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Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant associated with CD4+ T-cell activation and autoimmune disease. Prior studies showed that exposure to TCE in the drinking water of autoimmune-prone mice expanded effector/memory CD4+ T cells with an interferon-γ (IFN-γ)-secreting Th1-like phenotype. However, very little is known how TCE exposure skews CD4+ T cells towards this pro-inflammatory Th1 subset. As observed previously, TCE exposure was associated with hypermethylation of regions of the genome related to transcriptional repression in purified effector/memory CD4 T cells. We hypothesized that TCE modulates transcriptional and/or epigenetic programming of CD4+ T cells as they differentiate from a naive to effector phenotype. In the current study, purified naive CD4 T cells from both male and female autoimmune-prone MRL/MpJ mice were activated ex vivo and polarized towards a Th1 subset for 4 days in the presence or absence of the oxidative metabolite of TCE, trichloroacetaldehyde hydrate (TCAH) in vitro. An RNA-seq assessment and reduced representation bisulfite sequencing for DNA methylation were conducted on Th1 cells or activated, non-polarized cells. The results demonstrated TCAH's ability to regulate key genes involved in the immune response and autoimmunity, including Ifng, by altering the level of DNA methylation at the gene promoter. Intriguing sex differences were observed and for the most part, the effects were more robust in females compared to males. In conclusion, TCE via TCAH epigenetically regulates gene expression in CD4+ T cells. These results may have implications for mechanistic understanding or future therapeutics for autoimmunity.
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Metilação de DNA , Células Th1 , Tricloroetileno , Animais , Tricloroetileno/toxicidade , Metilação de DNA/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Feminino , Masculino , Camundongos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Camundongos Endogâmicos MRL lpr , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/genética , Epigênese Genética/efeitos dos fármacos , Autoimunidade/efeitos dos fármacosRESUMO
Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells.
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Quimiocina CXCL12 , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo , Receptor Notch3 , Transdução de Sinais , Microambiente Tumoral , Animais , Humanos , Camundongos , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.
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Rearranjo Gênico , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proto-Oncogenes , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.
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Melanoma , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Apoptose , Melanoma/patologia , Regiões Promotoras Genéticas , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Oncogenic overexpression of integrin-ß7 (ITGB7) in cases of high-risk multiple myeloma (MM) was reported to promote enhanced interactions between neoplastic plasma-B cells and stromal cells to develop cell-adhesion mediated drug resistance. METHODS: Expression profiles of adhesion related genes were analyzed in a cohort of MM patients containing major IgH translocations or hyperdiploidies (HY), diagnosed at the premalignant monoclonal gammopathy of undetermined significance (MGUS; n = 103), smoldering multiple myeloma; (SMM; n = 190) or MM (MM; n = 53) stage. Differential expression was integrated with loci-specific alterations in DNA-methylation and chromatin marks in MM patients. A CRISPR-based targeted induction of DNA-methylation at the ITGB7 super-enhancer (SE) in MM.1S cells was employed to intersect the impact of cis-regulatory elements on ITGB7 expression. RESULTS: ITGB7 was significantly (p < 0.05) upregulated in patients with t(14;16) and t(14;20) subgroups in all MGUS, SMM and MM stages, but sporadically upregulated in t(4;14) subgroup at the MM stage. We demonstrate a predetermined enhancer state on ITGB7 in primary-B cells that is maintained under bivalent chromatin, which undergoes a process of chromatin-state alterations and develops into an active enhancer in cases of the t(4;14) subgroup or SE in cases of the t(14;16) subgroup. We also demonstrate that while targeted induction of DNA-methylation at the ITGB7-SE further upregulated the gene, inhibition of ITGB7-SE-associated transcription factor bromodomain-4 downregulated expression of the gene. CONCLUSIONS: Our findings suggest an epigenetic regulation of oncogenic overexpression of ITGB7 in MM cells, which could be critical in MM progression and an attractive therapeutic target.
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Gamopatia Monoclonal de Significância Indeterminada , Mieloma Múltiplo , Humanos , Cromatina/genética , Análise Citogenética , Progressão da Doença , DNA/metabolismo , Metilação de DNA , Epigênese Genética , Cadeias beta de Integrinas , Integrinas/genética , Integrinas/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologiaRESUMO
Background: Orthopedic surgery is one of the least diverse medical specialties. Other medical specialties have employed diversity-related initiatives to increase the number of students underrepresented in medicine (URM). Furthermore, with the suspension of visiting student rotations during the COVID-19 pandemic, medical students used residency program Web sites as a main source of program-specific information. Aims/Purpose: The purpose of this study was to measure the extent to which orthopedic surgery residency program Web sites describe diversity and inclusion initiatives. Methods: The Electronic Residency Application Service (ERAS) was used to identify U.S. orthopedic surgery residency programs. The programs' Web sites were reviewed, and data on commitments to diversity and inclusion were collected. Descriptive statistics of these data were generated. Results: There were 192 residency programs identified and 3 were excluded from the analysis due to lack of Web sites. Of the remaining 189 residency program Web sites, only 55 (29.10%) contained information on diversity and inclusion. Information on a commitment to improving diversity and inclusion was the most prevalent data point found among program Web sites, although it was found on only 15% of program Web sites. Conclusion: Orthopedic surgery residency programs rarely address topics related to diversity and inclusion on their program Web sites. An emphasis on opportunities for URM students and initiatives related to diversity and inclusion on program Web sites may improve URM outreach and serve as one method for increasing URM matriculation into orthopedic surgery.
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High-risk Multiple Myeloma (MM) patients were found to maintain telomere length (TL), below the margin of short critical length, consistent with proactive overexpression of telomerase. Previously, DNA methylation has been shown as a determinant of telomere-related gene (TRG) expression and TL to assess risk in different types of cancer. We mapped genome-wide DNA methylation in a cohort of newly diagnosed MM (NDMM; n = 53) patients of major molecular subgroups, compared to age-matched healthy donors (n = 4). Differential methylation and expression at TRG-loci were analyzed in combination with overlapping chromatin marks and underlying DNA-sequences. We observed a strong correlation (R2 ≥ 0.5) between DNA methylation and expression amongst selective TRGs, such that demethylation at the promoters of DDX1 and TERF1 were associated to their oncogenic upregulation, while demethylation at the bodies of two key tumor suppressors ZNF208 and RAP1A led to downregulation of the genes. We demonstrated that TRG expression may be controlled by DNA methylation alone or in cooperation with chromatin modifications or CCCTC-binding factor at the regulatory regions. Additionally, we showed that hypomethylated DMRs of TRGs in NDMM are stabilized with G-quadruplex forming sequences, suggesting a crucial role of these epigenetically vulnerable loci in MM pathogenesis. We have identified a panel of five TRGs, which are epigenetically deregulated in NDMM patients and may serve as early detection biomarkers or therapeutic targets in the disease.
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Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.
Assuntos
Neoplasias Colorretais/genética , Epigênese Genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Regulação para Cima/genética , 5-Metilcitosina/metabolismo , Adenoma/genética , Sítios de Ligação/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The electronic structure of a series perovskites ABX3 (A = Cs; B = Ca, Sr, and Ba; X = F, Cl, Br, and I) in the presence and absence of antisite defect XB were systematically investigated based on density-functional-theory calculations. Both cubic and orthorhombic perovskites were considered. It was observed that for certain perovskite compositions and crystal structure, presence of antisite point defect leads to the formation of electronic defect state(s) within the band gap. We showed that both the type of electronic defect states and their individual energy level location within the bandgap can be predicted based on easily available intrinsic properties of the constituent elements, such as the bond-dissociation energy of the B-X and X-X bond, the X-X covalent bond length, and the atomic size of halide (X) as well as structural characteristic such as B-X-B bond angle. Overall, this work provides a science-based generic principle to design the electronic states within the band structure in Cs-based perovskites in presence of point defects such as antisite defect.
RESUMO
T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Experimentais/imunologia , Animais , Linhagem Celular Tumoral , Epigênese Genética/fisiologia , Camundongos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. METHODS: Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. RESULTS: We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. CONCLUSIONS: Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.
Assuntos
Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Mieloma Múltiplo/genética , Aneuploidia , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/ultraestrutura , Ciclina D1/biossíntese , Ciclina D1/genética , Ciclina D2/biossíntese , Ciclina D2/genética , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Código das Histonas , Histonas/metabolismo , Humanos , Mieloma Múltiplo/classificação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Plasmócitos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-maf/genética , Translocação GenéticaRESUMO
Primary plasma cell leukemia (pPCL) is a rare and aggressive form of multiple myeloma (MM) that is characterized by the presence of ≥20% circulating plasma cells. Overall survival remains poor despite advances of anti-MM therapy. The disease biology as well as molecular mechanisms that distinguish pPCL from non-pPCL MM remain poorly understood and, given the rarity of the disease, are challenging to study. In an attempt to identify key biological mechanisms that result in the aggressive pPCL phenotype, we performed whole-exome sequencing and gene expression analysis in 23 and 41 patients with newly diagnosed pPCL, respectively. The results reveal an enrichment of complex structural changes and high-risk mutational patterns in pPCL that explain, at least in part, the aggressive nature of the disease. In particular, pPCL patients with traditional low-risk features such as translocation t(11;14) or hyperdiploidy accumulated adverse risk genetic events that could account for the poor outcome in this group. Furthermore, gene expression profiling showed upregulation of adverse risk modifiers in pPCL compared to non-pPCL MM, while adhesion molecules and extracellular matrix proteins became increasingly downregulated. In conclusion, this is one of the largest studies to dissect pPCL on a genomic and molecular level.
Assuntos
Leucemia Plasmocitária/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genômica , Humanos , Leucemia Plasmocitária/patologia , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Transcriptoma , Translocação Genética , Sequenciamento do ExomaRESUMO
Phase-stability in a U-Zr-Te-Nd multi-component metallic fuel for advanced nuclear reactors is systematically investigated by taking into account binary, ternary and quaternary interactions between elements involved. Historically, the onset of fuel-cladding chemical interactions (FCCI) greatly limits the burnup potential of U-Zr fuels primarily due to interactions between lanthanide fission products and cladding constituents. Tellurium (Te) is evaluated as a potential additive for U-Zr fuels to bind with lanthanide fission products, e.g. neodymium (Nd), negating or mitigating the FCCI effect. Potential fresh fuel alloy compositions with the Te additive, U-Zr-Te, are characterized. Te is found to completely bind with Zr within the U-Zr matrix. Alloys simulating the formation of the lanthanide element Nd within U-Zr-Te are also evaluated, where the Te-Nd binary interaction dominates and NdTe is found to form as a high temperature stable compound. The experimental observations agree well with the trends obtained from density functional theory calculations. According to the calculated enthalpy of mixing, Zr-Te compound formation is favored in the U-Zr-Te alloy whereas NdTe compound formation is favored in the U-Zr-Te-Nd alloy. Further, the calculated charge density distribution and density of states provide sound understanding of the mutual chemical interactions between elements and phase-stability within the multi-component fuel.
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The power conversion efficiency of perovskite solar cells can be significantly improved if recombination losses and hysteresis effects, often caused by the presence of structural and chemical defects present at grain boundaries and interfaces, can be minimized during the processing of photoactive layers. As a crucial first step to address this issue, we performed density functional theory calculations to evaluate the electronic structure of the energetically favored (110) perovskite surface in the presence of the widely reported IPb antisite defects. Our calculations indicate that the nature of trap states formed is different for the perovskite surface with exposed methylammonium (MAI) and lead iodide (PbI2) terminating groups. While, in MAI terminated surfaces, IPb antisite defects lead to shallow states close to the valence band, both deep and shallow states are created in the bandgap region in the PbI2 terminated surface. Furthermore, we determined contribution from individual atoms to the trap states and inferred that the trap states originate from the clusters of iodine atoms that are formed near the defect site. The exact nature of the defect state is strongly correlated with the atomic structure of these clusters and can be potentially tuned by controlling the processing conditions of the perovskite film.
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Reduced representation bisulfite sequencing (RRBS) is one of the most comprehensive yet economic ways of mapping whole genome DNA-methylation. Here, we have substantially modified the RRBS protocol by combining end-repair and A-tailing steps, and by introducing a bead-based method for rapid and easy size selection of the library molecules. The method has been optimized for myeloma clinical samples, where the input DNA concentration can be as low as 100 ng. The method developed can be accomplished in 3 days, including the initial overnight MspI enzyme digestion. Although the protocol has been optimized in myeloma samples, it is broadly applicable to any clinical sample, which is restricted by very low input DNA concentrations.
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Mieloma Múltiplo/genética , Análise de Sequência de DNA , Ilhas de CpG , Código de Barras de DNA Taxonômico , Metilação de DNA , Biblioteca Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Controle de Qualidade , Análise de Sequência de DNA/métodosRESUMO
Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Epigênese Genética , Biologia Molecular/métodos , Células-Tronco Neurais , Optogenética/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Perfilação da Expressão Gênica , Luz , Camundongos , Regiões Promotoras GenéticasRESUMO
In this study we evaluated and correlated the cytotoxic effects of zinc oxide nanoparticles (ZnO-NPs) to the epigenetic modifications, using human embryonic kidney (HEK-293) cells as a model system. Imaging of singlet and total reactive oxygen species (ROS) in ZnO-NPs-treated live cells was performed followed by the evaluation of its effects on cytoskeletal, mitochondrial, and nuclear integrity, and on the expression of ROS responsive genes. Next, we determined the global and locus-specific changes in DNA-methylation at the 3 global genomic repeat sequences namely LINE-1, subtelomeric D4Z4 and pericentromeric NBL2, and at the promoter of selected ROS responsive genes (AOX1, HMOX1, NCF2, SOD3). Our studies revealed severe actin depolymerization, increased release of mitochondrial cytochrome C, and nuclear enlargement in ZnO-NPs-treated cells. At the epigenetic level, we observed global reduction in 5-methylcytosine and increase in 5-hydroxymethylcytosine content. Additionally, we observed significant increase in the expression of Ten-Eleven Translocation (TET)-methylcytosine dioxygenase genes but not in the expression of DNA-methyltransferases (DNMTs). Based on our findings, we suggest that ZnO-NPs induce abundant increase in ROS to promote multimodal structural and functional anomalies in cells. Most importantly, ZnO-NP-induced ROS may promote global hypomethylation in cells by triggering the expression of TET-enzymes, avoiding DNMT interferences. Global DNA demethylation is considered to be the hallmark of the majority of cancers and once acquired this could be propagated to future progenies. The present study, hence, can be used as a platform for the assessment of epigenomic toxicity of ZnO-NPs in humans in the light of its use in commercial products.
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Desmetilação do DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/agonistas , Óxido de Zinco/toxicidade , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Tamanho do Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Oxirredutases/genética , Oxirredutases/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sequências Repetitivas de Ácido Nucleico/efeitos dos fármacos , Óxido de Zinco/químicaRESUMO
Functionalized 3D nanographenes with controlled electronic properties have been synthesized through a multistep organic synthesis method and are further used as promising anode materials for lithium-ion batteries, exhibiting a much increased capacity (up to 950 mAh g-1 ), three times higher than that of the graphite anode (372 mAh g-1 ).
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Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Proteínas de Ligação ao Cálcio/metabolismo , Cromatina/química , Cromossomos/ultraestrutura , Criptocromos/metabolismo , DNA Metiltransferase 3A , Epigênese Genética , Células HeLa , Humanos , Optogenética , Domínios Proteicos , Telomerase/genética , Telômero/ultraestrutura , Homeostase do Telômero , Proteína 1 de Ligação a Repetições Teloméricas/metabolismoRESUMO
DNA hypermethylation at the promoter of tumour-suppressor genes is tightly correlated with their transcriptional repression and recognized as the hallmark of majority of cancers. Epigenetic silencing of tumour suppressor genes impairs their cellular functions and activates a cascade of events driving cell transformation and cancer progression. Here, we examine site-specific and spatiotemporal alteration in DNA methylation at a target region in BRCA1 gene promoter, a model tumour suppressor gene. We have developed a programmable CRISPR-Cas9 based demethylase tool containing the deactivated Cas9 (dCas9) fused to the catalytic domain (CD) of Ten-Eleven Translocation (TET) dioxygenase1 (TET1CD). The fusion protein selectively demethylates targeted regions within BRCA1 promoter as directed by the designed single-guide RNAs (sgRNA), leading to the transcriptional up-regulation of the gene. We also noticed the increment in 5-hydroxymethylation content (5-hmC) at the target DNA site undergoing the most profound demethylation. It confirms the catalytic activity of TET1 in TET1-dCas9 fusion proteins-mediated demethylation at these target sequences. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin or DNA modifying enzymes and for loci-specific targeting to uncover epigenetic regulatory pathways at gene promoters and other selected genomic regions.