Purification of Specific Chromatin Domains from Single-Copy Gene Loci in Saccharomyces cerevisiae

Abstract

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors

“…In brief, after sonicating 15 g cells in extraction buffer (25 mM HEPES-KOH, pH 7.4, 0.05% IGEPAL CA-630, 1 mM DTT, 100 mM NaCl, 2 mM MgCl 2 , 5 mM EGTA, 10% glycerol, proteinase inhibitor cocktail, and 1 mM PMSF), the nonspecific endonuclease Benzonase (50 units/mL extract) was added to degrade DNA and RNA. GS-tagged proteins were affinity purified using IgGcoupled magnetic beads (Hamperl et al, 2014), and eluted proteins were analyzed by SDS-PAGE and digested with trypsin. Peptides were separated by reverse-phase chromatography on an UltiMate 3000 RSLCnano System (Thermo Scientific) using a Reprosil-Pur Basic C18 nano column (75 mm i.d.…”

Genome-Wide Reconstitution of Chromatin Transactions: RSC Preferentially disrupts H2A.Z-Containing Nucleosomes

“…Conversely, chromatin studied in vivo obviously retains its natural composition, but is trapped in a context in which a process of interest is continuously subjected to the direct or indirect influence of the numerous biochemical reactions taking place inside the cell. Protocols in which chromatin is isolated directly from the host organism in a manner suitable for biochemical reconstitution have been described, but they are generally limited to the study of one or a few loci at a time (Griesenbeck et al 2003;Unnikrishnan et al 2012;Hamperl et al 2014;Ehrensberger et al 2015). As such, a system in which an entire genome is available in native form for the biochemical reconstitution of chromatin transactions would be very useful.…”

Nucleosomal promoter variation generates gene expression noise

“…[Promoter chromatin remodeling on PHO5 induction does not depend on the PHO5-TATA box (10,13).] The construct was flanked with recombination sequences (RS) recognized by the R recombinase of Zygosaccharomyces rouxii, which allowed for its release from the chromosome in the form of a chromatin ring (10,13,(19)(20)(21) (Fig. 1B).…”

“…4[pSH17], cultivated in a fermenter at 30°C with constant air supply, and analyzed by psoralen cross-linking and EM as previously described (10). The adapter was constitutively expressed under the control of the weak TEF2 promoter (21). Expression of the R recombinase was controlled by the inducible GAL1 promoter (28).…”