RESUMO
Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Epigênese Genética , Edição de Genes , Técnicas de Silenciamento de Genes , Humanos , Nucleossomos/metabolismo , Xenopus laevisRESUMO
Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing1-3. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation1,4,5. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A 6 -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH1-3. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ciclina T/química , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fosforilação , Fator B de Elongação Transcricional Positiva/metabolismo , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Ativação Transcricional , Quinases DyrkRESUMO
Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.
Assuntos
Proteína de Transporte de Acila/ultraestrutura , Infecções Bacterianas/microbiologia , Ácido Graxo Sintases/ultraestrutura , Conformação Proteica , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos/genética , Infecções Bacterianas/tratamento farmacológico , Borrelia burgdorferi/química , Borrelia burgdorferi/patogenicidade , Borrelia burgdorferi/ultraestrutura , Brucella melitensis/química , Brucella melitensis/patogenicidade , Brucella melitensis/ultraestrutura , Domínio Catalítico , Ácido Graxo Sintases/química , Ácido Graxo Sintases/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Lipídeo A/química , Lipídeo A/genética , Simulação de Dinâmica Molecular , Complexos Multienzimáticos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Percepção de Quorum/genética , Rickettsia prowazekii/química , Rickettsia prowazekii/patogenicidade , Rickettsia prowazekii/ultraestruturaRESUMO
Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.
Assuntos
Cromatina , Proteínas Inibidoras de STAT Ativados , Proteína com Valosina , Helicase da Síndrome de Werner , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Humanos , Animais , Cromatina/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Camundongos , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Instabilidade de Microssatélites , Proteólise/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , FemininoRESUMO
Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
RESUMO
Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain.
Assuntos
RNA Polimerase II , RNA Longo não Codificante , RNA Polimerase II/metabolismo , Heterocromatina , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação do Cromossomo X , Cromatina , RNA não Traduzido/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Single-particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we investigated methods inspired by Bayesian nonparametrics to infer distributions of state parameters from SPT data with short trajectories, variable localization precision, and absence of prior knowledge about the number of underlying states. We discuss the advantages and disadvantages of these approaches relative to other frameworks for SPT analysis.
Assuntos
Mamíferos , Imagem Individual de Molécula , Animais , Teorema de Bayes , Difusão , Imagem Individual de Molécula/métodosRESUMO
RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization.
Assuntos
Núcleo Celular/virologia , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Simplexvirus/crescimento & desenvolvimento , Replicação Viral , Animais , Linhagem Celular , Humanos , Ligação ProteicaRESUMO
Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.