Background

DNA-protein macrocomplexes are usually insoluble and lost while obtaining chromatin fractions (Lambert et al., 2009). To maximize DNA-protein recovery, cellular fractionation has been used in several organisms, such as Xenopus (Liang and Stillman, 1997; Khoudoli et al., 2008), Caenorhabditis elegans (Chu et al., 2006), humans (Du et al., 2006; Foltz et al., 2006; Foltz et al., 2009), rice (Tan et al., 2007) or budding yeast (Frei and Gasser, 2000; Kubota et al., 2011). In most cases, these studies have been accompanied by proteomic analyses conducted by mass spectrometry, including quantitative mass spectrometry (Kubota et al., 2011; Lambert et al., 2012). The chromatin immunoprecipitation method (ChIP) emerged as an alternative to the above-mentioned approaches and allows interactions to be identified between protein and genomic DNA regions (Aparicio et al., 2004). However, the use of formaldehyde for in vivo crosslinking renders this method less efficient for proteomic analyses (Metz et al., 2004). ChIP combined with mass spectrometry has also been used in mouse embryonic stem cells or other model organisms (Lambert et al., 2010; Engelen et al., 2015). Notably a method based on ChIP, termed mChIP, has also been developed including protein affinity purification from chromatin that is most useful for mass spectrometry analyses (Lambert et al., 2009 and 2010).

In budding yeast, many procedures use cell fractionation by including additional steps of zymolyase treatment to lyse cell wall of viable yeast cells and then, to obtain the spheroplast before nucleus and cytoplasm separation (Liang and Stillman, 1997; Frei and Gasser, 2000; Kubota et al., 2011). Finally, a method to obtain chromatin fractions to isolate nascent RNA has been recently developed in S. cerevisiae which, without following a prior zymolyase treatment, allows chromatin fractions depleted of rRNA and tRNA to be obtained (Carrillo Oesterreich et al., 2010).

However, the need of easy and scalable procedure to obtain highly chromatin-enriched fractions in S. cerevisiae led us to adapt and improve this methodology termed yChEFs for yeast Chromatin Enriched Fractions. This procedure does away with the need to perform the cell fractionation required to obtain the spheroplast by using zymolyase incubation and to achieve nucleus isolation, which greatly facilitates the procedure and makes chromatin isolation more efficient. In addition, yChEFs allows work to be done with small solution volumes, very useful if many samples need to be manipulated. This method, allows proteins to be isolated from non-crosslinked chromatin by avoiding later sonication steps after chromatin isolation, and can be combined with mass spectrometry for proteomic analyses.