Name: TEV Protease-Mediated Elution and Denaturation Elution of the Chromatin Locus Purified from Saccharomyces cerevisiae
Uploaded: 2023-11-17T13:20:00
Description: Watch this Scientific Journal Video about TEV Protease-Mediated Elution and Denaturation Elution of the Chromatin Locus Purified from Saccharomyces cerevisiae at JoVE.com

tev protease-mediated elution and denaturation elution of the chromatin locus purified from saccharomyces cerevisiae

Aislamiento de cromatina locus específico de células de levadura

The dynamic organization of eukaryotic genomes into chromatin compacts the DNA to fit within the confines of the nucleus while ensuring sufficient dynamics for gene expression and accessibility for regulatory factors. In part, this versatility is mediated by the nucleosome, the basic unit of chromatin, which comprises a core particle with 147 bp of DNA wrapped ~1.7 times around the histone octamer1. The nucleosome is a highly dynamic structure with respect to its composition, with numerous histone variants and posttranslational modifications (PTMs) on the N- and C-terminal histone tails. Furthermore, eukaryotic chromatin interacts with a multitude of other essential components, such as transcription factors, DNA and RNA processing machinery, architectural proteins, enzymes involved in chromatin remodeling and modification, and RNA molecules associated with chromatin. These crucial machineries involved in transcription, replication, and repair all require access to chromatin, which serves as the natural substrate for these processes. Consequently, comprehending the molecular mechanisms underlying these DNA transactions necessitates a precise definition of the collective alterations in chromatin structure at the specific genomic regions where these machineries converge and facilitate biological reactions.
Despite the identification of numerous chromatin factors through genetics and protein-protein interaction studies, performing direct, unbiased, and comprehensive analyses of chromatin interactions at particular genomic sites has remained a significant obstacle2,3. Initially, only highly abundant regions of the genome (i.e., repetitive loci) or multi-copy plasmids could be isolated in sufficient amounts and purity for mass spectrometric identification of the associated proteins4,5,6,7. A series of new approaches based on the direct hybridization of capture probes to chromatinized DNA, proximity biotinylation using the CRISPR-dCas9 system, or the binding of sequence-specific adapter proteins to the locus of interest have started to unravel the proteome of single-copy loci from yeast and mammalian genomes8,9,10. However, all these methods require formaldehyde crosslinking to stabilize the protein-DNA interactions and sonication to solubilize the chromatin for subsequent purification. Together, both manipulations exclude the possibility of subsequent structural and functional studies of the purified chromatin.
To overcome these limitations, we previously devised a methodology that employs site-specific recombination to extract targeted chromosomal domains from yeast11,12. In essence, the genomic region of interest is surrounded by recognition sites (RS) for the site-specific R-recombinase from Zygosaccharomyces rouxi while simultaneously incorporating a group of three DNA binding sites for the prokaryotic transcriptional repressor LexA protein (LexA) within the same region. The yeast cells contain an expression cassette for the simultaneous expression of R-recombinase and a LexA protein fused to a tandem affinity purification (TAP) tag. After the induction of R-recombinase, the enzyme efficiently excises the targeted region from the chromosome in the form of a circular chromatin domain. This domain can be purified via the LexA-TAP adapter protein, which binds to the LexA DNA binding sites, as well as to an affinity support. This method has been recently used to isolate distinct chromatin domains containing selected replication origins of yeast chromosome III13.
One major advantage of this ex vivo approach is that it allows for functional analyses of the isolated material. For example, replication origin domains purified with this method can be subjected to in vitro replication assays to assess the efficiency of origin firing in a test tube from native in vivo assembled chromatin templates. Ultimately, the biochemical and functional characterization of the isolated material may allow the reconstitution of nuclear processes using purified proteins together with the native chromatin template. In summary, this methodology opens an exciting avenue in chromatin research, as it will be possible to follow the collective compositional and structural chromatin changes of a specific genomic region undergoing a certain chromosomal transaction.

Autor destacado: Avance en la investigación de la cromatina y superación de las limitaciones con un protocolo de aislamiento de cromatina específico del locus de alto enriquecimiento 

Purificación de cromatina del locus del gen de copia única en Saccharomyces cerevisiae

A eukaryotic nucleus is very crowded with many biological processes occurring simultaneously. The scope of our research is to understand how all of these processes are coordinated on our genomes without major accidents that would otherwise lead to genomic instability and human diseases. This protocol allows us to purify a specific locus of the genome in its native state, allowing both the compositional and functional characterization of the isolated material, such as the measurements of protein DNA interactions.This protocol enables an enormous level of enrichment of a targeted locus, which overcomes many current limitations of locus-specific chromatin isolation. The material has not been cross-linked. That also allows many downstream functional analysis, such as in vitro transcription or in vitro replication.This protocol enabled us to excise and purify distinct replication origins from yeast chromosomes, allowing us to perform a proteomic analysis and identify novel origin interacting chromatin factors, and therefore, advance the research field of DNA replication. This protocol enables in vitro replication studies using the purified origin material to examine chromatin states, histone modifications, and perform structural analysis on the native nucleosomal templates from endogenous chromosomes, advancing chromatin research.

Watch this Scientific Journal Video about TEV Protease-Mediated Elution and Denaturation Elution of the Chromatin Locus Purified from Saccharomyces cerevisiae at JoVE.com

TEV Protease-Mediated Elution and Denaturation Elution of the Chromatin Locus Purified from Saccharomyces cerevisiae  

Elución mediada por proteasa TEV y elución por desnaturalización del locus de la cromatina purificada a partir de Saccharomyces cerevisiae

Libere los complejos de anillos de cromatina lexA CBP de las perlas magnéticas utilizadas para purificar el locus del gen de copia única de la cromatina incubando a las abejas con dos microlitros de virus de grabado de tabaco recombinante marcado con 6xHis o proteasa TEV. Después de separar las abejas del eluido final mediante una transferencia de rejilla magnética, el eluido que contiene anillos de cromatina escindidos a un nuevo tubo de reacción de 1,5 mililitros. Nuevamente, coloque los tubos en una rejilla magnética para separar las perlas residuales del eluido final.Vuelva a suspender las perlas en 750 microlitros de tampón de carbonato de amonio frío antes de almacenar algunas muestras a 20 grados centígrados para el análisis de ADN y proteínas. A partir del eluido final, sacar las muestras y almacenarlas a 20 grados centígrados para el análisis de ADN y proteínas. Además, tome muestras de las cuentas residuales.Comience la elución de desnaturalización lavando las perlas magnéticas dos veces en 750 microlitros de tampón de carbonato de amonio frío durante 20 minutos cada una, cuatro grados centígrados girando a 20 revoluciones por minuto. A continuación, agregue 500 microlitros de hidróxido de amonio 0,5 molar a las perlas para extraer los complejos de cromatina lexA unidos e incube durante 30 minutos a temperatura ambiente. Separe las perlas de la suspensión con una rejilla magnética y transfiera el eluido a un tubo de reacción de baja unión.Incubar el tubo de reacción de baja unión a temperatura ambiente durante 30 minutos y separar las perlas de la suspensión con una rejilla magnética. Una vez hecho esto, transfiera el eluido a un tubo de reacción de baja unión. Resuspenda las perlas en 750 microlitros de agua desionizada y recoja muestras para el análisis de ADN y proteínas.Por último, obtener muestras de análisis de ADN y proteínas del eluido final. En el estudio, se cuantificó la eficiencia de purificación del locus ARS316 y la cepa de recombinación y se comparó con el locus control PDC1. El locus ARS316 mostró una menor recuperación en el flujo en comparación con PDC1.Aunque una fracción de los anillos de cromatina permanecieron unidos a las perlas de TEV debido a la escisión incompleta de la proteasa de TEV, la elución por desnaturalización dio lugar a una recuperación del locus de ARS316 del 80 al 90%. Esto corresponde al alto enriquecimiento de las moléculas de ARS316 en las perlas de TEV y a las fracciones de elución de desnaturalización cuando se tiene en cuenta el tamaño del genoma de la levadura en comparación con la cepa de control que no mostró enriquecimiento del locus de ARS316 en ninguna de las fracciones cuantificadas. Después de la elución de TEV, las perlas mostraron un mayor porcentaje de recuperación del locus ARS316 ya que la eficiencia de escisión de TEV no fue del 100%, lo que dio como resultado que una fracción de los anillos de cromatina permanecieran unidos a las perlas.Sin embargo, la elusión desnaturalizante mostró una recuperación del 80 al 90% del locus ARS316 y la cepa de recombinación correspondiente al alto enriquecimiento de las moléculas de ARS316 cuando se tuvo en cuenta el tamaño del genoma de la levadura.

Research

JoVE Journal

Biochemistry

TEV Protease-Mediated Elution and Denaturation Elution of the Chromatin Locus Purified from Saccharomyces cerevisiae

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Investigación

Bioquímica

Yeast Cell Culture and Cell Harvesting for Single-Copy Gene Locus Chromatin Purification

Cultivo de células de levadura y recolección de células para la purificación de cromatina del locus del gen de copia única

Locus-Specific Chromatin Purification from Saccharomyces cerevisiae

Locus-Specific Chromatin Purification from Saccharomyces cerevisiae  

Purificación de cromatina específica de locus de Saccharomyces cerevisiae