RESUMO
Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to immunodeficiency, centromeric instability, facial anomalies syndrome, type 1 (ICF1). Although several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-derived induced pluripotent stem cells (iPSCs) and their CRISPR-Cas9-corrected clones to determine whether DNMT3B correction can globally overcome DNA methylation defects and related changes in the epigenome. Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1 patient peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are premarked during early development. CRISPR-corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions reacquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increases in H3K4me3 levels and/or abnormal CTCF binding, the epigenetic memory persists, and hypomethylation remains uncorrected. Overall, we demonstrate that restoring the catalytic activity of DNMT3B can reverse the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this rescue, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes Induzidas/metabolismo , Epigenoma , Memória Epigenética , Histonas/metabolismo , Metilação de DNA , DNA (Citosina-5-)-Metiltransferases/genéticaRESUMO
The most distal 2 kb region in the majority of human subtelomeres contains CpG-rich promoters for TERRA, a long non-coding RNA. When the function of the de novo DNA methyltransferase DNMT3B is disrupted, as in ICF1 syndrome, subtelomeres are abnormally hypomethylated, subtelomeric heterochromatin acquires open chromatin characteristics, TERRA is highly expressed, and telomeres shorten rapidly. In this study, we explored whether the regulation of subtelomeric epigenetic characteristics by DNMT3B is conserved between humans and mice. Studying the DNA sequence of the distal 30 kb of the majority of murine q-arm subtelomeres indicated that these regions are relatively CpG-poor and do not contain TERRA promoters similar to those present in humans. Despite the lack of human-like TERRA promoters, we clearly detected TERRA expression originating from at least seven q-arm subtelomeres, and at higher levels in mouse pluripotent stem cells in comparison with mouse embryonic fibroblasts (MEFs). However, these differences in TERRA expression could not be explained by differential methylation of CpG islands present in the TERRA-expressing murine subtelomeres. To determine whether Dnmt3b regulates the expression of TERRA in mice, we characterized subtelomeric methylation and associated telomeric functions in cells derived from ICF1 model mice. Littermate-derived WT and ICF1 MEFs demonstrated no significant differences in subtelomeric DNA methylation, chromatin modifications, TERRA expression levels, telomere sister chromatid exchange or telomere length. We conclude that the epigenetic characteristics of murine subtelomeres differ substantially from their human counterparts and that TERRA transcription in mice is regulated by factors others than Dnmt3b.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Face/anormalidades , Fibroblastos/patologia , Doenças da Imunodeficiência Primária/patologia , Telômero/fisiologia , Fatores de Transcrição/metabolismo , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Face/patologia , Fibroblastos/metabolismo , Humanos , Camundongos , Doenças da Imunodeficiência Primária/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Transcrição Gênica , DNA Metiltransferase 3BRESUMO
RBL2/p130, a member of the retinoblastoma family of proteins, is a key regulator of cell division and propagates irreversible senescence. RBL2/p130 is also involved in neuronal differentiation and survival, and eliminating Rbl2 in certain mouse strains leads to embryonic lethality accompanied by an abnormal central nervous system (CNS) phenotype. Conflicting reports exist regarding a role of RBL2/p130 in transcriptional regulation of DNA methyltransferases (DNMTs), as well as the control of telomere length. Here we describe the phenotype of three patients carrying bi-allelic RBL2-truncating variants. All presented with infantile hypotonia, severe developmental delay and microcephaly. Malignancies were not reported in carriers or patients. Previous studies carried out on mice and human cultured cells, associated RBL2 loss to DNA methylation and telomere length dysregulation. Here, we investigated whether patient cells lacking RBL2 display related abnormalities. The study of primary patient fibroblasts did not detect abnormalities in expression of DNMTs. Furthermore, methylation levels of whole genome DNA, and specifically of pericentromeric repeats and subtelomeric regions, were unperturbed. RBL2-null fibroblasts show no evidence for abnormal elongation by telomeric recombination. Finally, gradual telomere shortening, and normal onset of senescence were observed following continuous culturing of RBL2-mutated fibroblasts. Thus, this study resolves uncertainties regarding a potential non-redundant role for RBL2 in DNA methylation and telomere length regulation, and indicates that loss of function variants in RBL2 cause a severe autosomal recessive neurodevelopmental disorder in humans.
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Disfunção Cognitiva/genética , Metilação de DNA/genética , Proteína p130 Retinoblastoma-Like/genética , Encurtamento do Telômero/genética , Adolescente , Adulto , Alelos , Animais , Criança , Disfunção Cognitiva/complicações , Disfunção Cognitiva/fisiopatologia , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Fibroblastos/metabolismo , Predisposição Genética para Doença , Humanos , Masculino , Metiltransferases/genética , Camundongos , Microcefalia/complicações , Microcefalia/genética , Microcefalia/fisiopatologia , Atividade Motora/fisiologia , Hipotonia Muscular/complicações , Hipotonia Muscular/genética , Hipotonia Muscular/fisiopatologia , Telômero/genética , Sequenciamento do ExomaRESUMO
Human telomeres and adjacent subtelomeres are packaged as heterochromatin. Subtelomeric DNA undergoes methylation during development by DNA methyltransferase 3B (DNMT3B), including the CpG-rich promoters of the long non-coding RNA (TERRA) embedded in these regions. The factors that direct DNMT3B methylation to human subtelomeres and maintain this methylation throughout lifetime are yet unknown. The importance of subtelomeric methylation is manifested through the abnormal telomeric phenotype in Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome type 1 patients carrying mutations in DNMT3B. Patient cells demonstrate subtelomeric hypomethylation, accompanied by elevated TERRA transcription, accelerated telomere shortening and premature senescence of fibroblasts. ICF syndrome can arise due to mutations in at least three additional genes, ZBTB24 (ICF2), CDCA7 (ICF3) and HELLS (ICF4). While pericentromeric repeat hypomethylation is evident in all ICF syndrome subtypes, the status of subtelomeric DNA methylation had not been described for patients of subtypes 2-4. Here we explored the telomeric phenotype in cells derived from ICF2-4 patients with the aim to determine whether ZBTB24, CDCA7 and HELLS also play a role in establishing and/or maintaining human subtelomeric methylation. We found normal subtelomeric methylation in ICF2-4 and accordingly low TERRA levels and unperturbed telomere length. Moreover, depleting the ICF2-4-related proteins in normal fibroblasts did not influence subtelomeric methylation. Thus, these gene products are not involved in establishing or maintaining subtelomeric methylation. Our findings indicate that human subtelomeric heterochromatin has specialized methylation regulation and highlight the telomeric phenotype as a characteristic that distinguishes ICF1 from ICF2-4.
Assuntos
Anormalidades Múltiplas/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Helicases/genética , Metilação de DNA , Mutação , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Anormalidades Múltiplas/metabolismo , Adolescente , Adulto , Linhagem Celular , Centrômero , Criança , Pré-Escolar , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Helicases/metabolismo , Face/anormalidades , Fibroblastos , Heterocromatina/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Lactente , Recém-Nascido , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Telômero/metabolismo , Adulto Jovem , DNA Metiltransferase 3BRESUMO
OBJECTIVE: To utilise a community-based participatory approach in the design and implementation of an intervention targeting diet-related health problems on Navajo Nation. DESIGN: A dual strategy approach of community needs/assets assessment and engagement of cross-sectorial partners in programme design with systematic cyclical feedback for programme modifications. SETTING: Navajo Nation, USA. PARTICIPANTS: Navajo families with individuals meeting criteria for programme enrolment. Participant enrolment increased with iterative cycles. RESULTS: The Navajo Fruit and Vegetable Prescription (FVRx) Programme. CONCLUSIONS: A broad, community-driven and culturally relevant programme design has resulted in a programme able to maintain core programmatic principles, while also allowing for flexible adaptation to changing needs.
Assuntos
Dieta Saudável , Desertos Alimentares , Frutas , Verduras , Abastecimento de Alimentos , Promoção da Saúde , Humanos , Prescrições , Avaliação de Programas e Projetos de Saúde , Sudoeste dos Estados Unidos , Indígena Americano ou Nativo do AlascaRESUMO
Mutations in the de novo DNA methyltransferase DNMT3B lead to Immunodeficiency, Centromeric Instability and Facial anomalies (ICF) syndrome, type I. This syndrome is characterized, among other hypomethylated genomic loci, by severe subtelomeric hypomethylation that is associated with abnormally short telomere length. While it was demonstrated that the mean telomere length is significantly shorter in ICF type I cells, it is unknown whether all telomeres are equally vulnerable to shortening. To study this question we determined by combined telomere-FISH and spectral karyotyping the relative length of each individual telomere in lymphoblastoid cell lines (LCLs) generated from multiple ICF syndrome patients and control individuals. Here we confirm the short telomere lengths, and demonstrate that telomere length variance in the ICF patient group is much larger than in the control group, suggesting that not all telomeres shorten in a uniform manner. We identified a subgroup of telomeres whose relatively short lengths can distinguish with a high degree of certainty between a control and an ICF metaphase, proposing that in ICF syndrome cells, certain individual telomeres are consistently at greater risk to shorten than others. The majority of these telomeres display high sequence identity at the distal 2 kb of their subtelomeres, suggesting that the attenuation in DNMT3B methylation capacity affects individual telomeres to different degrees based, at least in part, on the adjacent subtelomeric sequence composition.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Telômero/genética , Anormalidades Múltiplas/genética , Linhagem Celular , Centrômero/genética , Centrômero/fisiologia , Aberrações Cromossômicas , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Face/anormalidades , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Masculino , Mutação , Linhagem , Doenças da Imunodeficiência Primária , Telômero/fisiologia , Encurtamento do Telômero/genética , DNA Metiltransferase 3BRESUMO
Health professionals, including social workers, community health workers, public health workers, and licensed health care providers, share common interests and responsibilities in promoting health equity and improving social determinants of health-the conditions in which people live, work, play, and learn. We summarize the underlying causes of health inequity and comparatively poor health outcomes in the United States. We describe barriers to realizing the hope embedded in the 2010 Patient Protection and Affordable Care Act, that moving away from fee-for-service payments will naturally drive care upstream as providers respond to greater financial risk by undertaking greater prevention efforts for the health of their patients. We assert that health equity should serve as the guiding framework for achieving the Triple Aim of health care reform and outline practical opportunities for improving care and promoting stronger efforts to address social determinants of health. These proposals include developing a dashboard of measures to assist providers committed to health equity and community-based prevention and to promote institutional accountability for addressing socioeconomic factors that influence health.
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Política Ambiental , Reforma dos Serviços de Saúde/organização & administração , Equidade em Saúde/organização & administração , Qualidade da Assistência à Saúde , Agentes Comunitários de Saúde , Feminino , Política de Saúde , Humanos , Masculino , Patient Protection and Affordable Care ActRESUMO
Human cells contain several hundred ribosomal genes (rDNA) that are clustered into nucleolar organizer regions (NORs) on the short arms of five different acrocentric chromosomes. Only approximately 50% of the gene copies are actually expressed in somatic cells. Here, we used a new cytological technique to demonstrate that rDNA is regulated allelically in a regional manner, with one parental copy of each NOR being repressed in any individual cell. This process is similar to that of X-chromosome inactivation in females. Early in development, one copy of each NOR becomes late-replicating, thus probably marking it for inactivation and subsequent targeted de novo methylation at rDNA promoter regions. Once established, this multichromosomal allelic pattern is then maintained clonally in somatic cells. This pathway may serve as an epigenetic mechanism for controlling the number of available rDNA copies during development.
Assuntos
Alelos , DNA Ribossômico/genética , Inativação Gênica , Animais , Linhagem Celular , Metilação de DNA , Período de Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Genes de RNAr/genética , Histonas/metabolismo , Humanos , Camundongos , Hibridização de Ácido NucleicoRESUMO
Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive subtelomeric DNA methylation and specific histone modifications. ICF (immunodeficiency, centromeric instability, facial anomalies) type I patients carry mutations in DNA methyltransferase 3B (DNMT3B) that methylates de novo repetitive sequences during early embryonic development. ICF type I patient fibroblasts display hypomethylated subtelomeres, abnormally short telomeres and premature senescence. In order to study the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells (iPSCs) from 3 ICF type I patients. Telomeres were elongated in ICF-iPSCs during reprogramming, and the senescence phenotype was abolished despite sustained subtelomeric hypomethylation and high TERRA levels. Fibroblast-like cells (FLs) isolated from differentiated ICF-iPSCs maintained abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts. These findings demonstrate that the abnormal telomere phenotype associated with subtelomeric hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The data in the current study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is linked to accelerated telomeric loss in this syndrome.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Face/anormalidades , Síndromes de Imunodeficiência/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Encurtamento do Telômero , Telômero/metabolismo , Células Cultivadas , Reprogramação Celular , Senescência Celular , Aberrações Cromossômicas , Cromossomos Humanos , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Face/fisiopatologia , Feminino , Fibroblastos/fisiologia , Humanos , Síndromes de Imunodeficiência/fisiopatologia , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Doenças da Imunodeficiência Primária , DNA Metiltransferase 3BRESUMO
Recent improvements in the accessibility of mapping tools and an increased recognition of the importance of leveraging data to inform public health operations has led to enthusiasm among public health departments to rapidly evolve their ability to analyze and apply data to programs. As the COVID-19 pandemic made evident, many health department data systems have been neglected for decades and data literacy among staff low. Significant federal dollars have been allocated to local health departments to modernize health systems. This case study recounts the effort to equip the Pima County Health Department with a highly sophisticated "COVID-19 Vaccines Solutions Dashboard" in 2021-2022, quantifying community vulnerability in the midst of the COVID-19 pandemic and shares key successes and challenges in process and outcomes that can guide other such dashboard initiatives. The experience informed the development of Pima' County Health Department's Data & Informatics Team as well as efforts to cultivate a more robust data culture throughout the department. Many health departments around the United States are in a similar position, and these lessons learned are widely applicable.
RESUMO
We describe two siblings from a consanguineous family with autosomal recessive Fanconi's syndrome and hypophosphatemic rickets. Genetic analysis revealed a homozygous in-frame duplication of 21 bp in SLC34A1, which encodes the renal sodium-inorganic phosphate cotransporter NaPi-IIa, as the causative mutation. Functional studies in Xenopus laevis oocytes and in opossum kidney cells indicated complete loss of function of the mutant NaPi-IIa, resulting from failure of the transporter to reach the plasma membrane. These findings show that disruption of the human NaPi-IIa profoundly impairs overall renal phosphate reabsorption and proximal-tubule function and provide evidence of the critical role of NaPi-IIa in human renal phosphate handling.
Assuntos
Raquitismo Hipofosfatêmico Familiar/genética , Síndrome de Fanconi/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Adulto , Animais , Calcitriol/sangue , Células Cultivadas , Consanguinidade , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Rim/citologia , Rim/metabolismo , Masculino , Mutação , Oócitos/metabolismo , Gambás , Linhagem , Irmãos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Xenopus laevisRESUMO
Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.
Assuntos
Síndromes de Imunodeficiência , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Mutação , Síndromes de Imunodeficiência/genética , Impressão GenômicaRESUMO
Mucosal melanoma remains a rare cancer with high mortality and a paucity of therapeutic options. This is due in significant part to its low incidence leading to limited patient access to expert care and downstream clinical/basic science data for research interrogation. Clinical challenges such as delayed and at times inaccurate diagnoses, and lack of consensus tumor staging have added to the suboptimal outcomes for these patients. Clinical trials, while promising, have been difficult to activate and accrue. While individual institutions and investigators have attempted to seek solutions to such problems, international, national, and local partnership may provide the keys to more efficient and innovative paths forward. Furthermore, a mucosal melanoma coalition would provide a potential network for patients and caregivers to seek expert opinion and advice. The Melanoma Research Foundation Mucosal Melanoma Meeting (December 16, 2022, New York, USA) highlighted the current clinical challenges faced by patients, providers, and scientists, identified current and future clinical trial investigations in this rare disease space, and aimed to increase national and international collaboration among the mucosal melanoma community in an effort to improve patient outcomes. The included proceedings highlight the clinical challenges of mucosal melanoma, global clinical trial experience, basic science advances in mucosal melanoma, and future directions, including the creation of shared rare tumor registries and enhanced collaborations.
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Melanoma , Humanos , New York , Melanoma/terapia , Melanoma/patologia , Mucosa/patologia , Terapia Combinada , Estadiamento de NeoplasiasRESUMO
Recent studies identified MYH9 as a major susceptibility gene for common forms of non-diabetic end-stage kidney disease (ESKD). A set of African ancestry DNA sequence variants comprising the E-1 haplotype, was significantly associated with ESKD. In order to determine whether African ancestry variants are also associated with disease susceptibility in admixed populations with differing genomic backgrounds, we genotyped a total of 1425 African and Hispanic American subjects comprising dialysis patients with diabetic and non-diabetic ESKD and controls, using 42 single nucleotide polymorphisms (SNPs) within the MYH9 gene and 40 genome-wide and 38 chromosome 22 ancestry informative markers. Following ancestry correction, logistic regression demonstrated that three of the E-1 SNPs are also associated with non-diabetic ESKD in the new sample sets of both African and Hispanic Americans, with a stronger association in Hispanic Americans. We also identified MYH9 SNPs that are even more powerfully associated with the disease phenotype than the E-1 SNPs. These newly associated SNPs, could be divided into those comprising a haplotype termed S-1 whose association was significant under a recessive or additive inheritance mode (rs5750248, OR 4.21, P < 0.01, Hispanic Americans, recessive), and those comprising a haplotype termed F-1 whose association was significant under a dominant or additive inheritance mode (rs11912763, OR 4.59, P < 0.01, Hispanic Americans, dominant). These findings strengthen the contention that a sequence variant of MYH9, common in populations with varying degrees of African ancestry admixture, and in strong linkage disequilibrium with the associated SNPs and haplotypes reported herein, strongly predisposes to non-diabetic ESKD.
Assuntos
Negro ou Afro-Americano/genética , Predisposição Genética para Doença/genética , Hispânico ou Latino/genética , Falência Renal Crônica/etnologia , Falência Renal Crônica/genética , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos/genética , Humanos , Padrões de Herança/genética , Desequilíbrio de Ligação , Modelos Logísticos , Cidade de Nova Iorque , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration.
Assuntos
Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina/fisiologia , Leucemia Experimental/metabolismo , Infecções por Retroviridae/metabolismo , Infecções Tumorais por Vírus/metabolismo , Montagem de Vírus , Animais , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/virologia , Células Cultivadas , Cromossomos de Mamíferos/genética , DNA Viral/genética , DNA Viral/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Imunofluorescência , Produtos do Gene gag/genética , Humanos , Imunoprecipitação , Hibridização in Situ Fluorescente , Rim/citologia , Rim/metabolismo , Rim/virologia , Leucemia Experimental/patologia , Leucemia Experimental/virologia , Camundongos , Mitose/fisiologia , Mutação/genética , Células NIH 3T3 , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/virologia , Filogenia , RNA Mensageiro/genética , Infecções por Retroviridae/patologia , Infecções por Retroviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia , Replicação ViralRESUMO
Although the relative risk of renal cell carcinoma associated with chronic kidney injury is particularly high among sub-Saharan African ancestry populations, it is unclear yet whether the APOL1 gene risk variants (RV) for kidney disease additionally elevate this risk. APOL1 G1 and G2 RV contribute to increased risk for kidney disease in black populations, although the disease mechanism has still not been fully deciphered. While high expression levels of all three APOL1 allelic variants, G0 (the wild type allele), G1, and G2 are injurious to normal human cells, renal carcinoma cells (RCC) naturally tolerate inherent high expression levels of APOL1. We utilized CRISPR/Cas9 gene editing to generate isogenic RCC clones expressing APOL1 G1 or G2 risk variants on a similar genetic background, thus enabling a reliable comparison between the phenotypes elicited in RCC by each of the APOL1 variants. Here, we demonstrate that knocking in the G1 or G2 APOL1 alleles, or complete elimination of APOL1 expression, has major effects on proliferation capacity, mitochondrial morphology, cell metabolism, autophagy levels, and the tumorigenic potential of RCC cells. The most striking effect of the APOL1 RV effect was demonstrated in vivo by the complete abolishment of tumor growth in immunodeficient mice. Our findings suggest that, in contrast to the WT APOL1 variant, APOL1 RV are toxic for RCC cells and may act to suppress cancer cell growth. We conclude that the inherent expression of non-risk APOL1 G0 is required for RCC tumorigenicity. RCC cancer cells can hardly tolerate increased APOL1 risk variants expression levels as opposed to APOL1 G0.
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Among the main metabolic pathways implicated in cancer cell proliferation are those of cholesterol and fatty acid synthesis, both of which are tightly regulated by sterol regulatory element-binding proteins (SREBPs). SREBPs are activated through specific cleavage by membrane-bound transcription factor protease 1 (MBTPS1), a serine protease that cleaves additional substrates (ATF6, BDNF, CREBs and somatostatin), some of which are also implicated in cell proliferation. The goal of this study was to determine whether MBTPS1 may serve as a master regulator in proliferation of colorectal cancer (CRC). Tumors from CRC patients showed variable levels of MBTPS1 mRNA, which were in positive correlation with the levels of SREBPs and ATF6, and in reverse correlation with BDNF levels. Chemical inhibition of MBTPS1 activity in two CRC-derived cell lines resulted in a marked decrease in the levels of SREBPs, but not of its other substrates and a marked decrease in cell proliferation, which suggested that MBTPS1 activity is critical for proliferation of these cells. In accordance, CRISPR/Cas9 targeted knockout (KO) of the MBTPS1 gene resulted in the survival of only a single clone that presented a phenotype of severely attenuated proliferation and marked downregulation of several energy metabolism pathways. We further showed that survival of the MBTPS1 KO clone was dependent upon significant upregulation of the type-1 interferon pathway, the inhibition of which halted proliferation entirely. Finally, rescue of the MBTPS1 KO cells, resulted in partial restoration of MBTPS1 levels, which was in accordance with partial recovery in proliferation and in SREBP levels. These finding suggest that MBTPS1 plays a critical role in regulating colon cancer proliferation primarily through SREBP-associated lipid metabolism, and as such may serve as a possible therapeutic target in CRC.
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Aim: This study explored uveal melanoma patient experiences and regret following molecular prognostic testing using a 15-gene expression profile (GEP) test. Materials & methods: A retrospective, cross-sectional survey study was conducted through an online questionnaire capturing patient-reported experiences with prognostic biopsy/molecular testing. Results: Of 177 respondents, 159 (90%) wanted prognostic information at diagnosis. Most 15-GEP-tested patients who shared their results (99%) reported gaining value from testing, as did patients tested with other methods. Patients who received prognostic testing experienced lower decision regret than those who opted out. Decision regret did not differ based on GEP class. Conclusion: Most uveal melanoma patients desire prognostic testing and gain value from the GEP, independent of a high- or low-risk result.
Uveal melanoma is a rare but aggressive eye cancer, resulting in distant metastasis in nearly 50% of patients. Molecular prognostic testing is often employed to determine who is at high or low risk of developing metastatic disease. A prognostic 15-gene expression profiling (GEP) test is commonly used throughout the USA and parts of Canada. The goal of this survey was to assess patient experiences with the 15-GEP and other prognostic methods. Of the 177 patients who participated in the survey, the majority reported that they wanted prognostic information at the time of diagnosis. Of patients who underwent 15-GEP testing, nearly all reported gaining value from their test result, regardless of their individual risk profile. This study supports prior findings using other prognostic methods that patients prefer information about their risk of metastasis and reinforces the importance of discussing prognostic testing options with newly diagnosed uveal melanoma patients.
RESUMO
BACKGROUND: Cellular senescence plays important roles in the aging process of complex organisms, in tumor suppression and in response to stress. Several markers can be used to identify senescent cells, of which the most widely used is the senescence-associated ß-galactosidase (SABG) activity. The main advantage of SABG activity over other markers is the simplicity of the detection assay and the capacity to identify in situ a senescent cell in a heterogeneous cell population. Several approaches have been introduced to render the SABG assay quantitative. However none of these approaches to date has proven particularly amenable to quantitative analysis of SABG activity in situ. Furthermore the role of cellular senescence (CS) in vivo remains unclear mainly due to the ambiguity of current cellular markers in identifying CS of individual cells in tissues. RESULTS: In the current study we applied a digital image analysis technique to the staining generated using the original SABG assay, and demonstrate that this analysis is highly reproducible and sensitive to subtle differences in staining intensities resulting from diverse cellular senescence pathways in culture. We have further validated our method on mouse kidney samples with and without diabetes mellitus, and show that a more accurate quantitative SABG activity with a wider range of values can be achieved at a pH lower than that used in the conventional SABG assay. CONCLUSIONS: We conclude that quantitative in situ SABG assay, is feasible and reproducible and that the pH at which the reaction is performed should be tailored and chosen, depending on the research question and experimental system of interest.
Assuntos
Senescência Celular , Ensaios Enzimáticos/métodos , beta-Galactosidase/metabolismo , Animais , Fibroblastos/enzimologia , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Rim/enzimologia , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Sinais Assistido por ComputadorRESUMO
Most human cancers circumvent senescence by activating a telomere length maintenance mechanism, most commonly involving telomerase activation. A minority of cancers utilize the recombination-based alternative lengthening of telomeres (ALT) pathway. The exact requirements for unleashing normally repressed recombination at telomeres are yet unclear. Epigenetic modifications at telomeric regions were suggested to be pivotal for activating ALT; however, conflicting data exist regarding their exact nature and necessity. To uncover common ALT-positive epigenetic characteristics, we performed a comprehensive analysis of subtelomeric DNA methylation, histone modifications, and TERRA expression in several ALT-positive and ALT-negative cell lines. We found that subtelomeric DNA methylation does not differentiate between the ALT-positive and ALT-negative groups, and most of the analyzed subtelomeres within each group do not share common DNA methylation patterns. Additionally, similar TERRA levels were measured in the ALT-positive and ALT-negative groups, and TERRA levels varied significantly among the members of the ALT-positive group. Subtelomeric H3K4 and H3K9 trimethylation also differed significantly between samples in the ALT-positive group. Our findings do not support a common route by which epigenetic modifications activate telomeric recombination in ALT-positive cells, and thus, different therapeutic approaches will be necessary to overcome ALT-dependent cellular immortalization.