ABSTRACT
Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.
Subject(s)
Celiac Disease , Diet, Gluten-Free , GTP-Binding Proteins , Gene Expression Profiling , Glutens , Intestinal Mucosa , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Celiac Disease/immunology , Humans , Glutens/immunology , Transglutaminases/metabolism , Transglutaminases/antagonists & inhibitors , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/genetics , Intestinal Mucosa/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/drug effects , Female , Male , Adult , Transcriptome , Duodenum/pathology , Duodenum/immunology , Duodenum/metabolism , Interferon-gamma/metabolism , Middle Aged , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , Young Adult , Adaptive Immunity/drug effectsABSTRACT
The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.
Subject(s)
Celiac Disease , Glutens , Humans , Protein Glutamine gamma Glutamyltransferase 2 , Enterocytes/metabolism , Gliadin , Transglutaminases/metabolism , PeptidesABSTRACT
Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18-K20, 75-95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.
Subject(s)
Carcinogenesis/metabolism , Carcinogenesis/pathology , Colon/pathology , Epithelial Cells/metabolism , Gene Deletion , Gene Targeting , Intestines/pathology , Keratin-8/genetics , Aging/pathology , Animals , Cell Differentiation , Cell Proliferation , Diarrhea/complications , Diarrhea/pathology , Down-Regulation , Fluorodeoxyglucose F18/metabolism , Goblet Cells/metabolism , Inflammation/pathology , Integrases/metabolism , Keratin-8/deficiency , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/metabolism , Permeability , Phenotype , Positron-Emission TomographyABSTRACT
Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens, and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, their development and differentiation remain yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development, and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; the absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell-associated markers such as Spi-B and Sox8. In vitro organoid analysis of RankL treated organoid showed an increase of mature marker GP2 expression and other M cell-associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. An increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mice with an increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that PRC2 regulated Atoh8 is one of the factors that regulate the population density of intestinal M cell in the Peyer's patch.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Animals , B-Lymphocytes/metabolism , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/immunology , Epithelial Cells/drug effects , Epithelial Cells/immunology , Immunity, Mucosal/genetics , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Mice , Mice, Knockout , Peyer's Patches/drug effects , Peyer's Patches/metabolism , Primary Cell Culture , RANK Ligand/pharmacology , Receptor Activator of Nuclear Factor-kappa B/pharmacology , T-Lymphocytes/metabolism , Transcytosis/geneticsABSTRACT
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
Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3' UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, the release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome.
Subject(s)
Chromatin/metabolism , Polycomb Repressive Complex 2/physiology , RNA, Messenger/metabolism , 3' Untranslated Regions , Animals , Cells, Cultured , Exons , Gene Expression Regulation , Introns , Mice , Mouse Embryonic Stem Cells/physiology , Nucleosomes/metabolism , Polycomb Repressive Complex 2/metabolism , Protein Binding , RNA StabilityABSTRACT
BACKGROUND: There is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease. The gold standard for successful treatment is a healed small intestinal mucosa, and therefore, the outcome measures in proof-of-concept studies should be based on evaluation of small intestine biopsies. We here evaluated morphometric, immunohistochemical and messenger RNA (mRNA) expression changes in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded biopsies. METHODS: Fifteen coeliac disease patients were challenged with 4 g of gluten per day for 10 weeks and 24 non-coeliac patients served as disease controls. A wide array of histological and immunohistochemical staining and mRNA-based gene expression tests (RT-qPCR and RNAseq) were carried out. RESULTS: Digital quantitative villous height: crypt depth ratio (VH: CrD) measurements revealed significant duodenal mucosal deterioration in all coeliac disease patients on gluten challenge. In contrast, the Marsh-Oberhuber class worsened in only 80% of coeliac patients. Measuring the intraepithelial CD3+ T-lymphocyte and lamina propria CD138+ plasma cell densities simultaneously proved to be a meaningful new measure of inflammation. Stainings for γδ T cells and IgA deposits, where previously frozen samples have been needed, were successful in PAXgene fixed paraffin-embedded samples. Messenger RNA extraction from the same paraffin-embedded biopsy block was successful and allowed large-scale qRT-PCR and RNAseq analyses for gene expression. Molecular morphometry, using the mRNA expression ratio of villous epithelium-specific gene APOA4 to crypt proliferation gene Ki67, showed a similar significant distinction between paired baseline and post-gluten challenge biopsies as quantitative histomorphometry. CONCLUSION: Rigorous digitally measured histologic and molecular markers suitable for gluten challenge studies can be obtained from a single paraffin-embedded biopsy specimen. Molecular morphometry seems to be a promising new tool that can be used in situations where assessing duodenal mucosal health is of paramount importance. In addition, the diagnostically valuable IgA deposits were now stained in paraffin-embedded specimens making them more accessible in routine clinics.
Subject(s)
Biopsy/methods , Celiac Disease/genetics , Celiac Disease/pathology , Duodenum/pathology , Gene Expression , Intestinal Mucosa/pathology , RNA, Messenger/genetics , Adult , Celiac Disease/diet therapy , Celiac Disease/immunology , Duodenum/immunology , Fixatives , Fluorescent Antibody Technique , Formaldehyde , Glutens/immunology , Humans , Intestinal Mucosa/immunology , Paraffin Embedding , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , T-Lymphocytes/pathologyABSTRACT
Canonical Wnt/ß-catenin signaling regulates the homeostasis of intestinal epithelium by controlling the balance between intestinal stem cell self-renewal and differentiation but epigenetic mechanisms enacting the process are not known. We hypothesized that epigenetic regulator, Polycomb Repressive Complex-2 (PRC2), is involved in Wnt-mediated epithelial homeostasis on the crypt-villus axis and aberrancies therein are implicated both in celiac disease and in intestinal malignancies. We found that PRC2 establishes repressive crypt and villus specific trimethylation of histone H3 lysine 27 (H3K27me3) signature on genes responsible for, for example, nutrient transport and cell killing in crypts and, for example, proliferation and differentiation in mature villi, suggesting that PRC2 facilitates the Wnt-governed intestinal homeostasis. When celiac patients are on gluten-containing diet PRC2 is out-of-bounds active and consequently its target genes were found affected in intestinal epithelium. Significant set of effective intestinal PRC2 targets are also differentially expressed in colorectal adenoma and carcinomas. Our results suggest that PRC2 gives rise and maintains polar crypt and villus specific H3K27me3 signatures. As H3K27me3 is a mark enriched in developmentally important genes, identified intestinal PRC2 targets are possibly imperative drivers for enterocyte differentiation and intestinal stem cell maintenance downstream to Wnt-signaling. Our work also elucidates the mechanism sustaining the crypt hyperplasia in celiac disease and suggest that PRC2-dependent fostering of epithelial stemness is a common attribute in intestinal diseases in which epithelial hyperplasia or neoplasia prevails. Finally, this work demonstrates that in intestine PRC2 represses genes having both pro-stemness and pro-differentiation functions, fact need to be considered when designing epigenetic therapies including PRC2 as a drug target. Stem Cells 2017;35:445-457.
Subject(s)
Homeostasis , Intestinal Mucosa/pathology , Intestinal Neoplasms/pathology , Polycomb Repressive Complex 2/metabolism , Stem Cells/pathology , Wnt Signaling Pathway , Animals , Celiac Disease/pathology , Cell Differentiation , Cell Proliferation , Enterocytes/pathology , Gene Expression Regulation, Neoplastic , Glutens , Histones/metabolism , Humans , Hyperplasia , Intestinal Neoplasms/genetics , Lysine/metabolism , Methylation , Mice, Inbred C57BL , Models, Biological , Stem Cell Niche , Stem Cells/metabolismABSTRACT
Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyzes trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3, but the significance of this remains unclear. Here, we identify a class of short RNAs, approximately 50-200 nucleotides in length, transcribed from the 5' end of polycomb target genes in primary T cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription, and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis, and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.
Subject(s)
RNA/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Animals , Base Sequence , Cells, Cultured , Chromatin/genetics , Chromatin/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Histones/genetics , Histones/metabolism , Lysine/metabolism , Mice , Molecular Sequence Data , Neurons/cytology , Neurons/physiology , Nucleic Acid Conformation , Polycomb-Group Proteins , Promoter Regions, Genetic , RNA/chemistry , RNA/genetics , Repressor Proteins/genetics , T-Lymphocytes/cytology , T-Lymphocytes/physiologyABSTRACT
UNLABELLED: The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging andin situproximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCE: Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy.
Subject(s)
Chromatin/virology , Genome, Viral , Histones/metabolism , Parvoviridae Infections/virology , Parvovirus, Canine/genetics , Promoter Regions, Genetic , Acetylation , Animals , Cats , Cell Line , DNA, Viral/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Viral , Lysine/metabolism , Microscopy, Confocal , Parvovirus, Canine/metabolism , Virus IntegrationABSTRACT
Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Autoantibodies/biosynthesis , Celiac Disease/immunology , GTP-Binding Proteins/immunology , Immunocompromised Host , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Transglutaminases/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , CHO Cells , Celiac Disease/genetics , Celiac Disease/pathology , Cricetulus , Female , GTP-Binding Proteins/genetics , Gene Expression , Glutens/chemistry , Glutens/immunology , Humans , Immunoglobulin A/biosynthesis , Immunohistochemistry , Inflammation , Injections, Intraperitoneal , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intestine, Small/immunology , Intestine, Small/pathology , Mice , Mice, Nude , Protein Glutamine gamma Glutamyltransferase 2 , Recombinant Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Transglutaminases/geneticsABSTRACT
Dermatitis herpetiformis (DH) is a blistering skin disease, which is regarded as an extra-intestinal manifestation of coeliac disease. Refractory cases of coeliac disease, that do not respond to a gluten-free diet and which carry an increased risk of lymphoma, are well-known in coeliac disease. To determine whether refractory cases of DH with active rash and persistent small bowel atrophy occur we analysed our series of 403 patients with DH. Seven (1.7%) patients, who had been on a gluten-free diet for a mean of 16 years, but who still required dapsone to treat the symptoms of DH, were identified. Of these, one patient died from mucinous adenocarcinoma before re-examination. At re-examination skin immunoglobulin A (IgA) deposits were found in 5/6 refractory and 3/16 control DH patients with good dietary response. Small bowel mucosa was studied at re-examination from 5 refractory and 8 control DH patients and was normal in all 5 refractory and 7/8 control DH patients. One refractory DH patient died from adenocarcinoma, but no lymphoma developed in any of the patients. This study documents for the first time refractory DH, in which the rash is non-responsive to a gluten-free diet, but the small bowel mucosa heals. This differs from refractory coeliac disease, in which the small bowel mucosa does not heal on a gluten-free diet.
Subject(s)
Celiac Disease/diet therapy , Dapsone/therapeutic use , Dermatitis Herpetiformis/therapy , Diet, Gluten-Free , Skin/drug effects , Adolescent , Adult , Atrophy , Biopsy , Celiac Disease/diagnosis , Celiac Disease/immunology , Child , Dermatitis Herpetiformis/diagnosis , Dermatitis Herpetiformis/diet therapy , Dermatitis Herpetiformis/immunology , Female , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intestine, Small/immunology , Intestine, Small/pathology , Male , Middle Aged , Retrospective Studies , Skin/immunology , Skin/pathology , Time Factors , Treatment Outcome , Wound Healing , Young AdultABSTRACT
Celiac disease pathogenesis, in addition to immune cell component, encompasses pathogenic events also in the duodenal epithelium. In celiac disease patients, exposure to dietary gluten induces drastic changes in epithelial differentiation and elicit cellular response to inflammatory cytokines. The autoantigen in celiac disease, transglutaminase 2 (TG2) enzyme, has been also suggested to play its pathogenic gliadin deamidation event in the intestinal epithelium. Therefore in vitro epithelial cell-line models have been exploited in the past to study these pathogenic mechanisms, but they are hampered by their simplistic nature lacking proper cell-type composition and intestinal environ. Moreover, these cell models harbor many cancer-related mutations in tumor suppressor genes making them unsuitable for studying cell differentiation. Intestinal organoids provide a near-native epithelial cell model to study pathogenic agents and mechanisms related to celiac disease. Here we describe protocols to initiate and maintain celiac patient-derived organoid cultures and how to grow them in alternative ways allowing their exploitation in different kind of experiments.
Subject(s)
Celiac Disease , Humans , Celiac Disease/genetics , Intestines , Organoids , Cell Differentiation , Cell LineABSTRACT
Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain gene repression and is essential for cell differentiation. In low-grade endometrial stromal sarcoma (LG-ESS), the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. We show that JAZF1-SUZ12 dysregulates PRC2 composition, genome occupancy, histone modification, gene expression, and cell differentiation. Loss of the SUZ12 N terminus in the fusion protein abrogates interaction with specific PRC2 accessory factors, reduces occupancy at PRC2 target genes, and diminishes H3K27me3. Fusion to JAZF1 increases H4Kac at PRC2 target genes and triggers recruitment to JAZF1 binding sites during cell differentiation. In human endometrial stromal cells, JAZF1-SUZ12 upregulated PRC2 target genes normally activated during decidualization while repressing genes associated with immune clearance, and JAZF1-SUZ12-induced genes were also overexpressed in LG-ESS. These results reveal defects in chromatin regulation, gene expression, and cell differentiation caused by JAZF1-SUZ12 that may underlie its role in oncogenesis.
Subject(s)
Co-Repressor Proteins , DNA-Binding Proteins , Histones , Neoplasm Proteins , Polycomb Repressive Complex 2 , Transcription Factors , Cell Differentiation/genetics , Co-Repressor Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Expression , Histones/metabolism , Humans , Neoplasm Proteins/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Transcription Factors/metabolismABSTRACT
The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.
ABSTRACT
Histological evaluation of the small intestinal mucosa is the cornerstone of celiac disease diagnostics and an important outcome in scientific studies. Gluten-dependent injury can be evaluated either with quantitative morphometry or grouped classifications. A drawback of mucosal readings is the subjective assessment of the border where the crypt epithelium changes to the differentiated villus epithelium. We studied potential immunohistochemical markers for the detection of the villus-crypt border: apolipoprotein A4 (APOA4), Ki-67, glucose transporter 2, keratin 20, cytochrome P450 3A4 and intestinal fatty-acid binding protein. Among these, villus-specific APOA4 was chosen as the best candidate for further studies. Hematoxylin-eosin (H&E)- and APOA4 stained duodenal biopsy specimens from 74 adult patients were evaluated by five observers to determine the villus-to-crypt ratio (VH : CrD). APOA4 delineated the villus to crypt epithelium transition clearly, and the correlation coefficient of VH : CrD values between APOA4 and H&E was excellent (r=0.962). The VH : CrD values were lower in APOA4 staining (p<0.001) and a conversion factor of 0.2 in VH : CrD measurements was observed to make the two methods comparable to each other. In the intraobserver analysis, the doubled standard deviations, representing the error ranges, were 0.528 for H&E and 0.388 for APOA4 staining, and the ICCs were 0.980 and 0.971, respectively. In the interobserver analysis, the average error ranges were 1.017 for H&E and 0.847 for APOA4 staining, and the ICCs were better for APOA4 than for H&E staining in all analyses. In conclusion, the reliability and reproducibility of morphometrical VH : CrD readings are improved with the use of APOA4 staining.
Subject(s)
Apolipoproteins A/genetics , Celiac Disease/etiology , Celiac Disease/pathology , Duodenum/metabolism , Duodenum/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Apolipoproteins A/metabolism , Biomarkers , Biopsy , Celiac Disease/metabolism , Disease Susceptibility , Female , Humans , Immunohistochemistry , Male , Middle Aged , Young AdultABSTRACT
BACKGROUND & AIMS: Gluten challenge studies are instrumental in understanding the pathophysiology of celiac disease. Our aims in this study were to reveal early gluten-induced transcriptomic changes in duodenal biopsies and to find tools for clinics. METHODS: Duodenal biopsies were collected from 15 celiac disease patients on a strict long-term gluten-free diet (GFD) prior to and post a gluten challenge (PGC) and from 6 healthy control individuals (DC). Biopsy RNA was subjected to genome-wide 3' RNA-Seq. Sequencing data was used to determine the differences between the three groups and was compared to sequencing data from the public repositories. The biopsies underwent morphometric analyses. RESULTS: In DC vs. GFD group comparisons, 167 differentially expressed genes were identified with 117 genes downregulated and 50 genes upregulated. In PGC vs. GFD group comparisons, 417 differentially expressed genes were identified with 195 genes downregulated and 222 genes upregulated. Celiac disease patients on a GFD were not "healthy". In particular, genes encoding proteins for transporting small molecules were expressed less. In addition to the activation of immune response genes, a gluten challenge induced hyperactive intestinal wnt-signaling and consequent immature crypt gene expression resulting in less differentiated epithelium. Biopsy gene expression in response to a gluten challenge correlated with the extent of the histological damage. Regression models using only four gene transcripts described 97.2% of the mucosal morphology and 98.0% of the inflammatory changes observed. CONCLUSIONS: Our gluten challenge trial design provided an opportunity to study the transition from health to disease. The results show that even on a strict GFD, despite being deemed healthy, patients reveal patterns of ongoing disease. Here, a transcriptomic regression model estimating the extent of gluten-induced duodenal mucosal injury is presented.
Subject(s)
Celiac Disease/immunology , Duodenum/pathology , Glutens/immunology , Intestinal Mucosa/pathology , Transcriptome/immunology , Adult , Biopsy , Celiac Disease/diagnosis , Celiac Disease/diet therapy , Celiac Disease/genetics , Datasets as Topic , Diet, Gluten-Free , Duodenum/immunology , Female , Glutens/administration & dosage , Humans , Intestinal Mucosa/immunology , Male , Middle Aged , RNA-Seq , Young AdultABSTRACT
BACKGROUND & AIMS: Microfold cells (M cells) are immunosurveillance epithelial cells located in the Peyer's patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related-receptor γ (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8. METHODS: Comparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor κ B ligand-induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8. RESULTS: chromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell-specific transcription factor acting on a receptor activator of nuclear factor κB ligand-receptor activator of nuclear factor κB-induced nuclear factor-κB pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression. CONCLUSIONS: PRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).
Subject(s)
Epithelial Cells/metabolism , Gene Expression Regulation , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Peyer's Patches/cytology , Peyer's Patches/metabolism , Polycomb Repressive Complex 2/metabolism , Animals , Biomarkers , Cell Differentiation/genetics , Gene Expression Profiling , Intestinal Mucosa/immunology , Mice , NF-kappa B/metabolism , Peyer's Patches/immunology , RANK Ligand/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Signal TransductionABSTRACT
Neural crest (NC) cells may be involved in kidney organogenesis by providing inductive signals and contributing to cells of the renal stroma. We show here that the lumbo-sacral NC cells fate mapped with the aid of Wnt-1 promoter in the mouse migrate close to the metanephros at the initiation of organogenesis but these cells remain superficial to the condensed Pax2-expressing mesenchymal cells. NC-derived cells enter later into the kidney proper from the midline region. The NC cells contribute also to development of the extra-adrenal para-aortic bodies, Zuckerkandl's bodies and the nerve cord of the sympathetic nervous system. Splotch (Sp(2H)/Sp(2H)) embryos, having a NC defect in the lumbo-sacral region, develop a normal metanephros even though the kidney does not express the NC markers Sox10, Phox2b and tyrosine hydroxylase. Consistent with the histological findings, the kidneys of Sp(2H)/Sp(2H) embryos also express the stromal genes Foxd1, Hoxa10 and RARbeta normally. Wnt-1 promoter-marked wild-type LacZ NC cells migrate intensely from the heterologous inducer tissue of the embryonic dorsal spinal cord (SPC) to the kidney mesenchyme, but tubule induction does not depend on NC migration, since the Sp(2H)/Sp(2H) SPC also induces tubulogenesis. The Sp(2H)/Sp(2H) mesenchyme also remains competent for tubulogenesis. We conclude that the NC cells fate mapped with the aid of Wnt-1 promoter migrate to the close to the metanephros and form later derivatives integrating with the kidney, but they may not be essential to the development of the stromal cells nor they may provide critical morphogenetic signals to regulate early kidney development in vivo.
Subject(s)
Kidney/growth & development , Lumbosacral Region , Neural Crest/cytology , Promoter Regions, Genetic , Wnt1 Protein/metabolism , Animals , Cell Line , Kidney/cytology , Mice , Wnt1 Protein/geneticsABSTRACT
BACKGROUND: Deacetylation of histones plays a fundamental role in gene silencing, and this is mediated by a corepressor complex containing Sin3 as an essential scaffold protein. In this report we examine the evolution of two proteins in this complex, the Sin3-associated proteins SAP30L and SAP30, by using an archive of protein sequences from 62 species. RESULTS: Our analysis indicates that in tetrapods SAP30L is more similar than SAP30 to the ancestral protein, and the two copies in this group originated by gene duplication which occurred after the divergence of Actinopterygii and Sarcopterygii about 450 million years ago (Mya). The phylogenetic analysis and biochemical experiments suggest that SAP30 has diverged functionally from the ancestral SAP30L by accumulating mutations that have caused attenuation of one of the original functions, association with the nuclear matrix. This function is mediated by a nuclear matrix association sequence, which consists of a conserved motif in the C-terminus and the adjacent nucleolar localization signal (NoLS). CONCLUSION: These results add further insight into the evolution and function of proteins of the SAP30 family, which share many characteristic with nuclear scaffolding proteins that are intimately involved in regulation of gene expression. Furthermore, SAP30L seems essential to eukaryotic biology, as it is found in animals, plants, fungi, as well as some taxa of unicellular eukaryotes.