ABSTRACT
Clonal expansion and immunological memory are hallmark features of the mammalian adaptive immune response and essential for prolonged host control of pathogens. Recent work demonstrates that natural killer (NK) cells of the innate immune system also exhibit these adaptive traits during infection. Here we demonstrate that differentiating and 'memory' NK cells possess distinct chromatin accessibility states and that their epigenetic profiles reveal a 'poised' regulatory program at the memory stage. Furthermore, we elucidate how individual STAT transcription factors differentially control epigenetic and transcriptional states early during infection. Finally, concurrent chromatin profiling of the canonical CD8+ T cell response against the same infection demonstrated parallel and distinct epigenetic signatures defining NK cells and CD8+ T cells. Overall, our study reveals the dynamic nature of epigenetic modifications during the generation of innate and adaptive lymphocyte memory.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Chromatin/metabolism , Herpesviridae Infections/immunology , Killer Cells, Natural/physiology , Muromegalovirus/physiology , STAT1 Transcription Factor/metabolism , STAT4 Transcription Factor/metabolism , Adaptive Immunity , Animals , Cells, Cultured , Chromatin/genetics , Clonal Selection, Antigen-Mediated , Epigenesis, Genetic , Gene Expression Profiling , Immunity, Innate , Immunologic Memory , Mice , Mice, Inbred C57BL , Mice, Knockout , STAT1 Transcription Factor/genetics , STAT4 Transcription Factor/geneticsABSTRACT
Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.
Subject(s)
Antineoplastic Agents , STAT5 Transcription Factor , Humans , Immunity, Innate , Cell Differentiation , Killer Cells, Natural , Inflammation , STAT4 Transcription Factor/geneticsABSTRACT
Although intergenic long noncoding RNAs (lincRNAs) have been linked to gene regulation in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identified 1,524 lincRNA clusters in 42 T cell samples, from early T cell progenitors to terminally differentiated helper T cell subsets. Our analysis revealed highly dynamic and cell-specific expression patterns for lincRNAs during T cell differentiation. These lincRNAs were located in genomic regions enriched for genes that encode proteins with immunoregulatory functions. Many were bound and regulated by the key transcription factors T-bet, GATA-3, STAT4 and STAT6. We found that the lincRNA LincR-Ccr2-5'AS, together with GATA-3, was an essential component of a regulatory circuit in gene expression specific to the TH2 subset of helper T cells and was important for the migration of TH2 cells.
Subject(s)
Gene Expression Regulation/immunology , Precursor Cells, T-Lymphoid/metabolism , RNA, Long Noncoding/genetics , Th1 Cells/metabolism , Th17 Cells/metabolism , Th2 Cells/metabolism , Animals , Cell Differentiation , Cell Movement , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/immunology , GATA3 Transcription Factor/metabolism , Genetic Loci , Mice , Mice, Inbred C57BL , Molecular Sequence Annotation , Precursor Cells, T-Lymphoid/cytology , Precursor Cells, T-Lymphoid/immunology , Protein Binding , RNA, Long Noncoding/immunology , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/immunology , STAT4 Transcription Factor/metabolism , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/immunology , STAT6 Transcription Factor/metabolism , Signal Transduction , T-Box Domain Proteins/genetics , T-Box Domain Proteins/immunology , T-Box Domain Proteins/metabolism , Th1 Cells/cytology , Th1 Cells/immunology , Th17 Cells/cytology , Th17 Cells/immunology , Th2 Cells/cytology , Th2 Cells/immunology , Transcriptome/immunologyABSTRACT
Innate lymphoid cells (ILCs) function to protect epithelial barriers against pathogens and maintain tissue homeostasis in both barrier and non-barrier tissues. Here, utilizing Eomes reporter mice, we identify a subset of adipose group 1 ILC (ILC1) and demonstrate a role for these cells in metabolic disease. Adipose ILC1s were dependent on the transcription factors Nfil3 and T-bet but phenotypically and functionally distinct from adipose mature natural killer (NK) and immature NK cells. Analysis of parabiotic mice revealed that adipose ILC1s maintained long-term tissue residency. Diet-induced obesity drove early production of interleukin (IL)-12 in adipose tissue depots and led to the selective proliferation and accumulation of adipose-resident ILC1s in a manner dependent on the IL-12 receptor and STAT4. ILC1-derived interferon-γ was necessary and sufficient to drive proinflammatory macrophage polarization to promote obesity-associated insulin resistance. Thus, adipose-resident ILC1s contribute to obesity-related pathology in response to dysregulated local proinflammatory cytokine production.
Subject(s)
Adipose Tissue/immunology , Insulin Resistance/immunology , Lymphocytes/immunology , Macrophages/immunology , Obesity/immunology , T-Box Domain Proteins/metabolism , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Differentiation , Cells, Cultured , Cytokines/metabolism , Humans , Immunity, Innate , Inflammation Mediators/metabolism , Interferon-gamma/metabolism , Interleukin-12/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolism , T-Box Domain Proteins/geneticsABSTRACT
Primary Sjögren's syndrome (pSS) is characterized by its autoimmune nature. This study investigates the role of the IFNγ SNP rs2069705 in modulating the susceptibility to pSS. Differential expression of IFNγ and BAFF was analyzed using the GEO database's mRNA microarray GSE84844. Genotyping of the IFNγ SNP rs2069705 was conducted via the dbSNP website. The JASPAR tool was used for predicting transcription factor bindings. Techniques such as dual-luciferase reporter assays, Chromatin immunoprecipitation, and analysis of a pSS mouse model were applied to study gene and protein interactions. A notable increase in the mutation frequency of IFNγ SNP rs2069705 was observed in MNCs from the exocrine glands of pSS mouse models. Bioinformatics analysis revealed elevated levels of IFNγ and BAFF in pSS samples. The model exhibited an increase in both CD20+ B cells and cells expressing IFNγ and BAFF. Knocking down IFNγ resulted in lowered BAFF expression and less lymphocyte infiltration, with BAFF overexpression reversing this suppression. Activation of the Janus kinase (JAK)/STAT1 pathway was found to enhance transcription in the BAFF promoter region, highlighting IFNγ's involvement in pSS. In addition, rs2069705 was shown to boost IFNγ transcription by promoting interaction between its promoter and STAT4. SNP rs2069705 in the IFNγ gene emerges as a pivotal element in pSS susceptibility, primarily by augmenting IFNγ transcription, activating the JAK/STAT1 pathway, and leading to B-lymphocyte infiltration in the exocrine glands.NEW & NOTEWORTHY The research employed a combination of bioinformatics analysis, genotyping, and experimental models, providing a multifaceted approach to understanding the complex interactions in pSS. We have uncovered that the rs2069705 SNP significantly affects the transcription of IFNγ, leading to altered immune responses and B-lymphocyte activity in pSS.
Subject(s)
B-Lymphocytes , Interferon-gamma , Polymorphism, Single Nucleotide , Sjogren's Syndrome , Transcriptional Activation , Animals , Female , Humans , Mice , B-Cell Activating Factor/genetics , B-Cell Activating Factor/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Disease Models, Animal , Genetic Predisposition to Disease , Interferon-gamma/genetics , Interferon-gamma/metabolism , Janus Kinases/metabolism , Janus Kinases/genetics , Polymorphism, Single Nucleotide/genetics , Signal Transduction , Sjogren's Syndrome/genetics , Sjogren's Syndrome/immunology , Sjogren's Syndrome/metabolism , Sjogren's Syndrome/pathology , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolismABSTRACT
The conversion of signal transducer and activator of transcription (STAT) proteins from latent to active transcription factors is central to cytokine signaling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers-STAT1, STAT3, STAT4, STAT5A, and STAT5B-and two heterodimers-STAT1:STAT2 and STAT5A:STAT5B-and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein-STAT6-was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation.
Subject(s)
Gene Expression Regulation , STAT Transcription Factors , Trans-Activators , Cytokines/metabolism , Phosphorylation , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/genetics , STAT2 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Trans-Activators/metabolism , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Protein MultimerizationABSTRACT
Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5 mC) conversion to 5-hydroxymethylcytosine (5 hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5 hmC in CD4(+) T cells and found that 5 hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5 hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.
Subject(s)
Cytokines/biosynthesis , DNA-Binding Proteins/immunology , Epigenesis, Genetic/immunology , Proto-Oncogene Proteins/immunology , Th1 Cells/immunology , Th17 Cells/immunology , 5-Methylcytosine/analogs & derivatives , Animals , Cell Differentiation , Cytokines/immunology , Cytosine/analogs & derivatives , Cytosine/immunology , Cytosine/metabolism , DNA/immunology , DNA/metabolism , DNA Methylation , DNA-Binding Proteins/genetics , Dioxygenases , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/immunology , Gene Expression Regulation , Genome , Humans , Mice , Mice, Transgenic , Proto-Oncogene Proteins/genetics , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/immunology , T-Box Domain Proteins/genetics , T-Box Domain Proteins/immunology , Th1 Cells/cytology , Th1 Cells/enzymology , Th17 Cells/cytology , Th17 Cells/enzymologyABSTRACT
Background: To uncover the potential significance of JAK-STAT-SOCS1 axis in penile cancer, our study was the pioneer in exploring the altered expression processes of JAK-STAT-SOCS1 axis in tumorigenesis, malignant progression and lymphatic metastasis of penile cancer. Methods: In current study, the comprehensive analysis of JAK-STAT-SOCS1 axis in penile cancer was analyzed via multiple analysis approaches based on GSE196978 data, single-cell data (6 cancer samples) and bulk RNA data (7 cancer samples and 7 metastasis lymph nodes). Results: Our study observed an altered molecular expression of JAK-STAT-SOCS1 axis during three different stages of penile cancer, from tumorigenesis to malignant progression to lymphatic metastasis. STAT4 was an important dominant molecule in penile cancer, which mediated the immunosuppressive tumor microenvironment by driving the apoptosis of cytotoxic T cell and was also a valuable biomarker of immune checkpoint inhibitor treatment response. Conclusions: Our findings revealed that the complexity of JAK-STAT-SOCS1 axis and the predominant role of STAT4 in penile cancer, which can mediate tumorigenesis, malignant progression, and lymphatic metastasis. This insight provided valuable information for developing precise treatment strategies for patients with penile cancer.
Subject(s)
Disease Progression , Janus Kinases , Lymphatic Metastasis , Penile Neoplasms , STAT4 Transcription Factor , Suppressor of Cytokine Signaling 1 Protein , Humans , Male , Penile Neoplasms/pathology , Penile Neoplasms/genetics , Penile Neoplasms/metabolism , Suppressor of Cytokine Signaling 1 Protein/genetics , Suppressor of Cytokine Signaling 1 Protein/metabolism , Lymphatic Metastasis/pathology , Lymphatic Metastasis/genetics , Janus Kinases/metabolism , STAT4 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , Gene Expression Regulation, Neoplastic , Carcinogenesis/genetics , Carcinogenesis/pathology , Signal Transduction , Tumor Microenvironment/immunology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacologyABSTRACT
Signal transducer and activator of transcription 4 (STAT4) plays a crucial role in the host immune response against Mycobacterium tuberculosis. This study investigates the association between STAT4 gene polymorphisms and pulmonary tuberculosis (TB) risk in the Moldavian population. A total of 272 TB patients and 251 community-matched controls underwent screening for functional single-nucleotide polymorphisms (SNPs) rs897200 and rs7574865 in the STAT4 gene. The minor T allele and the TT/CT genotype of rs897200 demonstrated a significant association with reduced pulmonary TB risk (allelic model: adjusted OR = .74, p = .025; log-additive model: adjusted OR = .72, p = .02; and dominant model: adjusted OR = .65, p = .023), indicating a protective effect. Similar associations, characterized by an even more pronounced reduction in risk, were observed among females and late-onset TB patients (>44 years). No significant associations were found for rs7574865. In addition, a combined genotype analysis incorporating 43 SNPs from our previous studies revealed potential associations, such as STAT4 rs897200 CT with IFNG rs2430561 AA (adjusted OR = .36, p = .0025) and STAT4 rs897200 CT with TNFA rs1800629 GA (adjusted OR = .33, p = .0012). This study emphasizes the significant association of STAT4 rs897200 with pulmonary TB risk in the Moldavian population, underscoring its role in the disease development.
Subject(s)
Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , STAT4 Transcription Factor , Tuberculosis, Pulmonary , Humans , STAT4 Transcription Factor/genetics , Tuberculosis, Pulmonary/genetics , Female , Male , Adult , Middle Aged , Genotype , Alleles , Moldova , Case-Control Studies , Genetic Association Studies , Gene Frequency , Mycobacterium tuberculosisABSTRACT
Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.
Subject(s)
Epidermal Growth Factor , Sjogren's Syndrome , Humans , Epidermal Growth Factor/pharmacology , Epidermal Growth Factor/metabolism , Sjogren's Syndrome/drug therapy , Sjogren's Syndrome/genetics , Interferon-alpha/pharmacology , Immunologic Factors , Cell Culture Techniques , RNA, Messenger/metabolism , Dietary Supplements , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Phosphorylation , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolismABSTRACT
Congenital amegakaryocytic thrombocytopenia (CAMT) is a recessive disorder characterized by severe reduction of megakaryocytes and platelets at birth, which evolves toward bone marrow aplasia in childhood. CAMT is mostly caused by mutations in MPL (CAMT-MPL), the gene encoding the receptor of thrombopoietin (THPO), a crucial cytokine regulating hematopoiesis. CAMT can be also due to mutations affecting the THPO coding region (CAMT-THPO). In a child with the clinical picture of CAMT, we identified the homozygous c.-323C>T substitution, affecting a potential regulatory region of THPO. Although mechanisms controlling THPO transcription are not characterized, bioinformatics and in vitro analysis showed that c.-323C>T prevents the binding of transcription factors ETS1 and STAT4 to the putative THPO promoter, impairing THPO expression. Accordingly, in the proband the serum THPO concentration indicates defective THPO production. Based on these findings, the patient was treated with the THPO-mimetic agent eltrombopag, which induced a significant increase in platelet count and stable remission of bleeding symptoms. Herein, we report a novel pathogenic variant responsible for CAMT and provide new insights into the mechanisms regulating transcription of the THPO gene.
Subject(s)
Receptors, Thrombopoietin , Thrombopoietin , Child , Infant, Newborn , Humans , Thrombopoietin/pharmacology , Receptors, Thrombopoietin/genetics , Mutation , Megakaryocytes/pathology , Promoter Regions, Genetic , Proto-Oncogene Protein c-ets-1/genetics , STAT4 Transcription Factor/geneticsABSTRACT
Single nucleotide polymorphisms in non-HLA genes are involved in the development of rheumatoid arthritis (RA). SNPS in genes: PADI4 (rs2240340), STAT4 (rs7574865), CD40 (rs4810485), PTPN22 (rs2476601), and TRAF1 (rs3761847) have been described as risk factors for the development of autoimmune diseases, including RA. This study aimed to assess the prevalence of polymorphisms of these genes in the Polish population of patients with rheumatoid arthritis as compared to healthy controls. 324 subjects were included in the study: 153 healthy subjects and 181 patients from the Department of Rheumatology, Medical University of Lodz who fulfilled the criteria of rheumatoid arthritis diagnosis. Genotypes were determined by Taqman SNP Genotyping Assay. rs2476601 (G/A, OR = 2.16, CI = 1.27-3.66; A/A, OR = 10.35, CI = 1.27-84.21), rs2240340 (C/T, OR = 4.35, CI = 2.55-7.42; T/T, OR = 2.80, CI = 1.43-4.10) and rs7574865 (G/T, OR = 1.97, CI = 1.21-3.21; T/T, OR = 3.33, CI = 1.01-11.02) were associated with RA in the Polish population. Rs4810485 was also associated with RA, however after Bonferroni's correction was statistically insignificant. We also found an association between minor alleles of rs2476601, rs2240340, and rs7574865 and RA (OR = 2.32, CI = 1.47-3.66; OR = 2.335, CI = 1.64-3.31; OR = 1.88, CI = 1.27-2.79, respectively). Multilocus analysis revealed an association between CGGGT and rare (below 0.02 frequency) haplotypes (OR = 12.28, CI = 2.65-56.91; OR = 3.23, CI = 1.63-6.39). In the Polish population, polymorphisms of the PADI4, PTPN22, and STAT4 genes have been detected, which are also known risk factors for RA in various other populations.
Subject(s)
Arthritis, Rheumatoid , Polymorphism, Single Nucleotide , Humans , TNF Receptor-Associated Factor 1/genetics , Poland/epidemiology , Genetic Predisposition to Disease , Arthritis, Rheumatoid/epidemiology , Arthritis, Rheumatoid/genetics , Genotype , Alleles , Case-Control Studies , Gene Frequency , Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics , STAT4 Transcription Factor/geneticsABSTRACT
Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT-qPCR analysis and Western blotting results showed that OCLN increased interleukin-8 (IL8) and p-signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual-luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.
Subject(s)
Interleukin-8 , Occludin , STAT4 Transcription Factor , Urinary Bladder Neoplasms , Animals , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Mice , Neovascularization, Pathologic/genetics , Occludin/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.
Subject(s)
HSC70 Heat-Shock Proteins/genetics , Heat Shock Transcription Factors/genetics , Heat-Shock Response/genetics , STAT4 Transcription Factor/genetics , Sumoylation/genetics , DNA-Binding Proteins/genetics , Heat-Shock Response/physiology , Homeostasis/genetics , Humans , Protein Folding , Protein Processing, Post-Translational/genetics , Stress, Physiological/genetics , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Signal transducer and activator of transcription 4 (STAT4) is closely related to liver diseases and affects the processes of inflammation and carcinogenesis by regulating immune responses. A single-nucleotide polymorphism rs7574865 (T > G) in STAT4 has been reported to be associated with the risk of hepatocellular carcinoma (HCC). In addition, hepatitis B virus (HBV) mutations are crucial risk factors for HBV-induced HCC. However, the effects of the interactions of STAT4 rs7574865 with HBV mutations on the risk of HCC remain unknown. Rs7574865 was genotyped in 846 healthy controls (HCs), 968 chronic hepatitis B (CHB) subjects, 316 liver cirrhosis (LC) subjects and 1021 HCC subjects using Sequenom MassArray. HBV mutations were detected via direct sequencing. Multivariate logistic regression analysis was used to evaluate the effects of the interactions of STAT4 rs7574865 with HBV mutations on the risk of HCC. We found that the rs7574865 TT genotype was significantly associated with HBeAg seroconversion (TT vs. GG, p = 0.012; TT vs. GT, p = 0.033). The rs7574865 GG genotype was significantly associated with increased risks of CHB (p = 0.048), LC (p = 0.005) and HCC (p < 0.001). The interaction term between rs7574865 and HBV C1913A significantly increased the risk of progression from CHB to HCC (p = 0.038), while the interaction term between rs7574865 and HBV T1674C significantly increased the risk of progression from LC to HCC (p = 0.023). STAT4 rs7574865 is significantly associated with the risks of CHB, LC and HCC. The interactions of rs7574865 with HBV C1913A and T1674C mutations significantly increase the risk of HCC, which have the potential to identify HBV-infected individuals who tend to progress from CHB or LC to HCC.
Subject(s)
Carcinoma, Hepatocellular , Hepatitis B, Chronic , Liver Neoplasms , STAT4 Transcription Factor , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Case-Control Studies , Genetic Predisposition to Disease , Genotype , Hepatitis B virus , Hepatitis B, Chronic/complications , Hepatitis B, Chronic/genetics , Humans , Liver Cirrhosis/genetics , Liver Neoplasms/genetics , Liver Neoplasms/virology , Mutation , Polymorphism, Single Nucleotide , STAT4 Transcription Factor/geneticsABSTRACT
A variant in signal transducer and activator of transcription 4 (STAT4) was reported to correlate with the response of interferon-α (IFN-α) in a retrospective study in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B virus (CHB) patients. Here, we conducted a prospective study to analyze the effect of STAT4 genetic polymorphism on the response of pegylated interferon-α-2a (PegIFN-α-2a) in HBeAg-positive patients. A prospective, multicenter, open-label, parallel cohort study was performed. One hundred and fifty treatment-naïve and 156 nucleos(t)ide analog (NA)-experienced HBeAg-positive CHB patients were enrolled, respectively. All patients received PegIFN-α-2a treatment for 48 weeks and 24-week follow-up post PegIFN-α-2a treatment. Before treatment, STAT4 genetic polymorphism was determined by PCR and DNA sequencing. Serological markers, serum HBV DNA levels, and adverse events were collected at each visit. We observed a larger reduction of HBV DNA load and a significantly higher HBeAg seroconversion rate in the GT/TT group than in the GG group at week 72 (p = 0.002 and p = 0.023) in treatment-naïve patients. In NA-experienced patients, the HBeAg seroconversion rate in the GT/TT group was higher than that in the GG group at week 72 (p = 0.005). STAT4 rs7574865 gene polymorphism was the strongest independent predictor of HBeAg seroconversion in both paralleled cohorts. Also, patients in the GT/TT group had a higher hepatitis B surface antigen loss rate than in the GG group in the study. There was no significant difference in adverse events between GG and GT/TT groups. This prospective cohort study confirmed that STAT4 rs7574865 gene polymorphism is associated with HBeAg seroconversion and HBsAg loss irrespective of naïve and NA-experienced HBeAg-positive CHB patients treated with PegIFN-α-2a.
Subject(s)
Antiviral Agents , Hepatitis B, Chronic , Interferon-alpha , STAT4 Transcription Factor , Antiviral Agents/therapeutic use , Cohort Studies , DNA, Viral , Hepatitis B Surface Antigens , Hepatitis B e Antigens , Hepatitis B, Chronic/drug therapy , Humans , Interferon-alpha/therapeutic use , Polyethylene Glycols/therapeutic use , Polymorphism, Genetic , Prospective Studies , Recombinant Proteins/therapeutic use , Retrospective Studies , STAT4 Transcription Factor/genetics , Seroconversion , Treatment OutcomeABSTRACT
STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modulating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimulation. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in modulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12-induced STAT4 activation to generate IFN-γ, with autocrine IFN-γ then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-γ- and IL-12-induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1 /STAT4-immune disease-associated variants modulate IFN-γ- and IL-12-associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis.
Subject(s)
Genetic Predisposition to Disease/genetics , Genetic Variation/immunology , Macrophages/metabolism , Receptors, Pattern Recognition/genetics , STAT1 Transcription Factor/genetics , STAT4 Transcription Factor/genetics , Alleles , Cell Line , Cytokines/genetics , Gene Expression/genetics , Humans , Inflammatory Bowel Diseases/genetics , Interferon-gamma/genetics , Interleukin-12/genetics , Myeloid Cells/metabolism , Phosphorylation/genetics , Signal Transduction/geneticsABSTRACT
Inflammation is an essential factor contributing to sepsis-induced endothelial cell (EC) activation. Interleukin-35 (IL-35) is an anti-inflammatory/immunosuppressive cytokine that exerts protective effects on many inflammatory diseases. In this study, we investigated the effects of IL-35 on lipopolysaccharide (LPS)-induced EC activation and the potential underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were incubated with LPS (1 µg/ml) for 24 h and then cocultured with different concentrations (0, 1, 10, or 100 ng/ml) of recombinant human IL-35 (rhIL-35) for 12 h. Flow cytometry analysis revealed that IL-35 inhibited LPS-induced HUVEC apoptosis in a dose-dependent manner. RT-qPCR and Western blot analyses showed significantly higher mRNA and protein levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the inflammatory factors IL-6 and IL-8 in the LPS group than in the control group. These changes were alleviated by IL-35 treatment, suggesting that IL-35 protects ECs by downregulating inflammation. Furthermore, IL-35 induced signal transducer and activator of transcription 1 (STAT1) and STAT4 activation and promoted their interaction. Blocking STAT1 or STAT4 expression by fludarabine (STAT1 inhibitor) treatment or siRNA-STAT4-interfering fragment transfection inhibited the protective effect of IL-35 on ECs. Moreover, we observed a similar protective effect of IL-35 treatment on ECs in a mouse sepsis model induced by intraperitoneal LPS injection. This study indicated that IL-35 exerts anti-inflammatory and antiapoptotic effects on LPS-induced EC activation by activating the STAT1 and STAT4 signaling pathways.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Apoptosis , Endothelium, Vascular/metabolism , Inflammation/prevention & control , Interleukins/metabolism , Lipopolysaccharides/toxicity , Sepsis/prevention & control , Animals , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Gene Expression Regulation , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/pathology , Interleukins/administration & dosage , Interleukins/genetics , Male , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , NF-kappa B/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolism , Sepsis/chemically induced , Sepsis/metabolism , Sepsis/pathology , Signal TransductionABSTRACT
In genomic fine-mapping studies, some approaches leverage annotation data to prioritize likely functional polymorphisms. However, existing annotation resources can present challenges as many lack information for novel variants and/or may be uninformative for non-coding regions. We propose a novel annotation source, sequence-dependent DNA topology, as a prioritization metric for fine-mapping. DNA topology and function are well-intertwined, and as an intrinsic DNA property, it is readily applicable to any genomic region. Here, we constructed and applied Minor Groove Width (MGW) as a prioritization metric. Using an established MGW-prediction method, we generated a MGW census for 199 038 197 SNPs across the human genome. Summarizing a SNP's change in MGW (ΔMGW) as a Euclidean distance, ΔMGW exhibited a strongly right-skewed distribution, highlighting the infrequency of SNPs that generate dissimilar shape profiles. We hypothesized that phenotypically-associated SNPs can be prioritized by ΔMGW. We tested this hypothesis in 116 regions analyzed by a Massively Parallel Reporter Assay and observed enrichment of large ΔMGW for functional polymorphisms (P = 0.0007). To illustrate application in fine-mapping studies, we applied our MGW-prioritization approach to three non-coding regions associated with systemic lupus erythematosus. Together, this study presents the first usage of sequence-dependent DNA topology as a prioritization metric in genomic association studies.
Subject(s)
Chromosome Mapping/methods , DNA/chemistry , Genome, Human , Genome-Wide Association Study/methods , Genomics/methods , Base Sequence , Bayes Theorem , Black People/genetics , DNA-Binding Proteins/genetics , Databases, Genetic , Hispanic or Latino/genetics , Humans , Lupus Erythematosus, Systemic/genetics , Molecular Sequence Annotation/methods , Polymorphism, Single Nucleotide , Proteins/genetics , Quantitative Trait Loci , STAT4 Transcription Factor/genetics , White People/genetics , src-Family Kinases/geneticsABSTRACT
BACKGROUND: Signaling pathways in the STAT4 gene play an essential role in interferon-mediated antiviral effects. OBJECTIVE: This study was aimed at investigating the role of rs7574865, a single nucleotide polymorphism (SNP) in STAT4, in patients with chronic hepatitis B (CHB) treated with pegylated interferon (PEG-IFN). METHODS: A total 261 Thai patients (115 HBeAg-positive and 146 HBeAg-negative CHB) treated with 48-week PEG-IFN were recruited. Virological response (VR) at 48 weeks post treatment was defined as HBeAg seroconversion plus HBV DNA < 2,000 IU/mL for HBeAg-positive CHB and HBV DNA < 2,000 IU/mL for HBeAg-negative CHB. The SNP was analyzed by TaqMan PCR assay. RESULTS: The distribution of GG, GT and TT genotypes of rs7574865 was 41.8%, 42.9% and 15.3%, respectively. There was no different in its distribution according to HBeAg status. Overall, patients with TT genotype, compared with non-TT genotype, achieved higher VR (64.3% vs. 30.5%; P < 0.001) and HBsAg clearance (23.8% vs. 5.0%; P < 0.001). There was the same trend in the HBeAg-positive group (VR, 52.4% vs. 30.9%; P = 0.077; HBsAg clearance, 23.8% vs. 6.4%; P = 0.028) and in the HBeAg-negative group (VR, 68.4% vs. 32.3%; P = 0.004; HBsAg clearance, 21.1% vs. 4.7%; P = 0.026). Multiple regression analysis demonstrated that low baseline HBsAg level and TT genotype were factors independently associated with VR and HBsAg clearance. CONCLUSIONS: Our data support that SNP rs7574865 is associated with response to PEG-IFN therapy in Thai patients with CHB, regardless of baseline HBeAg status. Thus, the determination of this SNP could maximize cost-effectiveness of PEGIFN in patients with CHB.