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1.
Mol Cell ; 82(1): 140-158.e12, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34890565

RESUMO

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Neoplasias/enzimologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Complexos Multienzimáticos , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ratos
2.
Chromosome Res ; 24(2): 175-81, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26685864

RESUMO

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Reagentes de Ligações Cruzadas/química , DNA Topoisomerases Tipo I/química , DNA/química , Formaldeído/química , Proteínas de Fluorescência Verde/química , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Células HCT116 , Humanos , Fatores de Tempo
3.
Proc Natl Acad Sci U S A ; 105(47): 18296-301, 2008 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19015535

RESUMO

The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.


Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Consenso , DNA Helicases/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genes myc , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mutação Puntual , Proteínas de Ligação a RNA , Técnica de Seleção de Aptâmeros
4.
Mol Cell Biol ; 26(17): 6584-97, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16914741

RESUMO

The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal alpha-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , DNA/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Proteínas Repressoras/metabolismo , Frações Subcelulares , Transativadores/química , Fatores de Transcrição
5.
Nat Struct Mol Biol ; 11(11): 1092-100, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15502847

RESUMO

The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.


Assuntos
DNA/metabolismo , Transcrição Gênica , Sequência de Bases , Cromatina/química , DNA/química , DNA Super-Helicoidal/química , Eletroforese em Gel Bidimensional , Escherichia coli/metabolismo , Genes myc , Guanosina Trifosfato/metabolismo , Magnésio/química , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , RNA/química , Recombinação Genética
6.
Nat Struct Mol Biol ; 15(2): 146-54, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18193062

RESUMO

Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation.


Assuntos
Cromatina/química , Cromatina/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica/fisiologia , Imunoprecipitação da Cromatina , DNA Topoisomerases/metabolismo , Eletroforese em Gel de Ágar , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transativadores/metabolismo
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