RESUMO
The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.
Assuntos
Instabilidade Genômica , Regiões Promotoras Genéticas , Estruturas R-Loop , Terminação da Transcrição Genética , Humanos , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica/genética , Mutação , Estruturas R-Loop/genética , RNA Polimerase II/metabolismo , Regiões Promotoras Genéticas/genética , Genoma Humano , Proteínas de Ligação a DNA/metabolismoRESUMO
G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type-specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.
Assuntos
Quadruplex G , Iniciação da Transcrição Genética , Cromatina , DNA/química , Ligantes , Regiões Promotoras GenéticasRESUMO
The NAD-dependent deacetylase Sirtuin 1 (SIRT1) plays a vital role in leukemogenesis. Nicotinamide (NAM) is the principal NAD+ precursor and a noncompetitive inhibitor of SIRT1. In our study, we showed that NAM enhanced the sensitivity of chronic myeloid leukemia (CML) to doxorubicin (DOX) via SIRT1. We found that SIRT1 high expression in CML patients was associated with disease progression and drug resistance. Exogenous NAM efficiently repressed the deacetylation activity of SIRT1 and induced the apoptosis of DOX-resistant K562 cells (K562R) in a dose-dependent manner. Notably, the combination of NAM and DOX significantly inhibited tumor cell proliferation and induced cell apoptosis. The knockdown of SIRT1 in K562R cells enhanced NAM+DOX-induced apoptosis. SIRT1 rescue in K562R reduced the NAM+DOX-induced apoptosis. Mechanistically, the combinatory treatment significantly increased the cleavage of caspase-3 and PARP in K562R in vitro and in vivo. These results suggest the potential role of NAM in increasing the sensitivity of CML to DOX via the inhibition of SIRT1.
Assuntos
Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Niacinamida/farmacologia , Sirtuína 1/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos SCID , Sirtuína 1/genética , Sirtuína 1/metabolismo , Células Tumorais Cultivadas , Complexo Vitamínico B/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastatic disease remains the primary cause of death for individuals with T cell acute lymphoblastic leukemia (T-ALL). microRNAs (miRNAs) play important roles in the pathogenesis of T-ALL by inhibiting gene expression at posttranscriptional levels. The goal of the current project is to identify any significant miRNAs in T-ALL metastasis. We observed miR-146b-5p to be downregulated in T-ALL patients and cell lines, and bioinformatics analysis implicated miR-146b-5p in the hematopoietic system. miR-146b-5p inhibited the migration and invasion in T-ALL cells. Interleukin-17A (IL-17A) was predicted to be a target of miR-146b-5p; this was confirmed by luciferase assays. Interestingly, T-ALL patients and cell lines secreted IL-17A and expressed the IL-17A receptor (IL-17RA). IL-17A/IL-17RA interactions promoted strong T-ALL cell migration and invasion responses. Gene set enrichment analysis (GSEA) and quantitative polymerase chain reaction (qPCR) analysis indicated that matrix metallopeptidase-9 (MMP9), was a potential downstream effector of IL-17A activation, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was also implicated in this process. Moreover, IL-17A activation promoted T-ALL cell metastasis to the liver in IL17A -/- mouse models. These results indicate that reduced miR-146b-5p expression in T-ALL may lead to the upregulation of IL-17A, which then promotes T-ALL cell migration and invasion by upregulating MMP9 via NF-κB signaling.
Assuntos
Interleucina-17/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Linfócitos T/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Biologia Computacional , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Interleucina-17/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs , Reação em Cadeia da Polimerase em Tempo Real , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.
Assuntos
Falência Hepática Aguda , Precursores de RNA , Fatores de Processamento de RNA , Splicing de RNA , Proteínas de Ligação a RNA , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genética , Animais , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Splicing de RNA/genética , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Masculino , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Modelos Animais de Doenças , Ligação Proteica , TransativadoresRESUMO
Tumor cells must rewire nucleotide synthesis to satisfy the demands of unbridled proliferation. Meanwhile, they exhibit augmented reactive oxygen species (ROS) production which paradoxically damages DNA and free deoxy-ribonucleoside triphosphates (dNTPs). How these metabolic processes are integrated to fuel tumorigenesis remains to be investigated. MYC family oncoproteins coordinate nucleotide synthesis and ROS generation to drive the development of numerous cancers. We herein perform a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based functional screen targeting metabolic genes and identified nudix hydrolase 1 (NUDT1) as a MYC-driven dependency. Mechanistically, MYC orchestrates the balance of two metabolic pathways that act in parallel, the NADPH oxidase 4 (NOX4)-ROS pathway and the Polo like kinase 1 (PLK1)-NUDT1 nucleotide-sanitizing pathway. We describe LC-1-40 as a potent, on-target degrader that depletes NUDT1 in vivo. Administration of LC-1-40 elicits excessive nucleotide oxidation, cytotoxicity and therapeutic responses in patient-derived xenografts. Thus, pharmacological targeting of NUDT1 represents an actionable MYC-driven metabolic liability.
Assuntos
Nucleotídeos , Nudix Hidrolases , Humanos , Espécies Reativas de Oxigênio/metabolismo , Oxirredução , Nucleotídeos/metabolismoRESUMO
The epithelial-mesenchymal transition (EMT) is a fundamental process governing morphogenesis in multicellular organisms and has recently been implicated in promoting carcinoma invasion and metastasis. Besides their therapeutic effects, accumulating evidences suggest that chemotherapeutic agents also induced EMT and enhanced the malignancy of treated cancer cells; however, the mechanism(s) still remains unclear. Here, we investigated the role of ß-catenin signaling in doxorubicin (Dox)-induced EMT in human gastric cancer cell line BGC-823. We found that the transient treatment of Dox induced EMT and enhanced the in vitro migration ability of cancer cells. We also found that ß-catenin signaling was activated upon Dox treatment. Inhibition of ß-catenin by indomethacin (Indo) or siRNA suppressed Dox-induced EMT and decreased cancer cell migration ability. Our results showed that ß-catenin signaling was critical to Dox-induced EMT. Indo and other ß-catenin inhibitors may have a potential implication in prevention of gastric cancer metastasis.
Assuntos
Movimento Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , beta Catenina/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Indometacina/farmacologia , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
MYCN amplification in neuroblastoma leads to aberrant expression of MYCN oncoprotein, which binds active genes promoting transcriptional amplification. Yet, how MYCN coordinates transcription elongation to meet productive transcriptional amplification and which elongation machinery represents MYCN-driven vulnerability remain to be identified. We conducted a targeted screen of transcription elongation factors and identified the super elongation complex (SEC) as a unique vulnerability in MYCN-amplified neuroblastomas. MYCN directly binds EAF1 and recruits SEC to enhance processive transcription elongation. Depletion of EAF1 or AFF1/AFF4, another core subunit of SEC, leads to a global reduction in transcription elongation and elicits selective apoptosis of MYCN-amplified neuroblastoma cells. A combination screen reveals SEC inhibition synergistically potentiates the therapeutic efficacies of FDA-approved BCL-2 antagonist ABT-199, in part due to suppression of MCL1 expression, both in MYCN-amplified neuroblastoma cells and in patient-derived xenografts. These findings identify disruption of the MYCN-SEC regulatory axis as a promising therapeutic strategy in neuroblastoma.
Assuntos
Neuroblastoma , Proteínas Nucleares , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fatores de TranscriçãoRESUMO
Cyclin-dependent kinase 12 (CDK12) interacts with cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). To gain a comprehensive understanding of CDK12's cellular function, we used chemical genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the polymerase-associated factor 1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. Moreover, we discovered that LEO1 interacts with and is dephosphorylated by the Integrator-PP2A complex (INTAC) and that INTAC depletion promotes the association of PAF1C with Pol II. Together, this study reveals an uncharacterized role for CDK12 and INTAC in regulating LEO1 phosphorylation, providing important insights into gene transcription and its regulation.
Assuntos
Quinases Ciclina-Dependentes , RNA Polimerase II , Humanos , Fosforilação/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , RNA Polimerase II/metabolismo , Núcleo Celular/metabolismo , Transcrição Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The HIV-1 Tat protein hijacks the Super Elongation Complex (SEC) to stimulate viral transcription and replication. However, the mechanisms underlying Tat activation and inactivation, which mediate HIV-1 productive and latent infection, respectively, remain incompletely understood. Here, through a targeted complementary DNA (cDNA) expression screening, we identify PRMT2 as a key suppressor of Tat activation, thus contributing to proviral latency in multiple cell line latency models and in HIV-1-infected patient CD4+ T cells. Our data reveal that the transcriptional activity of Tat is oppositely regulated by NPM1-mediated nucleolar retention and AFF4-induced phase separation in the nucleoplasm. PRMT2 preferentially methylates Tat arginine 52 (R52) to reinforce its nucleolar sequestration while simultaneously counteracting its incorporation into the SEC droplets, thereby leading to its functional inactivation to promote proviral latency. Thus, our studies unveil a central and unappreciated role for Tat methylation by PRMT2 in connecting its subnuclear distribution, liquid droplet formation, and transactivating function, which could be therapeutically targeted to eradicate latent viral reservoirs.
Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Linhagem Celular , Provírus/genética , Linfócitos T/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Latência Viral/genética , Infecções por HIV/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
The reports on the origin of human CD8(+) Valpha24(+) T-cell receptor (TCR) natural killer T (NKT) cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV)-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8(+) NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8(+) NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8(+) NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells) and CD8(+) NKT cells ( approximately 25% of NKT cells) is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8(+) NKT cells undetectable, respectively). The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8(+) NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8(+) NKT cells display an activated memory phenotype (CD69(+)CD45RO(hi)CD161(+)CD62L(lo)). After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8(+) NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or reaggregated thymic organ cultures. Thymic antigen-presenting EBV-infected dendritic cells are required for this process. IL-7, produced mainly by thymic dendritic cells, is a major and essential factor for CD8(+) NKT cell differentiation in EBV-challenged human-thymus/liver-SCID chimeras and fetal thymic organ cultures. Additionally, these EBV-induced CD8(+) NKT cells produce remarkably more perforin than that in counterpart CD4(+) NKT cells, and predominately express CD8alphaalpha homodimer in their co-receptor. Thus, upon interaction with certain viruses, CD8 lineage-specific NKT cells are developed, differentiated and matured intrathymically, a finding with potential therapeutic importance against viral infections and tumors.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/virologia , Infecções por Vírus Epstein-Barr/imunologia , Mononucleose Infecciosa/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/virologia , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Células Cultivadas , Quimera , Feminino , Citometria de Fluxo , Expressão Gênica/imunologia , Doença de Hodgkin/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-7/genética , Células Matadoras Naturais/imunologia , Camundongos , Camundongos SCID , Técnicas de Cultura de Órgãos , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Timo/imunologia , Timo/virologiaRESUMO
Lipopolysaccharide (LPS) plays an important role in the activation of innate immune cells, leading to secretion of proinflammatory factors and bridging the adaptive immune system. Exposing total mouse thymic cells culture to LPS induced a unique expression profile of cytokines (IL-17A, IL-17F, and IL-22) and the essential ROR-γt master transcription factor, which suggested a preferential differentiation of thymocytes towards the Th17 cell phenotype. Th17-polarizing molecules (IL-23, IL-23R, IL-6, and TGF-ß) and IL-17A(+)CD4(+) thymocytes were also specifically produced by the in vitro LPS-stimulation of thymic cells. Furthermore, both the expression of Th17 differentiation-related molecules and the frequency of Th17 cells were significantly up-regulated with increasing doses of LPS, as evidenced by quantitative RT-PCR and flow cytometric analysis, respectively. The expressions and frequency reached maximum levels when LPS exposure had been maintained at an extremely high concentration (100 µg/mL) for 48 h. On the other hand, depletion of thymic dendritic cells (DCs) blocked the LPS-induced polarization of thymus-derived Th17 cell lineage. Addition of bone marrow-derived DCs (BMDCs) to the purified immature CD4(+) CD62L(low) thymocytes culture recovered the switch towards Th17 cells, which synergistically prompted the cytotoxic activity of CD8(+) T cells. Taken together, our data indicates that high doses of LPS can promote the differentiation of mouse thymus-derived Th17 cells by a mechanism involving components associated with mature DCs.
Assuntos
Células Dendríticas/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Células Th17/imunologia , Timócitos/imunologia , Animais , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Feminino , Interleucina-17/biossíntese , Interleucina-23/biossíntese , Interleucina-6/biossíntese , Interleucinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Timócitos/citologia , Fator de Crescimento Transformador beta/biossíntese , Interleucina 22RESUMO
BACKGROUND: G-quadruplexes (G4s) are unique noncanonical nucleic acid secondary structures, which have been proposed to physically interact with transcription factors and chromatin remodelers to regulate cell type-specific transcriptome and shape chromatin landscapes. RESULTS: Based on the direct interaction between G4 and natural porphyrins, we establish genome-wide approaches to profile where the iron-liganded porphyrin hemin can bind in the chromatin. Hemin promotes genome-wide G4 formation, impairs transcription initiation, and alters chromatin landscapes, including decreased H3K27ac and H3K4me3 modifications at promoters. Interestingly, G4 status is not involved in the canonical hemin-BACH1-NRF2-mediated enhancer activation process, highlighting an unprecedented G4-dependent mechanism for metabolic regulation of transcription. Furthermore, hemin treatment induces specific gene expression profiles in hepatocytes, underscoring the in vivo potential for metabolic control of gene transcription by porphyrins. CONCLUSIONS: These studies demonstrate that G4 functions as a sensor for natural porphyrin metabolites in cells, revealing a G4-dependent mechanism for metabolic regulation of gene transcription and chromatin landscapes, which will deepen our knowledge of G4 biology and the contribution of cellular metabolites to gene regulation.
Assuntos
Quadruplex G , Porfirinas , Cromatina , Hemina/química , Transcrição GênicaRESUMO
Murine immune-responsive gene 1 (IRG1) plays significant roles in embryonic implantation and neurodegeneration. The expression pattern of the human IRG1 gene, however, has not yet been established, and the predicted gene sequence has been revised several times according to computed expressed sequence tags (ESTs). To determine the human IRG1 gene expression profile, human fetal tissue samples, peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the human leukemia cell lines THP-1 and K-562 challenged with lipopolysaccharide (LPS) were subjected to RT-PCR using degenerate primers. The results indicated that the IRG1 gene is differentially expressed in human fetal PBMCs and LPS-stimulated adult PBMCs. The amplified gene fragment was cloned into the pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system. After affinity chromatography, human IRG1h fusion proteins were isolated by SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen to immunize rabbits. The titer and specificity of the purified rabbit antiserum were sufficient to measure human IRG1 gene expression in various tissues and cultures. This purified polyclonal antiserum will allow us to initiate studies to elucidate the biological roles of the human IRG1 gene.
Assuntos
Perfilação da Expressão Gênica , Soros Imunes/imunologia , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Carboxiliases , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Dados de Sequência Molecular , Filogenia , Proteínas/classificação , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase-hexahistidine-2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.
Assuntos
Estruturas R-Loop , RNA , DNA/química , Humanos , RNA/química , RNA/genéticaRESUMO
Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells, are characterized by their immunosuppressive abilities through the secretion of various cytokines such as inducible nitric oxide synthase, nitric oxide, reactive oxygen species, transforming growth factor-ß, and arginase-1. Accumulating evidence highlights its potential role in maintaining immune tolerance in solid organ and hematopoietic stem cell transplantation. Mechanistically, MDSCs-induced transplant tolerance is mainly dependent on direct suppression of allogeneic reaction or strengthened cross-talk between MDSCs and Treg or NKT cells. Adopted transfer of in vitro- or in vivo-induced MDSCs by special drugs therefore becomes a potential strategy for maintaining transplantation tolerance. In this review, we will summarize the previously published data about the role of MDSCs in the biology of transplantation tolerance and gain insights into the possible molecular mechanism governing this process.
Assuntos
Transferência Adotiva/métodos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Células Supressoras Mieloides/imunologia , Tolerância ao Transplante , Animais , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Células Supressoras Mieloides/transplante , Transplante de Órgãos/efeitos adversosRESUMO
Mixed lineage leukemia (MLL) is an aggressive and refractory blood cancer that predominantly occurs in pediatric patients and is often associated with poor prognosis and dismal outcomes. Thus far, no effective target therapy for the treatment of MLL leukemia is available. MLL leukemia is caused by the rearrangement of MLL genes at 11q23, which generates various MLL chimeric proteins that promote leukemogenesis through transcriptional misregulation of MLL target genes. Biochemical studies on MLL chimeras have identified that the most common partners exist in the superelongation complex (SEC) and DOT1L complex, which activate or sustain MLL target gene expression through processive transcription elongation. The results of these studies indicate a transcription-related mechanism for MLL leukemogenesis and maintenance. In this study, we first review the history of MLL leukemia and its related clinical features. Then, we discuss the biological functions of MLL and MLL chimeras, significant cooperating events, and transcriptional addiction mechanisms in MLL leukemia with an emphasis on potential and rational therapy development. Collectively, we believe that targeting the transcriptional addiction mediated by SEC and the DOT1L complex will provide new avenues for target therapies in MLL leukemia and serve as a novel paradigm for targeting transcriptional addiction in other cancers.
RESUMO
Natural fiber reinforced polymer-based composites have been growing into a type of green composites. The properties of natural fiber reinforced polymer-based composites are closely related to the structure of natural fibers. Bagasse fiber (BF) is one of the most used natural fibers for preparing natural fiber reinforced polymer-based composites. However, few examples of previous research touch on the quantitatively characterization of structure of BF and its effect on the properties of BF reinforced polymer-based composites. In this work, four kinds of BF including untreated BF (UBF), alkali treated BF (ABF), BF modified by silane coupling agent (SBF), and BF modified combining alkali treatment with silane coupling agent (ASBF) were prepared and melting blended with polylactic acid (PLA) to prepare PLA/BF composites. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), thermogravimetry (TGA) and mechanical properties testing were used to characterize and analyze the structure and properties of modified BF and its reinforced PLA-based composites. Results showed that the used methods changed the structure of BF and their bonding modes. The surface energies of UBF, ABF, SBF, and ASBF were 19.8 mJ/m2, 34.7 mJ/m2, 12.3 mJ/m2, and 21.6 mJ/m2, respectively. The O/C ratios of UBF, ABF, SBF and, ASBF are 0.48, 0.53, 0.47, and 0.51. Due to the synergistic effect of alkali treatment and silane coupling agent modification on the surface chemical properties, the content of silicon elements on the surface of ASBF (4.15%) was higher than that of ASBF (2.38%). However, due to the destroying of alkali treatment on the microstructure of BF, the alkali treatment had no prominently synergetic effect with coupling agent modification on the mechanical properties of PLA/BF composites. Alkali treatment removed the small molecular compounds from BF, decreased its thermal stability, and increased the crystalline region and crystallinity of cellulose. Meanwhile, alkali treatment made BF fibrillated and increased its contactable active area with the coupling agents, but destructed the nature structure of BF. The silane coupling agent played a more important role than alkali treatment did in improving the interfacial compatibility of PLA/BF composites.
RESUMO
Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the only curative treatment for multiple hematologic malignancies and non-malignant hematological diseases. However, graft-vs.-host disease (GVHD), one of the main complications after allo-HSCT, remains the major reason for morbidity and non-relapse mortality. Emerging evidence has demonstrated that innate lymphoid cells (ILCs) play a non-redundant role in the pathophysiology of GVHD. In this review, we will summarize previously published data regarding the role of ILCs in the pathogenesis of GVHD.
Assuntos
Suscetibilidade a Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/metabolismo , Imunidade Inata , Linfócitos/imunologia , Linfócitos/metabolismo , Biomarcadores , Plasticidade Celular/imunologia , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: ZWINT is a crucial component of the mitotic checkpoint. However, its possible role in lung cancer is unclear. In this study, we determined its correlation with lung cancer. METHODS: Real-time PCR and immunohistochemistry (IHC) were used to determine 40 collected clinical lung cancer samples. Chi-square test was used to examine possible correlations between ZWINT expression and clinicopathological factors. The prognostic significance of mRNA expression of ZWINT in lung cancer was evaluated using the Kaplan-Meier plotter. Univariate and multivariate Cox proportional hazards regression analysis were performed to determine whether ZWINT is an independent risk factor for overall survival (OS) and disease-free survival (DFS) of lung cancer patients. Additionally, STRING database was used to analyze protein-protein interactions. RESULTS: In this study, we screened 13 GSE datasets and detected that ZWINT is highly expressed in multiple carcinomas including lung, melanoma, prostate, nasopharyngeal, gastric, pancreatic, colon, esophageal, ovarian, renal, breast and liver cancer. Real-time PCR and IHC results of collected clinical lung cancer samples confirmed that ZWINT is highly expressed in tumor tissues compared with adjacent non-tumor tissues. Additionally, high expression of ZWINT might predict poor OS and DFS in lung cancer patients. Moreover, disease stage and expression level of ZWINT were correlated with recurrence-free survival and OS in lung cancer. Analysis of protein-protein interaction based on STRING database gained 8 top genes which could interact with ZWINT, including PMF1, MIS12, DSN1, ZW10, BUB1, BUB1B, CASC5, NDC80, NSL1 and NUF2. CONCLUSION: ZWINT is aberrantly highly expressed in lung tumor tissues and might be involved in the pathogenesis of lung cancer.