A novel yeast chromatin-enriched fractions purification approach, yChEFs, for the chromatin-associated protein analysis used for chromatin-associated and RNA-dependent chromatin-associated proteome studies from Saccharomyces cerevisiae

Abstract

We adapted and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. yChEFs allows the easy, reproducible and scalable recovery of proteins associated with chromatin, and bypasses subcellular fractionation requirements that involve using zymolyase to obtain the spheroplast. The use of a small amount of culture cells and small volumes of solutions during the procedure greatly facilitates high-enriched chromatin purification, which is very useful when many samples need to be manipulated. It also reduces costs and efforts. yChEFs can be combined with mass spectrometry for proteomic analyses. We used yChEFs and mass spectrometry to identify the chromatin-associated proteome in Saccharomyces cerevisiae, and detected about 750 chromatin-bound proteins, many of which can be detected by other previous approaches. We also used this methodology to identify the RNA-dependent chromatin-associated proteome and identified about 500 proteins, including: RNA polymerase subunits, transcription regulators, such as helicases Sen1 and Rat1 that participate in transcription termination, components of nucleosome modifying complexes, chromatin remodelers complexes, and some proteins involved in RNA processing, among them, Dcp2, Xrn1 and Dhh1, which act in mRNA processing. Finally, yChEFs identified weak or transient chromatin-associated proteins.