RESUMO
Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdhα, -ß, and -γ). This diversity is generated by a combination of stochastic promoter choice and alternative pre-mRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdhα promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdhα isoforms αc1 and αc2. We propose that CTCF and the cohesin complex initiate and maintain Pcdhα promoter choice by mediating interactions between Pcdhα promoters and enhancers.
Assuntos
Caderinas/genética , Caderinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Processamento Alternativo/genética , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosfoproteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , CoesinasRESUMO
Starting from a random RNA library expressed in yeast cells, we evolved an RNA-based transcriptional silencing domain with potency comparable to that observed when Sir1, a known silencing protein, is localized to a promoter. Using secondary-structure predictions and site-directed mutagenesis, we dissected the functional domains of the most active evolved RNA transcriptional silencer. Observed RNA-based silencing was general, rather than gene specific, and the origin recognition complex was required for full activity of the evolved RNA. Using genetic studies, we demonstrated that the RNA-based silencer acts through a Sir protein-dependent mechanism. Our results highlight the value of evolving RNA libraries as probes of biological processes and suggest the possible existence of natural RNA-based, RNAi-independent gene silencers.
Assuntos
Evolução Molecular Direcionada , Inativação Gênica , RNA/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , RNA/química , Relação Estrutura-AtividadeRESUMO
From random RNA libraries expressed in yeast, we evolved RNA-based transcriptional activators that are comparable in potency to the strongest natural protein activation domains. The evolved RNAs activated transcription up to 53-fold higher than a three-hybrid positive control using the Gal4 activation domain and only 2-fold lower than the highly active VP16 activation domain. Using a combination of directed evolution and site-directed mutagenesis, we dissected the functional elements of the evolved transcriptional activators. A surprisingly large fraction of RNAs from our library are capable of activating transcription, suggesting that nucleic acids may be well suited for binding transcriptional machinery elements normally recruited by proteins. In addition, our work demonstrates an RNA evolution-based approach to perturbing natural cellular function that may serve as a general tool for studying selectable or screenable biological processes in living cells.
Assuntos
Biblioteca Gênica , RNA/genética , Transativadores/genética , Motivos de Aminoácidos , Sequência Consenso , Proteínas de Ligação a DNA , Mutagênese Sítio-Dirigida , RNA/biossíntese , RNA/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Transativadores/biossíntese , Transativadores/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We have developed a versatile tool for the delivery of inhibitors of carbonic anhydrase II, which allows modification of a hydrophobic drug with either a water-solubilizing, photolabile cage or a hydrophobic, photolabile cage. The former mask is useful for direct delivery of hydrophobic molecules in an aqueous prodrug form. The latter may find application if delivery from a surface is desirable. In our system, where the target enzyme is found in the eye, both approaches may be useful for the delivery of hydrophobic drugs having subnanomolar dissociation constants from the enzyme.