Name: Yeast Cell Culture and Cell Harvesting for Single-Copy Gene Locus Chromatin Purification
Uploaded: 2023-11-17T13:20:00
Description: Watch this Scientific Journal Video about Yeast Cell Culture and Cell Harvesting for Single-Copy Gene Locus Chromatin Purification at JoVE.com

yeast cell culture and cell harvesting for single-copy gene locus chromatin purification

عزل الكروماتين الخاص بالموقع من خلايا الخميرة

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.

تسليط الضوء على المؤلف: تطوير أبحاث الكروماتين والتغلب على القيود باستخدام بروتوكول عزل الكروماتين عالي التخصيب الخاص بموضع 

نسخة واحدة من تنقية الكروماتين لموضع الجين في 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 Yeast Cell Culture and Cell Harvesting for Single-Copy Gene Locus Chromatin Purification at JoVE.com

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

زراعة خلايا الخميرة وحصاد الخلايا لتنقية الكروماتين في موضع الجين أحادي النسخة

ابدأ بتلقيح سلالات الخميرة المختصة بالتحكم وإعادة التركيب من ألواح YPD إلى خمسة ملليلتر من وسط YPR ، وقم بتنمية الثقافة بين عشية وضحاها عند 30 درجة مئوية و 200 دورة في الدقيقة. بعد ذلك ، قم بتوسيع نطاق الثقافة عن طريق تلقيح ملليلتر إلى 100 ملليلتر من وسط YPR الطازج وزراعته بين عشية وضحاها عند 30 درجة مئوية و 200 دورة في الدقيقة. لكل سلالة ، قم بتوزيع 1،800 ملليلتر من وسط خميرة الأوتوكلاف بيبتون ، و 200 ملليلتر من الأوتوكلاف 20٪ محلول رافينوز في قارورتين Erlenmeyer سعة 5 لتر.قم بتلقيح كل سلالة من الخميرة بكثافة بصرية 0.2 عند 600 نانومتر في الوسط المعني ، واحتضانها لمدة ست ساعات عند 200 دورة في الدقيقة ، أو حتى تصل الخلايا إلى الكثافة البصرية المطلوبة 1.0. أضف 200 ملليلتر من 2٪ جالاكتوز و 110 ميكرولتر من عامل تزاوج الخميرة ألفا أو YMFA في وقت واحد لإيقاف الخلايا في المرحلة G1 واحتضانها لمدة ساعتين. نقل تعليق الخلية إلى دلاء الطرد المركزي لتر واحد وأجهزة الطرد المركزي دلو في 6،000 غرام وأربع درجات مئوية لمدة 10 دقائق.تخلص من المادة الطافية وأعد تعليق كريات الخلية في 10 إلى 15 ملليلتر من الماء المقطر. بعد ذلك ، أغلق حقنة سعة 25 ملليلتر بسدادة إغراء وضعها داخل أنبوب مخروطي سعة 50 ملليلتر مملوء بالماء. نقل تعليق الخلية إلى مجموعة حقنة.اغسل دلاء الطرد المركزي بخمسة ملليلتر من الماء المقطر لجمع الخلايا المتبقية. ثم ، أجهزة الطرد المركزي التجميع في 2،397 G لمدة 10 دقائق في درجة حرارة الغرفة وتجاهل طاف. ثم قم بإزالة سدادة الإغراء من المحقنة وقذف الخلايا إلى نيتروجين سائل لتشكيل معكرونة خلوية.أخيرا ، انقل السباغيتي الخلوية المجمدة إلى أنبوب مخروطي سعة 50 ملليلتر وقم بتخزينه في درجة حرارة 80 درجة مئوية تحت الصفر حتى الاستخدام مرة أخرى.

Research

JoVE Journal

Biochemistry

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

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 ...