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1.
J Clin Exp Hematop ; 61(3): 120-125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34511544

RESUMO

Post-transplant lymphoproliferative disorder (PTLD) and other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) are iatrogenic lymphoproliferative disorders (LPD) that develop in association with immunosuppressive treatment in the setting of organ transplantation and autoimmune disease, respectively. Each has a spectrum of pathologies ranging from lymphoid hyperplasia to lymphoma. To clarify the characteristics of the diffuse large B-cell lymphoma (DLBCL) subtype in a cohort of 25 patients with PTLD or OIIA-LPD from our institute, we selected 13 with a histological subtype of DLBCL, including 2 cases of PTLD and 11 of OIIA-LPD. The median patient age at diagnosis was 70 years, with a female predominance. Both PTLD cases developed after kidney transplant. Of the patients with OIIA-LPD, 10 had rheumatoid arthritis, 1 had mixed connective tissue disease, and 8 were treated using methotrexate. Both of the PTLD patients and 6 of the OIIA-LPD patients had extranodal manifestations. All patients except for one were classified as having the non-germinal center B-cell (non-GCB) subtype according to the Hans algorithm. Tissue samples from 8 patients were positive for CD30 and 8 were positive for Epstein-Barr virus (EBV)-encoded small RNA. Seven patients had MYC-positive tissue samples, but none had MYC translocation. Our study suggests that extranodal manifestations and the non-GCB subtype are common, that EBV is associated with the DLBCL subtype of PTLD and OIIA-LPD, and that anti-CD30 therapy is applicable. In addition, our patients with the DLBCL subtype of PTLD and OIIA-LPD exhibited MYC overexpression without MYC translocation, suggesting an alternative mechanism of MYC upregulation.


Assuntos
Regulação da Expressão Gênica , Genes myc , Doença Iatrogênica , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/etiologia , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/etiologia , Idoso , Idoso de 80 Anos ou mais , Suscetibilidade a Doenças , Infecções por Vírus Epstein-Barr/complicações , Humanos , Hospedeiro Imunocomprometido , Imunossupressores/efeitos adversos , Pessoa de Meia-Idade , Transplante de Órgãos/efeitos adversos
2.
Mol Cell ; 81(15): 3048-3064.e9, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216543

RESUMO

RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYC-driven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m6A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a RNA/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Morte Celular/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Camundongos Nus , Camundongos Transgênicos , Biossíntese de Proteínas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Methods Mol Biol ; 2328: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251628

RESUMO

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas Repressoras/metabolismo , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Primers do DNA , Genes myc/genética , Luciferases/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Proteínas Repressoras/genética , Tabaco/genética
4.
Zhonghua Bing Li Xue Za Zhi ; 50(6): 604-608, 2021 Jun 08.
Artigo em Chinês | MEDLINE | ID: mdl-34078047

RESUMO

Objective: To investigate the clinicopathological features, molecular genetics, treatment and prognosis of Burkitt-like lymphoma with 11q aberration (BLL-11q). Methods: Six cases of BLL-11q diagnosed at the First Affiliated Hospital of Zhengzhou University, from January 2016 to January 2020 were reviewed and analyzed using hematoxylin-eosin staining, immunohistochemistry, EBER in situ hybridization and fluorescence in situ hybridization. Clinical information including follow-up data was collected and analyzed. Results: The median age of the six immunocompetent patients was 29 years (range 20-38 years) and the male to female ratio was 5∶1. All patients had nodal disease in the head and neck region. Five patients had Ann Arbor stage Ⅰ-Ⅱ disease, while one patient had stage Ⅳ disease. Lymph nodes showed partial or total architectural effacement by a diffuse proliferation of monomorphic lymphocytes. Four cases were morphologically similar to Burkitt lymphoma, and two cases were unclassified with histological features between Burkitt lymphoma and diffuse large B-cell lymphoma. Mitotic figures, apoptosis and necrosis were conspicuous. Five cases exhibited the"starry sky"pattern. CD20, CD10 and bcl-6 were diffusely and strongly positive. The Ki-67 index was more than 95%. The follicular-dendritic-cell meshwork was noted in one case using CD21 stain. C-MYC was expressed variably. CD3, bcl-2, MUM-1, CD30 and TDT were negative in all cases. EBER in situ hybridization was also all negative. FISH analyses using C-MYC, bcl-2 and bcl-6 break-apart probes were all negative. All cases had the 11q23.3 gain/11q24.3 loss pattern, and 11q23.3 amplification was found in one case. IgH and IRF4 break-apart probes analysis was also negative. All patients were alive with no disease after a follow-up of 4 to 19 months. Conclusion: BLL-11q is a rare lymphoma that resembles Burkitt lymphoma morphologically and phenotypically, but lacks C-MYC gene rearrangements. Instead, it has a chromosome-11q alteration characterized by proximal gains and telomeric losses. It's necessary to improve our understanding of BLL-11q to avoid misdiagnosis and missed diagnosis.


Assuntos
Linfoma de Burkitt , Linfoma Difuso de Grandes Células B , Adulto , Linfoma de Burkitt/genética , Aberrações Cromossômicas , Feminino , Genes myc/genética , Humanos , Hibridização in Situ Fluorescente , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Masculino , Biologia Molecular , Translocação Genética , Adulto Jovem
5.
Zhonghua Bing Li Xue Za Zhi ; 50(6): 598-603, 2021 Jun 08.
Artigo em Chinês | MEDLINE | ID: mdl-34078046

RESUMO

Objective: To investigate the genetic abnormality and protein expression of C-MYC and PD-L1 in the patients with ALK-negative anaplastic large cell lymphoma (ALK-ALCL), and to explore their roles in the pathogenesis of ALK-ALCL and their relationship with clinicopathological characteristics. Methods: Thirty-seven cases of ALK-ALCL diagnosed at Fujian Provincial Hospital from January 2003 to January 2017 were selected. Fluorescence in situ hybridization (FISH) was used to detect the genetic abnormality of C-MYC and PD-L1. The expression of C-MYC and PD-L1 proteins was detected by immunohistochemistry. The relationship between C-MYC and PD-L1 genes' abnormalities and protein expression was analyzed, as well as their associations with various clinicopathological parameters. Results: Among the 37 ALK-ALCL patients, 17 (45.9%) were positive for C-MYC protein, and 14 (37.8%) were positive for PD-L1 protein. There was a significant correlation between C-MYC protein and PD-L1 protein (r=0.990,P=0.014). The protein expression of C-MYC and PD-L1 (versus negative) was associated with the clinical stage of ALK-ALCL, respectively. The international prognosis index (IPI) in high-risk group was higher than that in the low-risk group (P<0.05). FISH test showed that 9 (24.3%) of the 37 cases had amplification of C-MYC gene, and no translocation of C-MYC gene was found in any of the cases. Amplification of PD-L1 gene was found in only 2 cases (5.4%). The 3-year overall survival rate of the C-MYC or PD-L1 immunohistochemistry-positive cases was significantly lower than those of the C-MYC or PD-L1 negative cases (P<0.01 and P<0.05), respectively. Conclusion: The expression of C-MYC and PD-L1 proteins are related to the clinical stage, IPI and overall survival rate of ALK-ALCL. Thus, it can be used to assess the disease's aggressiveness and to predict the prognosis of ALK-ALCL. The expression of PD-L1 in ALK-ALCL may be regulated by C-MYC, thus suggesting a possible design of combined C-MYC targeted therapy and immune checkpoint blocking for some ALK-ALCL patients.


Assuntos
Linfoma Anaplásico de Células Grandes , Quinase do Linfoma Anaplásico/genética , Antígeno B7-H1/genética , Genes myc , Humanos , Hibridização in Situ Fluorescente , Linfoma Anaplásico de Células Grandes/genética , Proteínas Proto-Oncogênicas c-myc , Receptores Proteína Tirosina Quinases/genética
6.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065149

RESUMO

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21Waf1/Cip1, p27Kip1, or p16Ink4a. c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin's anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Tricodermina/farmacologia , Animais , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias Ovarianas , Tricodermina/química , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 12(1): 3361, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099699

RESUMO

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma de Células B/genética , Linfoma não Hodgkin/genética , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Translocação Genética , Biologia Computacional/métodos , Rearranjo Gênico , Genes bcl-2/genética , Genes myc/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Linfoma de Células B/diagnóstico , Linfoma não Hodgkin/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-6/genética , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade
8.
Nat Commun ; 12(1): 3964, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172720

RESUMO

The regulation of bone vasculature by chronic diseases, such as heart failure is unknown. Here, we describe the effects of myocardial infarction and post-infarction heart failure on the bone vascular cell composition. We demonstrate an age-independent loss of type H endothelium in heart failure after myocardial infarction in both mice and humans. Using single-cell RNA sequencing, we delineate the transcriptional heterogeneity of human bone marrow endothelium, showing increased expression of inflammatory genes, including IL1B and MYC, in ischemic heart failure. Endothelial-specific overexpression of MYC was sufficient to induce type H bone endothelial cells, whereas inhibition of NLRP3-dependent IL-1ß production partially prevented the post-myocardial infarction loss of type H vasculature in mice. These results provide a rationale for using anti-inflammatory therapies to prevent or reverse the deterioration of bone vascular function in ischemic heart disease.


Assuntos
Osso e Ossos/irrigação sanguínea , Células Endoteliais/patologia , Insuficiência Cardíaca/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Idoso , Animais , Osso e Ossos/fisiopatologia , Estudos de Casos e Controles , Células Endoteliais/metabolismo , Feminino , Furanos/farmacologia , Genes myc , Insuficiência Cardíaca/etiologia , Células-Tronco Hematopoéticas/patologia , Humanos , Indenos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Sulfonamidas/farmacologia
9.
Cancer Discov ; 11(5): 1011-1013, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33947717

RESUMO

In this issue of Cancer Discovery, Welti and colleagues demonstrate a positive correlation between the expression of the histone acetyltransferase paralogs CBP and p300 with increased androgen receptor (AR) signaling and androgen deprivation therapy resistance in advanced prostate cancer. CCS1477, a selective inhibitor of p300/CBP bromodomain, disrupts AR- and MYC-regulated gene expression, suppresses tumor growth in vivo in multiple castration-resistant prostate cancer xenograft models, and modulates biomarker expression in early clinical evaluation, providing a novel therapeutic approach for AR-addicted advanced prostate cancer.See related article by Welti et al., p. 1118.


Assuntos
Antagonistas de Androgênios , Neoplasias da Próstata , Linhagem Celular Tumoral , Genes myc , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Receptores Androgênicos/genética
10.
Methods Mol Biol ; 2318: 69-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019287

RESUMO

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.


Assuntos
Imunoprecipitação/métodos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-myc/genética , Imunofluorescência , Genes myc/genética , Genes myc/fisiologia , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sumoilação , Ubiquitinação
11.
Rinsho Ketsueki ; 62(4): 245-250, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33967147

RESUMO

Acute myeloid leukemia (AML) associated with double-minute chromosomes (dmin) is a rare condition and has a poor prognosis. A 68-year-old man with leukocytosis and thrombocytopenia was admitted to our hospital. Bone marrow aspiration showed that 79.5% of myeloblasts were positive for myeloperoxidase. The patient was diagnosed with acute myeloid leukemia (French-American-British classification: M2, World Health Organization classification: AML, not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of dmin: 45, X, -Y, 5-33 dmin. Fluorescence in situ hybridization revealed multiple MYC signals, and spectral karyotyping showed that dmin was derived from chromosome 8. These findings indicated resistance to chemotherapy alone. After the standard induction therapy with daunorubicin and cytarabine, the number of myeloblasts in the bone marrow decreased, and the amplified MYC signals disappeared. Then, the patient achieved complete remission. Reportedly, most patients with AML correlated with dmin have a complex karyotype, except for this case. Owing to the absence of a complex karyotype, the patient had good sensitivity to chemotherapy. Further studies with a larger population of patients with AML associated with dmin, but without complex karyotypes, should be conducted to accurately predict prognosis in such cases.


Assuntos
Genes myc , Leucemia Mieloide Aguda , Idoso , Cromossomos , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente , Quimioterapia de Indução , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Indução de Remissão
12.
Nucleic Acids Res ; 49(10): 5943-5955, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33999211

RESUMO

DNA binding proteins recognize DNA specifically or non-specifically using direct and indirect readout mechanisms like sliding, hopping, and diffusion. However, a common difficulty in explicitly elucidating any particular mechanism of site-specific DNA-protein recognition is the lack of knowledge regarding target sequences and inadequate account of non-specific interactions, in general. Here, we decipher the structural basis of target search performed by the key regulator of expression of c-myc proto-oncogene, the human RBMS1 protein. In this study, we have shown the structural reorganization of this multi-domain protein required for recognizing the specific c-myc promoter sequence. The results suggest that a synergy between structural re-organization and thermodynamics is necessary for the recognition of target sequences. The study presents another perspective of looking at the DNA-protein interactions.


Assuntos
Proteínas de Ligação a DNA/química , Genes myc , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a RNA/química , Sítios de Ligação , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes , Termodinâmica
13.
Methods Mol Biol ; 2318: 231-239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019293

RESUMO

The MYC gene regulates normal cell growth and is deregulated in many human cancers, contributing to tumor growth and progression. The MYC transcription factor activates RNA polymerases I, II, and III target genes that are considered housekeeping genes. These target genes are largely involved in ribosome biogenesis, fatty acid, protein and nucleotide synthesis, nutrient influx or metabolic waste efflux, glycolysis, and glutamine metabolism. MYC's function as a driver of cell growth has been revealed through RNA sequencing, genome-wide chromatin immunoprecipitation, proteomics, and importantly metabolomics, which is highlighted in this chapter.


Assuntos
Carcinogênese/metabolismo , Metabolômica/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Carcinogênese/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , DNA/genética , Genes myc/genética , Genes myc/fisiologia , Glucose/metabolismo , Glicólise , Humanos , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase I/metabolismo , Ribossomos/metabolismo
14.
Methods Mol Biol ; 2318: 13-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019284

RESUMO

The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/química , DNA/genética , Dimerização , Escherichia coli/genética , Expressão Gênica , Genes myc , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo
15.
Methods Mol Biol ; 2318: 1-11, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019283

RESUMO

The MYC transcription factor coordinates a wide range of intra- and extracellular processes associated with tissue proliferation and regeneration. While these processes are typically tightly regulated in physiological conditions, they become deregulated in cancer, where MYC is oncogenically activated.The last decade has seen MYC progress from a renowned undruggable target to a hot topic in the cancer therapy field, as proof emerged from mouse models that its inhibition constitutes an effective and broadly applicable approach to fight cancer. However, there are several aspects of MYC biology that still appear to be elusive and maintain the interest in further studying this intriguing protein. Since MYC's discovery, more than four decades ago, multiple strategies have been developed to study it, related to the many and varied facets of its biology. This new version of The Myc gene: Methods and Protocols provides valuable tips from key "inhabitants of the MYC world," which significantly increase the reach of our investigative tools to shed light on the mysteries still surrounding MYC.


Assuntos
Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Genes myc , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
16.
Methods Mol Biol ; 2318: 21-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019285

RESUMO

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Cristalografia por Raios X/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Sequências Hélice-Alça-Hélice/genética , Sequências Hélice-Alça-Hélice/fisiologia , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo
17.
Methods Mol Biol ; 2318: 45-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019286

RESUMO

By identifying MYC protein-protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein-protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo-in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Sítios de Ligação , Biologia Computacional/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Humanos , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo
18.
Methods Mol Biol ; 2318: 87-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019288

RESUMO

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Alelos , Sequência de Aminoácidos , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Evolução Molecular , Genes myc/genética , Genes myc/fisiologia , Humanos , Mutação , Neoplasias/patologia , Oncogenes , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética
19.
Methods Mol Biol ; 2318: 161-185, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019290

RESUMO

Here, we present a strategy to map and quantify the interactions between Myc and chromatin using a calibrated Myc ChIP-seq approach. We recommend the use of an internal spike-in control for post-sequencing normalization to enable detection of broad changes in Myc binding as can occur under conditions with varied Myc abundance. We also highlight a range of bioinformatic analyses that can dissect the downstream effects of Myc binding. These methods include peak calling, mapping Myc onto an integrated metagenome, juxtaposing ChIP-seq data with matching RNA-seq data, and identifying gene ontologies enriched for genes with high Myc binding. Our aim is to provide a guided strategy, from cell harvest through to bioinformatic analysis, to elucidate the global effects of Myc on transcription.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Imunoprecipitação da Cromatina/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Sítios de Ligação , Cromatina/genética , Biologia Computacional/métodos , DNA/genética , Proteínas de Ligação a DNA , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Sequência de DNA/métodos
20.
Methods Mol Biol ; 2318: 119-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019289

RESUMO

The c-MYC oncogene is activated in ~50% of all tumors and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, we describe the validation of single c-MYC targets identified by NGS . Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts. Ultimately, these analyses will further illuminate the role of this important oncogene.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas Proto-Oncogênicas c-myc/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Genes myc , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/genética , RNA Mensageiro/genética , Análise de Sequência de DNA/métodos
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