factor 4 alpha (HNF4α). Analysis of variance was performed to compare groups. We found that DTX1 was decreased in D-GalN/TNF-α-induced LO2 cells. DTX1 overexpression significantly inhibited D-GalN/TNF-α-induced cell pyroptosis and inflammation. DTX1 interacted with NLRP3 and induced NLRP3 ubiquitination and degradation. Furthermore, by targeting NLRP3, DTX1 knockdown significantly induced cell pyroptosis and inflammation. In addition, HNF4α promoted DTX1 transcription by binding with its promoter. Our study revealed that DTX1 suppressed D-GalN/TNF-α-induced hepatocyte pyroptosis and inflammation by regulating NLRP3 ubiquitination.

The associations between Deltex1 and clinical characteristics of breast cancer.

Deltex 1 (DTX1) is a single transmembrane protein with ubiquitin E3 ligase activity which has been found to play a role in the development of several cancers. We aimed to investigate the associations between DTX1 and breast cancer (BC). We explored the roles and mechanisms of DTX1 in BC by using BC cell lines in vitro. Levels of DTX1 in serum and tissues were determined in 316 patients with BC, 102 patients with fibroadenoma, and 113 healthy controls by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). The associations between DTX1 and clinical characteristics of BC were analyzed using multivariate analysis and Cox regression survival analysis. Lower levels of DTX1 promoted BC cell proliferation, migration, and invasion. The cell growth and survival of BC might be regulated by DTX1 via the Notch signaling pathway. Levels of DTX1 in BC tissues were lower compared to fibroadenoma tissues and peri-neoplastic breast tissues (P<0.01). A lower level of DTX1 was shown to be associated with advanced tumor grade (P=0.017), advanced clinical stage (P=0.031), positive lymph node metastasis (LNM) (P=0.009), and high Ki-67 index (P=0.023). Lower DTX1 expression was recognized as an impact factor for metastasis-free survival (MFS) in BC. Lower levels of DTX1 could promote BC cell proliferation and migration, and are associated with advanced BC. There is potential for DTX1 as a marker to assist the selection of new BC treatment.

Effect of genetic variation in Notch regulator DTX1 on SCLC prognosis compared with the effect on NSCLC prongosis.

Deltex‐1 (DTX1) is a negative regulator of the Notch signaling pathway. Here, we investigated the clinical effect of DTX1 rs1732786A > G, which is associated with better prognosis in patients with early‐stage non‐small cell lung cancer (NSCLC), in 261 patients with small cell lung cancer (SCLC). DTX1 rs1732786A > G was associated with a significantly worse chemotherapy response and lower overall survival in the codominant model (odds ratio = 0.42, 95% confidence interval [CI]: 0.26–0.66, P = 2 × 10−4; hazard ratio = 1.47, 95% CI: 1.17–1.84, P = 0.001, respectively). An in vitro luciferase assay was performed, and the 1732786G allele demonstrated significantly higher promoter activity than the 1732786A allele (P = 2 × 10−7). In summary, DTX1 rs1732786A > G was associated with poor prognosis in patients with SCLC as opposed to patients with NSCLC.Key pointsSignificant findings of the study DTX1 rs1732786A > G was associated with better prognosis in patients with early‐stage non‐small cell lung cancer (NSCLC) in our previous study.What this study adds DTX1 rs1732786A > G was associated with a significantly worse chemotherapy response and lower overall survival in small cell lung cancer (SCLC).

Genetic Variant of Notch Regulator DTX1 Predicts Survival After Lung Cancer Surgery.

We evaluated the association between genetic variants in the Notch pathway and survival outcomes of patients with surgically resected NSCLC. Sixty-four single nucleotide polymorphisms (SNPs) in the Notch pathway genes were evaluated in the discovery study (n = 354) and two sequential validation studies (n = 772 and n = 746, respectively). The association of genotype with overall survival (OS) and disease-free survival (DFS) was evaluated. Of the 64 SNPs analyzed in the discovery study, 9 were significantly associated with OS or DFS. Among them, the association remained significant only for Deltex-1 (DTX1) rs1732786A>G in the first validation study. The second validation study confirmed again the association between DTX1 rs1732786A>G and survival outcomes. In the combined analysis, rs1732786A>G was significantly associated with better OS and DFS (adjusted HR ·aHR· for OS, 0.75; 95% CI 0.64-0.87; P = 0.0002; aHR for DFS, 0.79; 95% CI 0.71-0.89; P = 0.0001). In vitro luciferase assay showed that the rs1732786G allele was associated with higher promoter activity compared to rs1732786A allele. Consistently, relative mRNA expression level of DTX1 showed significant positive correlation with rs1732786 A-to-G change (Ptrend = 0.02) in tumor tissues. These results suggest that DTX1 rs1732786 is a potential prognostic factor that may have clinical utility in the management of early stage NSCLC.

Deltex1 suppresses T cell function and is a biomarker for diagnosis and disease activity of systemic lupus erythematosus.

OBJECTIVE Deletion of Deltex1 (DTX1) in mice caused hyperactivation of T cells and lupus-like autoimmune syndromes, however, the association of DTX1 with human autoimmune diseases is totally unknown. This study investigated the role of DTX1 in human T cell functions and its correlation with disease activity in patients with SLE. METHODS The influence of DTX1 on T cell function was evaluated using human primary cells. DTX1 expression in peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients was measured by quantitative real-time PCR and the SLEDAI was used to assess disease activity. RESULTS After stimulation with anti-CD3 and anti-CD28, silencing of DTX1 expression enhanced IFN-γ secretion by human T cells. The expression of DTX1 in PBMCs was significantly lower in 100 SLE patients than in 50 age- and sex-matched healthy controls (DTX1/glyceraldehyde 3-phosphate dehydrogenase, 0.452 vs 1.269, P < 0.001). The area under the receiver operator characteristics curve of the model was 0.737 (95% CI 0.658, 0.815). Intriguingly, a low DTX1 level in T cells led to high IFN-γ production in SLE patients and had a correlation with severe disease activity. In addition, low DTX1 expression in SLE patients was associated with active LN, lung involvement or hypocomplementaemia. CONCLUSION Knockdown DTX1 expression in human T cells reduced IFN-γ secretion. DTX1 expression in the PBMCs was significantly lower in SLE patients and had an inverse correlation with disease activity, indicating that the DTX1 level may be a good disease marker of SLE.

Deltex1 suppresses T cell function and is a biomarker for diagnosis and disease activity of systemic lupus erythematosus.

Deletion of Deltex1 (DTX1) in mice caused hyperactivation of T cells and lupus-like autoimmune syndromes, however, the association of DTX1 with human autoimmune diseases is totally unknown. This study investigated the role of DTX1 in human T cell functions and its correlation with disease activity in patients with SLE. The influence of DTX1 on T cell function was evaluated using human primary cells. DTX1 expression in peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients was measured by quantitative real-time PCR and the SLEDAI was used to assess disease activity. After stimulation with anti-CD3 and anti-CD28, silencing of DTX1 expression enhanced IFN-γ secretion by human T cells. The expression of DTX1 in PBMCs was significantly lower in 100 SLE patients than in 50 age- and sex-matched healthy controls (DTX1/glyceraldehyde 3-phosphate dehydrogenase, 0.452 vs 1.269, P < 0.001). The area under the receiver operator characteristics curve of the model was 0.737 (95% CI 0.658, 0.815). Intriguingly, a low DTX1 level in T cells led to high IFN-γ production in SLE patients and had a correlation with severe disease activity. In addition, low DTX1 expression in SLE patients was associated with active LN, lung involvement or hypocomplementaemia. Knockdown DTX1 expression in human T cells reduced IFN-γ secretion. DTX1 expression in the PBMCs was significantly lower in SLE patients and had an inverse correlation with disease activity, indicating that the DTX1 level may be a good disease marker of SLE.

C-FLIP is a target of the E3 ligase deltex1 in gastric cancer

The ubiquitin E3 ligase DELTEX1 (DTX1) is specifically downregulated in gastric cancer tissues, and expression of DTX1 is linked to better prognoses and survival in gastric cancer. Cellular FLICE inhibitory protein (c-FLIP) is known for its pivotal role in the resistance of cancer cells to death receptor-induced cell death. Here, we show that DTX1 is an E3 ligase for c-FLIP in gastric cancer cells. DTX1 promoted c-FLIP downregulation. Overexpression of DTX1 sensitized gastric cancer cells to TRAIL-induced apoptosis, whereas DTX1-knockdown attenuated apoptosis induction. DTX1 binds c-FLIPL and directs it into the endosome-lysosomal pathway for proteasome-independent degradation. Moreover, induction of DTX1 in AGS cells by geldanamycin conferred susceptibility of those cells to TRAIL-induced apoptosis. Our results reveal a tumor-suppressive role for DTX1 and suggest a new approach to increasing TRAIL efficacy by raising DTX1 levels in gastric cancer therapy. DTX1 also enhanced c-FLIP degradation and FasL-induced and TRAIL-induced apoptosis in T cells, suggesting that DTX1 constitutes one of the physiological mechanisms regulating c-FLIP stability.

In Vivo MR Imaging of Dual MRI Reporter Genes and Deltex-1 Gene-modified Human Mesenchymal Stem Cells in the Treatment of Closed Penile Fracture.

The purpose of this study was to investigate the feasibility of dual magnetic resonance imaging (MRI) reporter genes, including ferritin heavy subunit (Fth) and transferrin receptor (TfR), which provide sufficient MRI contrast for in vivo MRI tracking, and the Deltex-1 (DTX1) gene, which promotes human mesenchymal stem cell (hMSC) differentiation to smooth muscle cells (SMCs), to treat closed penile fracture (CPF). Multi-gene co-expressing hMSCs were generated. The expression of mRNA and proteins was assessed, and the original biological properties of hMSCs were determined and compared. The intracellular uptake of iron was evaluated, and the ability to differentiate into SMCs was detected. Fifty rabbits with CPF were randomly transplanted with PBS, hMSCs, Fth-TfR-hMSCs, DTX1-hMSCs, and Fth-TfR-DTX1-hMSCs. In vivo MRI was performed to detect the distribution and migration of the grafted cells and healing progress of CPF, and the results were correlated with histology. The mRNA and proteins of the multi-gene were highly expressed. The transgenes could not influence the original biological properties of hMSCs. The dual MRI reporter genes increased the iron accumulation capacity, and the DTX1 gene promoted hMSC differentiation into SMCs. The distribution and migration of the dual MRI reporter gene-modified hMSCs, and the healing state of CPF could be obviously detected by MRI and confirmed by histology. The dual MRI reporter genes could provide sufficient MRI contrast, and the distribution and migration of MSCs could be detected in vivo. The DTX1 gene can promote MSC differentiation into SMCs for the treatment of CPF and effectively inhibit granulation tissue formation.

Deltex1 Polymorphisms Are Associated with Hepatitis B Vaccination Non-Response in Southwest China.

BackgroundHepatitis B vaccination is the most important tool available for preventing hepatitis B virus (HBV) infection and reducing the prevalence of infection. However, epidemiological studies have demonstrated that morethan 5% of patients exhibit a non- or hypo-response to the HBV vaccine. Genetic variations associated with T cell immunity contribute to the immune response to HBV vaccination. The deltex 1 (DTX1) gene is involved in T cell anergy, which may also be associated with the immune response to the HBV vaccination.MethodsWe detected 10 single nucleotide polymorphisms (SNPs) in or around the DTX1 gene in 601 infants out of a population from Southwest China, including 299 high responders(HRs; HBsAb > 100 mIU/mL) and 302 non-responders (NRs; HBsAb < 10 mIU/mL). An additional validation study was performed, comprising 230 adult patients(135 HRs and 95 NRs) from Southwest China.ResultsThis study found that the minor allele ‘G’ of rs2384077 (adjusted p = 2.63E-04,) and the minor allele ‘C’ of rs10744794 (adjusted p = 3.69E-04) in the first intron of the DTX1 gene were remarkably associated with the immune response to HBV vaccination in both infant and adult populations. Moreover, a subsequent analysis indicated that haplotypes (A-T, G-C) of the two SNPs were significantly associated with the immune response to HBV vaccination.ConclusionsTwo SNPs (rs2384077 and rs10744794) in an intron of DTX1 and the linkage disequilibrium (LD) block are significantly associated with the immune response to HBV vaccination. The functional element annotation of the LD block between the two SNPs contains four transcriptional regulatory elements. The results suggest that these two SNPs may be involved in the immune response to HBV vaccination.

Aberrant expression of Notch1, HES1, and DTX1 genes in glioblastoma formalin-fixed paraffin-embedded tissues.

Glioblastoma is the most common malignant brain tumor accounting for more than 54% of all gliomas. Despite aggressive treatments, median survival remains less than 1year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2(-ΔΔCt) method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85% of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis.

Deltex1 antagonizes HIF-1α and sustains the stability of regulatory T cells in vivo.

Application of regulatory T cells (Tregs) in transplantation, autoimmunity and allergy has been extensively explored, but how Foxp3 and Treg stability is regulated in vivo is incompletely understood. Here, we identify a requirement for Deltex1 (DTX1), a contributor to T-cell anergy and Foxp3 protein level maintenance in vivo. Dtx1−/− Tregs are as effective as WT Tregs in the inhibition of CD4+CD25− T-cell activation in vitro. However, the suppressive ability of Dtx1−/− Tregs is greatly impaired in vivo. We find that Foxp3 expression is diminished when Dtx1−/− Tregs are co-transferred with effector T cells in vivo. DTX1 promotes the degradation of HIF-1α. Knockout of HIF-1α restores the Foxp3 stability and rescues the defective suppressive activity in Dtx1−/− Treg cells in vivo. Our results suggest that DTX1 exerts another level of control on Treg stability in vivo by sustaining the expression of Foxp3 protein in Tregs.

Somatic Mutations in E3 Ubiquitin Ligase Deltex 1 Are Associated with Survival in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both biologically and clinically. Although a number of genetic alterations have been associated with the pathogenesis of DLBCL, survival association of most of the identified mutations remains to be shown. Here we have used exome and RNA sequencing (RNAseq) data to identify novel genetic events associated with treatment outcome after chemoimmunotherapy.Initial screen was performed with eight primary tumor samples and matched normal DNA using exome sequencing. The patients had clinically high risk DLBCL, and were treated in a Nordic phase II protocol with dose dense chemoimmunotherapy and CNS prophylaxis. After stringent filtering (VarScan2 p < 0.05), and removal of known germline variants, 967 somatic point mutations and indels in 561 different genes were identified. Of these, 436 were non-synonymous, and 27 were detected in more than one patient. These included both novel and several previously described DLBCL associated mutations, such as MYD88, CD79B, B2M and PTEN.To correlate the identified mutations with survival, we used RNAseq and survival data of 92 DLBCL samples from the Cancer Genome Characterization Initiative (CGCI) repository. The mutational status of the 436 genes identified in our screen was assessed with Bambino, which confirmed 405 of these genes also to be mutated in the CGCI set. Kaplan-Meier survival estimates per gene with at least one single nucleotide variant (SNV; with criteria of minimum 3 reads and 10% coverage and known polymorphisms filtered out) revealed 17 genes that were associated with overall survival (OS, p<0.05, minimum 10 samples mutated).We highlight mutations affecting Deltex1 (DTX1), which functions as an E3 ubiquitin ligase and targets Notch1 intracellular domain for degradation. In the study cohort, 14% of the patients had a non-synonymous DTX1 mutation. Mutations were equally distributed between GCB and ABC DLBCLs and low (0-2) and high (3-5) International Prognostic Index (IPI) risk groups. According to Kaplan-Meier analysis, the 5-year PFS rate for the patients with DTX1 mutations was significantly worse as compared to ones without mutations (46% vs 78%, p=0.010). The corresponding OS values were 54% and 85% (p=0.006). In multivariate analysis with IPI, DTX1 mutation status remained as an independent prognostic factor for both PFS (RR, 2.83; 95% CI, 1.17-6.85, p=0.021) and OS (RR, 3.51; 95% CI, 1.41-8.71, p=0.007). Mutational status was also found to correlate with the expression levels; the patients with mutations had lower DTX1 mRNA levels (p=0.006). When DTX1 mutations were analyzed according to different functional domains, 11 out of 16 (65%) were located in the first exon encoding the WWE 1 domain and a binding site for intracellular domain of Notch1. Interestingly, the patients with mutations in exon 1 had a significantly worse survival in comparison to other patients (Figure 1).Taken together, the results demonstrate that DTX1 mutations in DLBCL are an independent predictor of survival after chemoimmunotherapy, and mutations enriched in exon 1 identify cases with dismal prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deltex1 promotes protein kinase Cθ degradation and sustains Casitas B-lineage lymphoma expression.

The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b.

Ubiquitin E3 ligase deltex1 promotes PKCθ degradation for full T cell anergy (P1109)

Abstract The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Cbl-b, Itch, and GRAIL. Both Cbl-b and Itch contribute to the downregulation of PKCθ in anergic T cells. Recently, we identified deltex1 (DTX1) as another E3 ligase that is upregulated in T cell anergy. DTX1-deficiency prevents T cell anergy production and leads to autoimmunity. In the present study, we found that DTX1 also targeted to PKCθ for degradation. DTX1 overexpression stimulated the degradation of PKCθ. There was a direct interaction between DTX1 and PKCθ. Because DTX1 also promotes Cbl-b expression, we showed that Cbl-b knockdown did not affect the ability of DTX1 to downregulate PKCθ, excluding an involvement of Cbl-b in DTX1-mediated PKCθ degradation. We also found that DTX1-induced PKCθ degradation was not prevented by proteasome inhibitor. Instead, DTX1 directed PKCθ into lysosomal pathway for degradation. In addition, T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1-/- T cells, supporting the essential role of DTX1 in PKCθ downregulation. Our results suggest that DTX1 acts together with Cbl-b and Itch to assure a complete silencing of PKCθ for T cell anergy and immune tolerance.

Deltex-1 Activates Mitotic Signaling and Proliferation and Increases the Clonogenic and Invasive Potential of U373 and LN18 Glioblastoma Cells and Correlates with Patient Survival

Glioblastoma (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including microRNA-21- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low DTX1 levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets.

Deltex1 Is a Target of the Transcription Factor NFAT that Promotes T Cell Anergy

The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.

Notch1 Signaling Regulates Radial Glia Differentiation through Multiple Transcriptional Mechanisms

Signaling by the Notch1 receptor is critical for the formation of radial glia in the developing nervous system. We have shown previously that Notch1 regulates the molecular and morphological differentiation of radial glia through the transcriptional activation of at least two genes, brain lipid binding protein (BLBP) and the erbB2 receptor tyrosine kinase. However, the mechanisms by which this occurs remained undefined. Here we demonstrate that Notch1 effects on radial glia gene expression are mediated by two downstream mechanisms, one that the depends on Suppressor of Hairless [Su(H)] and the other on Deltex1 (DTX1). These two Notch1-binding proteins contribute to the regulation of BLBP and erbB2 expression, respectively. Importantly, our results suggest that, although these events can occur simultaneously, a hierarchical relationship might exist between DTX1 and Su(H), because overexpression of DTX1 or a dominant-negative form of this protein inhibits Su(H)-mediated events but not vice versa. In contrast to the effects of DTX1 overexpression, interference RNA-mediated knock-down of DTX1 blocks Notch1-induced erbB2 promoter activation and radial glia formation selectively, without affecting Su(H)-dependent pathways, indicating that loss of DTX1 expression and expression of dominant-negative DTX1 result in different alterations in cell differentiation and gene expression. Together, these results show that Notch1 regulates radial glia formation through two distinct transcriptional mechanisms and that the outcomes of Notch1 signaling may depend on the relative expression levels of its coregulators.

Role of Deltex-1 as a Transcriptional Regulator Downstream of the Notch Receptor

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.