A Detailed Protocol for Chromatin Immunoprecipitation in the Yeast Saccharomyces cerevisiae

Abstract

Critical cellular processes such as DNA replication, DNA damage repair, and transcription are mediated and regulated by DNA-binding proteins. Many efforts have been invested therefore in developing methods that monitor the dynamics of protein-DNA association. As older techniques such as DNA footprinting, and electrophoretic mobility shift assays (EMSA) could be applied mostly in vitro, the development of the chromatin immunoprecipitation (ChIP) method, which allows quantitative measurement of protein-bound DNA most accurately in vivo, revolutionized our capabilities of understanding the mechanisms underlying the aforementioned processes. Furthermore, this powerful tool could be applied at the genomic-scale providing a global picture of the protein-DNA complexes at the entire genome.The procedure is conceptually simple; involves rapid crosslinking of proteins to DNA by the addition of formaldehyde to the culture, shearing the DNA and immunoprecipitating the protein of interest while covalently bound to its DNA targets. Following decrosslinking, DNA that was coimmunoprecipitated could be amplified by PCR or could serve as a probe of a genomic microarray to identify all DNA fragments that were bound to the protein.Although simple in principle, the method is not trivial to implement and the results might be misleading if proper controls are not included in the experiment. In this chapter, we provide therefore a highly detailed protocol of ChIP assay as is applied successfully in our laboratory. We pay special attention to describe every small detail, in order that any investigator could readily and successfully apply this important and powerful technology.

A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

“…These combined ChIP analyses provide important information about DNA-binding motifs and putative target genes, as well as the biological roles of proteins of interest, through functional analysis of their target sequences [ 9 – 12 ]. In addition to its utility for the study of transcriptional regulation, ChIP can be also used to map genome-wide epigenetic modifications via the histone modifiers [ 13 , 14 ].…”

Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

“…ChIP combined with microarray or massively parallel sequencing is used to study gene regulatory networks active during development and/or in response to the environment. ChIP is also a valuable tool for mapping genome wide epigenetic modifications such as histone marks, and has been used to characterize several eukaryotic genomes [7,8]. However, no ChIP protocol has been reported for marine phytoplankton.…”

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

“…The method is an adoption of a chromatin immuno-precipitation protocol of Grably and Engelberg (Grably and Engelberg, 2010). In brief, selected human targets and NRTKs were picked from an open reading frame (ORF) collection of gateway entry clones and were shuttled into the yeast expression vectors pRS425_GDP_TAP (Addgene).…”