RESUMO
Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.
RESUMO
Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Mutação , Proteínas de Peixe-Zebra , Peixe-Zebra , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Mutação/genética , Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt/genética , Desenvolvimento Embrionário/genética , Dosagem de Genes , Mesoderma/metabolismo , Mesoderma/embriologiaRESUMO
Zebrafish offer an excellent tool for studying the vertebrate hematopoietic system thanks to a highly conserved and rapidly developing hematopoietic program, genetic amenability, optical transparency, and experimental accessibility. Zebrafish studies have contributed to our understanding of hematopoiesis, a complex process regulated by signaling cues, inflammation being crucial among them. Hematopoietic stem cells (HSCs) are multipotent cells producing all the functional blood cells, including immune cells. HSCs respond to inflammation during infection and malignancy by proliferating and producing the blood cells in demand for a specific scenario. We first focus on how inflammation plays a crucial part in steady-state HSC development and describe the critical role of the inflammasome complex in regulating HSC expansion and balanced lineage production. Next, we review zebrafish studies of inflammatory innate immune mechanisms focusing on interferon signaling and the downstream JAK-STAT pathway. We also highlight insights gained from zebrafish models harbouring genetic perturbations in the role of inflammation in hematopoietic disorders such as bone marrow failure, myelodysplastic syndrome, and myeloid leukemia. Indeed, inflammation has been recently identified as a potential driver of clonal hematopoiesis and leukemogenesis, where cells acquire somatic mutations that provide a proliferative advantage in the presence of inflammation. Important insights in this area come from mutant zebrafish studies showing that hematopoietic differentiation can be compromised by epigenetic dysregulation and the aberrant induction of signaling pathways.
RESUMO
Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3, and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organization. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a, and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 h post-fertilization. Homozygous loss of either paralogue alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.
RESUMO
Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top 'hits' was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of ß-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.
Assuntos
Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Mutações Sintéticas Letais/genética , Via de Sinalização Wnt/fisiologia , Animais , Divisão Celular , Linhagem Celular , Humanos , Peixe-Zebra , CoesinasRESUMO
The precise molecular mechanisms by which urate-associated genetic variants affect urate levels are unknown. Here, we tested for functional linkage of the maximally associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression. We performed expression quantitative trait loci (eQTL) and likelihood analyses and gene expression assays. Zebrafish were used to evaluate tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude. Probabilistic Annotation Integrator analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. Small interfering RNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T allele of rs1967017 gains enhancer activity relative to the urate-decreasing C allele, with T allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A. Computational prediction of increased motif strength, together with our functional assays, suggests that the urate-increasing T allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. Our and other data predict that the urate-raising T allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.
Assuntos
Proteínas de Transporte/genética , Fator 4 Nuclear de Hepatócito/genética , Locos de Características Quantitativas/genética , Ácido Úrico/sangue , Animais , Sítios de Ligação , Regulação da Expressão Gênica/genética , Células HEK293 , Células Hep G2 , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs) that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding. Replication domains are established simultaneously with TADs during the cell cycle and the two correlate well in terms of characteristic features, such as lamin association and histone modifications. CCCTC-binding factor (CTCF) and cohesin cooperate across different cell types to regulate genes and genome organization. CTCF elements that demarcate TAD boundaries are commonly disrupted in cancer and promote oncogene activation. Chromatin looping facilitates interactions between distant promoters and enhancers, and the resulting enhanceosome complex promotes gene expression. Deciphering the chromatin tangle requires comprehensive integrative analyses of DNA- and protein-dependent factors that regulate genomic organization.
RESUMO
Over 70 different genetic variants with a significant association with rheumatoid arthritis (RA) have been discovered. Anti-citrullination protein antibodies (ACPA)-positive RA variants are more well-defined than their ACPA-negative counterparts. The human leukocyte antigen, HLA-DRB1 locus remains the prime suspect in anti-citrullination protein antibodies (ACPA)-positive RA. Different HLA-DRB1 alleles are linked to RA susceptibility across different ethnicities. With evolving techniques, like genome-wide association studies (GWAS) and single nucleotide polymorphism (SNP) arrays, more non-HLA susceptibility loci have been identified for both types of RA. However, the functional significance of only a handful of these variants is known. Their roles include increasing susceptibility to RA or in determining the speed at which the disease progresses. Additionally, a couple of variations are associated with protection from RA. Defining such clear-cut biological functions can aid in the clinical diagnosis and treatment of RA. Recent research has focused on the implication of microRNAs, with miR-146a widely studied. In addition to disease susceptibility, genetic variations that influence the efficacy and toxicity of anti-RA agents have also been identified. Polymorphisms in the MTHFR gene influence the effectiveness of methotrexate, the first line of therapy in RA. Larger studies are, however, needed to identify potential biomarkers for early disease identification and monitoring disease progression.
Assuntos
Artrite Reumatoide/diagnóstico , Artrite Reumatoide/etiologia , Pesquisa Translacional Biomédica , Artrite Reumatoide/terapia , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , MicroRNAs/genética , Farmacogenética , Medicina de PrecisãoRESUMO
BACKGROUND AND AIM: Mutations in the NKX2.5 gene, a cardiac transcription factor, have been implicated in various types of congenital heart defects (CHD) and it is known that optimal expression levels of this gene are crucial for proper cardiogenesis. However, most of the mutations have been identified in cases of syndromic CHD, and the functional significance of other mutations in this gene has not been studied. We describe in this study the mutational and expression analysis of the NKX2.5 gene in nonsyndromic CHD patients. METHODS: In this study, exon 1 of the NKX2.5 gene was sequenced from 50 probands with sporadic CHD and 50 healthy volunteers. NKX2.5 gene expression levels in blood and cardiac tissue samples were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in the probands. RESULTS: No new mutations were identified; however, a previously reported variant A63G (rs2277923) was found to be present at significantly higher levels in the CHD population than in the control group. Changes in expression between the blood and tissue samples were seen in 37 out of the 50 CHD patients. CONCLUSION: Multiple factors, in addition to NKX2.5 gene mutations, may cause CHDs. NKX2.5 gene mutations may be mosaic in nature, therefore warranting investigation in both blood and tissue samples.