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1.
Methods Mol Biol ; 2173: 171-188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32651918

RESUMO

Blue light-induced chromatin recruitment (BLInCR) is a versatile optogenetic tool to enrich effector proteins at specific loci within the nucleus using illumination in the 400-500 nm range. The resulting chromatin binding reaction is reversible on the time scale of minutes. BLInCR is advantageous over ligand-binding induced methods since it does not require a change of growth medium for the relatively slow depletion of the inducer from the nucleus. However, applying BLInCR for reversibility experiments is challenging because of the need to spectrally separate light-induced activation from visualization of the chromatin locus and effector and/or reader proteins by light microscopy. Here, we describe an improved BLInCR protocol for light-dependent association and dissociation of effectors using the near-infrared fluorescent protein iRFP713. Due to its spectral properties, iRFP713 can be detected separately from the red fluorescent protein mCherry. Thus, it becomes possible to trace two proteins labeled with iRFP713 and mCherry independently of the light activation reaction. This approach largely facilitates applications of the BLInCR system for experiments that test the reversibility, persistence, and memory of chromatin states.


Assuntos
Cromatina/metabolismo , Luz , Optogenética/métodos , Humanos , Software , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
2.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
3.
Methods Mol Biol ; 2038: 251-270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31407290

RESUMO

Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.


Assuntos
Luz , Microscopia Confocal , Microscopia de Fluorescência , Optogenética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Fatores de Tempo , Fatores de Transcrição/genética
4.
J Cell Sci ; 130(24): 4213-4224, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29122982

RESUMO

Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.


Assuntos
Cromatina/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Transcrição Genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Cromatina/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Genes Reporter/genética , Humanos , Cinética , Luz
5.
Science ; 356(6335): 256, 2017 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-28428389
6.
Science ; 355(6320): 34, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-28059733
7.
Mol Syst Biol ; 10: 746, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25134515

RESUMO

The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.


Assuntos
Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Inativação Gênica , Marcadores Genéticos , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Mitose , Células NIH 3T3 , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade
8.
Lab Chip ; 12(5): 916-22, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22252585

RESUMO

Despite its tremendous high-throughput screening capabilities, widespread applications of droplet-based microfluidics are still limited by the poor availability of appropriate analytical assays. Here we report on a novel sensor method that exploits the osmosis-driven change in droplet size as a quantitative and label-free marker for reactions inside the droplets. We present an analysis of the underlying mechanism and apply the method for monitoring metabolic activity at a single-cell level.


Assuntos
Microfluídica/métodos , Nanocápsulas/química , Células Cultivadas , Cinética , Osmose , Tamanho da Partícula , Coloração e Rotulagem , Tensoativos/química , Leveduras/metabolismo
9.
New York; Oxford University Press for the World Bank; 2000. 343 p. ilus.(Voices of the poor).
Monografia em Inglês | PAHO | ID: pah-51284
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