ABSTRACT
Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression.
Subject(s)
Forkhead Transcription Factors , Interleukin-2 Receptor alpha Subunit , RNA, Long Noncoding , T-Lymphocytes, Regulatory , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Cell Differentiation/geneticsABSTRACT
AIMS/HYPOTHESIS: Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. METHODS: Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4+ T cell, CD8+ T cell and CD4-CD8- cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. RESULTS: We identified 79, 56 and 45 differentially methylated regions in CD4+ T cells, CD8+ T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4+ T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4+ T cells. CONCLUSIONS/INTERPRETATION: These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.
Subject(s)
DNA Methylation , Diabetes Mellitus, Type 1 , Autoantibodies/genetics , Autoimmunity/genetics , CD8-Positive T-Lymphocytes , Child , CpG Islands , DNA Methylation/genetics , Diabetes Mellitus, Type 1/genetics , Epigenesis, Genetic/genetics , Humans , Leukocytes, MononuclearABSTRACT
AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.
Subject(s)
Diabetes Mellitus, Type 1 , Autoantibodies , Child , Child, Preschool , DNA Methylation/genetics , Female , Fetal Blood/metabolism , Glutamate Decarboxylase , Humans , PregnancyABSTRACT
Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19+/CD20+-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Genetic Loci , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/chemistry , Core Binding Factor Alpha 2 Subunit/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Genome, Human , Humans , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Type 1 diabetes is a complex heterogenous autoimmune disease without therapeutic interventions available to prevent or reverse the disease. This study aimed to identify transcriptional changes associated with the disease progression in patients with recent-onset type 1 diabetes. METHODS: Whole-blood samples were collected as part of the INNODIA study at baseline and 12 months after diagnosis of type 1 diabetes. We used linear mixed-effects modelling on RNA-seq data to identify genes associated with age, sex, or disease progression. Cell-type proportions were estimated from the RNA-seq data using computational deconvolution. Associations to clinical variables were estimated using Pearson's or point-biserial correlation for continuous and dichotomous variables, respectively, using only complete pairs of observations. FINDINGS: We found that genes and pathways related to innate immunity were downregulated during the first year after diagnosis. Significant associations of the gene expression changes were found with ZnT8A autoantibody positivity. Rate of change in the expression of 16 genes between baseline and 12 months was found to predict the decline in C-peptide at 24 months. Interestingly and consistent with earlier reports, increased B cell levels and decreased neutrophil levels were associated with the rapid progression. INTERPRETATION: There is considerable individual variation in the rate of progression from appearance of type 1 diabetes-specific autoantibodies to clinical disease. Patient stratification and prediction of disease progression can help in developing more personalised therapeutic strategies for different disease endotypes. FUNDING: A full list of funding bodies can be found under Acknowledgments.
Subject(s)
Autoimmune Diseases , Diabetes Mellitus, Type 1 , Humans , Transcriptome , Disease Progression , AutoantibodiesABSTRACT
The Sin3A-associated proteins SAP30 and SAP30L share 70% sequence identity and are part of the multiprotein Sin3A corepressor complex. They participate in gene repression events by linking members of the complex and stabilizing interactions among the protein members as well as between proteins and DNA. While most organisms have both SAP30 and SAP30L, the zebrafish is exceptional because it only has SAP30L. Here we demonstrate that SAP30L is expressed ubiquitously in embryonic and adult zebrafish tissues. Knockdown of SAP30L using morpholino-mediated technology resulted in a morphant phenotype manifesting as cardiac insufficiency and defective hemoglobinization of red blood cells. A microarray analysis of gene expression in SAP30L morphant embryos revealed changes in the expression of genes involved in regulation of transcription, TGF-beta signaling, Wnt-family transcription factors, and nuclear genes encoding mitochondrial proteins. The expression of the heart-specific nkx2.5 gene was markedly down-regulated in SAP30L morphants, and the cardiac phenotype could be partially rescued by nkx2.5 mRNA. In addition, changes were detected in the expression of genes known to be important in hemoglobin synthesis and erythropoiesis. Our results demonstrate that SAP30L regulates several transcriptional pathways in zebrafish embryos and is involved in the development of cardiac and hematopoietic systems.
Subject(s)
Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Heart/embryology , Hematopoiesis , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Embryo, Nonmammalian/cytology , Erythrocytes/metabolism , Erythrocytes/pathology , Gene Knockdown Techniques , Heart/anatomy & histology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.
Subject(s)
DNA Methylation , Fetal Blood , Infant, Newborn , Pregnancy , Female , Humans , Fetal Blood/metabolism , Data Analysis , Sequence Analysis, DNAABSTRACT
Acute lymphoblastic leukemia is marked by aberrant transcriptional features that alter cell differentiation, self-renewal, and proliferative features. We sought to identify the transcription factors exhibiting altered and subtype-specific expression patterns in B-ALL and report here that SOX11, a developmental and neuronal transcription factor, is aberrantly expressed in the ETV6-RUNX1 and TCF3-PBX1 subtypes of acute B-cell leukemias. We show that a high expression of SOX11 leads to alterations of gene expression that are typically associated with cell adhesion, migration, and differentiation. A high expression is associated with DNA hypomethylation at the SOX11 locus and a favorable outcome. The results indicate that SOX11 expression marks a group of patients with good outcomes and thereby prompts further study of its use as a biomarker.
Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Regulation, Leukemic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , SOXC Transcription Factors/metabolism , Adolescent , Biopsy , Bone Marrow/pathology , Cell Adhesion/genetics , Cell Differentiation/genetics , Cell Line, Tumor , Cell Movement/genetics , Child , Child, Preschool , Core Binding Factor Alpha 2 Subunit/genetics , DNA Methylation , Disease-Free Survival , Female , Follow-Up Studies , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Infant , Male , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prognosis , SOXC Transcription Factors/geneticsABSTRACT
Main hurdles of lignin valorization are its diverse chemical composition, recalcitrance, and poor solubility due to high-molecular weight and branched structure. Controlled fragmentation of lignin could lead to its use in higher value products such as binders, coatings, fillers, etc. Oxidative enzymes (i.e., laccases and peroxidases) have long been proposed as a potentially promising tool in lignin depolymerization. However, their application was limited to ambient pH, where lignin is poorly soluble in water. A Finnish biotechnology company, MetGen Oy, that designs and supplies industrial enzymes, has developed and brought to market several lignin oxidizing enzymes, including an extremely alkaline lignin oxidase MetZyme® LIGNO™, a genetically engineered laccase of bacterial origin. This enzyme can function at pH values as high as 10-11 and at elevated temperatures, addressing lignin at its soluble state. In this article, main characteristics of this enzyme as well as its action on bulk lignin coming from an industrial process are demonstrated. Lignin modification by MetZyme® LIGNO™ was characterized by size exclusion chromatography, UV spectroscopy, and dynamic light scattering for monitoring particle size of solubilized lignin. Under highly alkaline conditions, laccase treatment not only decreased molecular weight of lignin but also increased its solubility in water and altered its dispersion properties. Importantly, organic solvent-free soluble lignin fragmentation allowed for robust industrially relevant membrane separation technologies to be applicable for product fractionation. These enzyme-based solutions open new opportunities for biorefinery lignin valorization thus paving the way for economically viable biorefinery business.
ABSTRACT
Cell signalling, which is often derailed in cancer, is a network of multiple interconnected pathways with numerous feedback mechanisms. Dynamics of cell signalling is intimately regulated by addition and removal of phosphate groups by kinases and phosphatases. We examined expression of members of the PTP4A family of phosphatases across acute leukemias. While expression of PTP4A1 and PTP4A2 remained relatively unchanged across diseases, PTP4A3 showed marked overexpression in ETV6-RUNX1 and BCR-ABL1 subtypes of precursor B cell acute lymphoblastic leukemia. We show that PTP4A3 is regulated by the ETV6-RUNX1 fusion, but noticed no marked impact on cell viability either after PTP4A3 silencing or treatment with a PTP4A3 inhibitor. Regulation of PTP4A3 expression is altered in specific subgroups of acute leukemias and this is likely brought about by expression of the aberrant fusion genes.
Subject(s)
Core Binding Factor Alpha 2 Subunit , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein Tyrosine Phosphatases/genetics , Cell Line , Cell Survival , Fusion Proteins, bcr-abl , HumansABSTRACT
The zebrafish has proven to be a valuable vertebrate model in which to elucidate the molecular mechanisms of various diseases. A high degree of genetic and morphological similarity in hematopoiesis between the zebrafish and human indicates that zebrafish can provide valuable knowledge about the mechanisms behind pathogenesis of leukemia. To date, a small number of zebrafish leukemia models have been published and they have already provided some interesting information. However, the full potential of these models, especially the identification of contributing genetic factors and high-throughput drug screens, is yet to be fulfilled. Further transgenic or mutant animals are needed, especially for modeling high-risk leukemias, such as MLL rearranged infant leukemias.
Subject(s)
Disease Models, Animal , Leukemia/etiology , Zebrafish/physiology , Animals , Animals, Genetically Modified , Cell Transplantation/methods , Drug Screening Assays, Antitumor/methods , Humans , Leukemia/genetics , Leukemia/pathology , Research/trends , Xenograft Model Antitumor Assays/methods , Zebrafish/geneticsABSTRACT
Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of â¼25 µM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.