FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

The Ubiquitin-Associated and SH3 Domain-Containing Proteins (UBASH3) Family in Mammalian Development and Immune Response.

UBASH3A and UBASH3B are protein families of atypical protein tyrosine phosphatases that function as regulators of various cellular processes during mammalian development. As UBASH3A has only mild phosphatase activity, its regulatory effects are based on the phosphatase-independent mechanisms. On the contrary, UBASH3B has strong phosphatase activity, and the suppression of its receptor signalling is mediated by Syk and Zap-70 kinases. The regulatory functions of UBASH3A and UBASH3B are particularly evident in the lymphoid tissues and kidney development. These tyrosine phosphatases are also known to play key roles in autoimmunity and neoplasms. However, their involvement in mammalian development and its regulatory functions are largely unknown and are discussed in this review.

UBASH3A Interacts with PTPN22 to Regulate IL2 Expression and Risk for Type 1 Diabetes.

UBASH3A is a negative regulator of T cell activation and IL-2 production and plays key roles in autoimmunity. Although previous studies revealed the individual effects of UBASH3A on risk for type 1 diabetes (T1D; a common autoimmune disease), the relationship of UBASH3A with other T1D risk factors remains largely unknown. Given that another well-known T1D risk factor, PTPN22, also inhibits T cell activation and IL-2 production, we investigated the relationship between UBASH3A and PTPN22. We found that UBASH3A, via its Src homology 3 (SH3) domain, physically interacts with PTPN22 in T cells, and that this interaction is not altered by the T1D risk coding variant rs2476601 in PTPN22. Furthermore, our analysis of RNA-seq data from T1D cases showed that the amounts of UBASH3A and PTPN22 transcripts exert a cooperative effect on IL2 expression in human primary CD8+ T cells. Finally, our genetic association analyses revealed that two independent T1D risk variants, rs11203203 in UBASH3A and rs2476601 in PTPN22, interact statistically, jointly affecting risk for T1D. In summary, our study reveals novel interactions, both biochemical and statistical, between two independent T1D risk loci, and suggests how these interactions may affect T cell function and increase risk for T1D.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

CRKL, AIFM3, AIF, BCL2, and UBASH3A during Human Kidney Development.

We aimed to investigate the spatio-temporal expression of possible CAKUT candidate genes CRKL, AIFM3, and UBASH3A, as well as AIF and BCL2 during human kidney development. Human fetal kidney tissue was stained with antibodies and analyzed by fluorescence microscopy and RT-PCR. Quantification of positive cells was assessed by calculation of area percentage and counting cells in nephron structures. Results showed statistically significant differences in the temporal expression patterns of the examined markers, depending on the investigated developmental stage. Limited but strong expression of CRKL was seen in developing kidneys, with increasing expression up to the period where the majority of nephrons are formed. Results also lead us to conclude that AIFM3 and AIF are important for promoting cell survival, but only AIFM3 is considered a CAKUT candidate gene due to the lack of AIF in nephron developmental structures. Our findings imply great importance of AIFM3 in energy production in nephrogenesis and tubular maturation. UBASH3A raw scores showed greater immunoreactivity in developing structures than mature ones which would point to a meaningful role in nephrogenesis. The fact that mRNA and proteins of CRKL, UBASH3A, and AIFM3 were detected in all phases of kidney development implies their role as renal development control genes.

TULA-family proteins: Jacks of many trades and then some.

UBASH3/STS/TULA is a novel two-member family, which exerts several key regulatory effects in multiple cell types. UBASH3B/STS-1/TULA-2 is a highly active protein tyrosine phosphatase; its major target appears to be a specific regulatory site of protein tyrosine kinases of the Syk family, dephosphorylation of which inhibits Syk and Zap-70 kinases and suppresses receptor signaling mediated by these kinases. UBASH3A/STS-2/TULA exhibits substantial homology to UBASH3B/STS-1/TULA-2, but possesses only a small fraction of phosphatase activity of UBASH3B/STS-1/TULA-2, and thus, its regulatory effect may be based also on the phosphatase-independent mechanisms. Critical physiologic effects of these proteins have been demonstrated in T lymphocytes, platelets, stem cells, and other important cell types. These proteins have also been shown to play a key role in such pathologic conditions as autoimmunity, cancer, and thrombosis. The review focuses on the recent studies of this important family of cellular regulators.

Replication of association at the LPP and UBASH3A loci in a UK autoimmune Addison's disease cohort.

Autoimmune Addison's disease (AAD) arises from a complex interplay between multiple genetic susceptibility polymorphisms and environmental factors. The first genome wide association study (GWAS) with patients from Scandinavian Addison's registries has identified association signals at four novel loci in the genes LPP, SH2B3, SIGLEC5, and UBASH3A. To verify these novel risk loci, we performed a case-control association study in our independent cohort of 420 patients with AAD from the across the UK. We report significant association of alleles of the LPP and UBASH3A genes [odds ratio (95% confidence intervals), 1.46 (1.21-1.75)and 1.40 (1.16-1.68), respectively] with AAD in our UK cohort. In addition, we report nominal association of AAD with SH2B3 [OR 1.18 (1.02-1.35)]. We confirm that variants at the LPP and UBASH3A loci confer susceptibility to AAD in a UK population. Further studies with larger patient cohorts are required to robustly confirm the association of SH2B3 and SIGLEC5/SPACA6 alleles.

IL-6 production through repression of UBASH3A gene via epigenetic dysregulation of super-enhancer in CD4+ T cells in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with immune dysfunction. UBASH3A as a negative regulator of T cell receptors (TCRs) signaling is a susceptible factor in RA. The aim of this study was to determine the role of UBASH3A in RA pathogenesis, by assessing the role of super-enhancer (SE) in the control of UBASH3A expression in CD4+ T cells and the contribution of the latter in proinflammatory cytokine production in patients with RA. METHODS: UBASH3A mRNA and protein levels were quantified by PCR and western blotting, respectively. The cells were treated with a locked nucleic acid to inhibit enhancer RNA (eRNA) expression. Chromatin immunoprecipitation was used to identify the factors recruited to UBASH3A loci displaying SE architecture. CD4+ T cells were transfected with UBASH3A plasmids, and cytokine levels were measured by a cytometric bead array. RESULTS: UBASH3A was extracted as a RA susceptibility gene associated with SNPs in the SEs that are highly expressed in CD4+ T cells by in silico screening. UBASH3A mRNA and protein expression levels were lower in CD4+ T cells of RA patients than in the control. eRNA_1 and eRNA_3 knockdown reduced UBASH3A mRNA levels. RA patients exhibited accumulation of BTB and CNC homology 2 (BACH2), the silencing transcription factor, at the UBASH3A loci in CD4+ T cells, but not the SE-defining factor, mediator complex subunit 1 (MED1)/bromodomain 4 (BRD4). However, opposite changes were observed in the control. Stimulation of TCRs expressed on CD4+ T cells of RA patients resulted in interleukin (IL)-6 production, while UBASH3A over-expression significantly inhibited the production. CONCLUSIONS: In RA, transcription of UBASH3A is suppressed via epigenetic regulation of SE in CD4+ T cells. Low UBASH3A levels result in excessive TCR signal activation with subsequent enhancement of IL-6 production.

TULA proteins as signaling regulators.

Two members of the UBASH3/STS/TULA family exhibit a unique protein domain structure, which includes a histidine phosphatase domain, and play a key role in regulating cellular signaling. UBASH3A/STS-2/TULA is mostly a lymphoid protein, while UBASH3B/STS-1/TULA-2 is expressed ubiquitously. Dephosphorylation of tyrosine-phosphorylated proteins by TULA-2 and, probably to a lesser extent, by TULA critically contribute to the molecular basis of their regulatory effect. The notable differences between the effects of the two family members on cellular signaling and activation are likely to be linked to the difference between their specific enzymatic activities. However, these differences might also be related to the functions of their domains other than the phosphatase domain and independent of their phosphatase activity. The down-regulation of the Syk/Zap-70-mediated signaling, which to-date appears to be the best-studied regulatory effect of TULA family, is discussed in detail in this publication.

UBASH3A gene polymorphisms and expression profile in rheumatoid arthritis.

OBJECTIVES: Recent evidence has demonstrated that UBASH3A play a pivotal role in multiple autoimmune diseases. In this study, we explored the association between UBASH3A gene single-nucleotide polymorphisms (SNPs) and rheumatoid arthritis (RA) in a Chinese Han population. We also comparatively evaluated the UBASH3A expression profile in peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls. METHODS: Four UBASH3A polymorphisms (rs1893592, rs11203203, rs2277798, and rs3788013) were studied in 553 patients with RA and 587 controls in a Chinese population. Genotyping was performed using the Fluidigm 192.24 Dynamic Array Integrated Fluidic Circuit (IFC). For gene expression study, UBASH3A mRNA levels of 30 RA patients and 31 healthy individuals were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). Data were analyzed by SPSS 19.0 software. RESULTS: A significant association between rs1893592 polymorphism and RA was found under all genetic models (all p<.05). We also discovered a significant association between rs3788013 polymorphism and RA in the allele and genotype distributions, as well as the recessive model (all p<.05). Moreover, we found the genotype distribution and allele frequency of rs1893592 were significantly associated with RF phenotype in the RA patients (χ2 = 6.786, p=.034; χ2 = 4.534, p=.033; respectively). We also found the genotype distribution and allele frequency of rs2277798 were significantly associated with anti-CCP phenotype in the RA patients (χ2 = 7.873, p=.020; χ2 = 4.473, p=.034; respectively). However, we did not detect any significant associations between rs11203203 and RA susceptibility and autoantibody profiles (all p>.05). The mRNA expression of UBASH3A was increased in PBMCs of patients with RA when compared to healthy controls (p=.001). CONCLUSIONS: Our observations suggested that the dysregulation of UBASH3A might be associated with the pathogenesis of RA, and UBASH3A gene polymorphisms (rs1893592 and rs3788013) might contribute to RA susceptibility in Chinese Han population.

Evaluation of the association of UBASH3A and SYNGR1 with rheumatoid arthritis and disease activity and severity in Han Chinese.

Rheumatoid arthritis (RA) is a common complex autoimmune disorder. UBASH3A and SYNGR1 were identified recently as susceptibility genes for RA risk in Korean and European populations, but the genetic aetiology and pathogenesis of RA have not been fully elucidated. We designed a two-stage case-control study including 916 RA patients and 2,266 unrelated healthy controls to identify common genetic variants in UBASH3A and SYNGR1 that predispose Han Chinese individuals to RA. We also evaluated the role of associated variants in clinical manifestations of RA, which may provide clues to the mechanisms involved in the aetiology of RA. We successfully identified two SNPs, rs1893592 in UBASH3A and rs909685 in SYNGR1, as significantly associated with the disease status of RA using our two-stage strategy. The rs1893592 SNP in UBASH3A was related with DAS28, CRP level and bone erosion. In summary, our results indicate that genetic variants in UBASH3A and SYNGR1 may modify individual susceptibility to RA in the Han Chinese population and support the role of the UBASH3A gene in RA disease activity and severity.

Association of UBASH3A gene polymorphisms and systemic lupus erythematosus in a Chinese population.

Recent evidence has demonstrated that UBASH3A gene was associated with multiple autoimmune diseases. The aim of this study was to explore the association between UBASH3A gene single-nucleotide polymorphisms (SNPs) and systemic lupus erythematosus (SLE) in a Chinese Han population. Four UBASH3A polymorphisms (rs11203203, rs3788013, rs2277798, and rs1893592) were genotyped using the Fluidigm 192.24 Dynamic Array™ Integrated Fluidic Circuit (IFC). Data were analyzed by SPSS 11.5 software. A total of 792 SLE patients and 777 healthy controls were included in this study. The CC genotype and C allele of rs3788013 polymorphism were more frequent in the patient group than in controls (OR=1.583, 95% CI=1.095-2.287; OR=1.258, 95% CI=1.083-1.461, respectively). We also found a statistical significance under the recessive model (OR=1.298, 95% CI=1.049-1.607, p=0.017). The frequency of variant genotype AC of rs3788013 was associated with the phenotype of vasculitis (p=0.012). A statistically significant association was observed between UBASH3A rs1893592 C allele and skin rash, oral ulcer and arthritis (p<0.05). Moreover, we found that the genotype distribution of rs2277798 was significantly associated with hematuria in the SLE patients (p=0.003). However, UBASH3A rs11203203, rs2277798, and rs1893592 polymorphisms were not associated with the risk of SLE (p>0.05). The findings suggest that UBASH3A gene might contribute to SLE susceptibility and influence the clinical phenotype of the disease. Further studies are necessary to elucidate the exact role of UBASH3A gene in the pathogenesis of SLE.

Lack of association between polymorphisms in the UBASH3A gene and autoimmune thyroid disease: a case control study / Falta de associação entre polimorfismos do gene UBASH3A e doença tiroidiana autoimune: estudo caso controle

Objective: The aim of this study was to investigate UBASH3A gene variation association with autoimmune thyroid disease and clinical features in a Chinese Han population. Subjects and methods: A total of 667 AITD patients (417 GD and 250 HT) and 301 healthy controls were genotyped for two single nucleotide polymorphisms (SNPs) rs11203203, rs3788013 of UBASH3A gene, utilizing the Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) Platform. Results: Between the control group and AITD, GD and HT group, no statistically significant difference was observed in the genotypic and allelic frequencies of the two SNPs. There was no significant difference in allelic frequencies of the two SNPs between GD with and without ophthalmopathy. There was no significant difference in haplotype distributions between the control group and AITD, GD or HT group. Conclusion: Rs11203203 and rs3788013 in UBASH3A gene may not be associated with AITD patients in Chinese Han population. .

Objetivo: O objetivo deste estudo foi investigar a variação no gene UBASH3A com a doença tiroidiana autoimune e características clínicas na população chinesa Han. Sujeitos e métodos: Um total de 667 pacientes com DTAI (417 com DG e 250 com TH) e 301 controles saudáveis foi genotipado para dois polimorfismos de nucleotídeo simples (SNPs) rs11203203, rs3788013 do gene UBASH3A, usando-se a plataforma MALDI-TOF-MS (Ionização/Dessorção de Matriz Assistida por Laser – Tempo de Voo/Espectrômetro de Massa). Resultados: Não foram observadas diferenças significativas entre as frequências genotípicas e alélicas dos dois SNPs nos grupos controle e DTAI, DG e TH. Não houve diferenças significativas entre as frequências alélicas dos dois SNPs em pacientes com DG com ou sem olftalmopatia. Não houve diferenças significativas nas distribuições de haplótipos no grupo controle e nos grupos DTAI, DG e TH. Conclusão: Os SNPs rs11203203 e rs3788013 do gene UBASH3A podem não estar associados a pacientes com DTAI na população chinesa Han. .