ABSTRACT
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique for genome-wide mapping of protein-DNA interactions and epigenetic marks in vivo. Recent studies have suggested an important role of heat shock protein 90 (Hsp90) at chromatin. This molecular chaperone assists other proteins to acquire their mature and functional conformation and helps in the assembly of many complexes. In this chapter, we provide specific details on how to perform Hsp90 ChIP-seq from Drosophila Schneider (S2) cells. Briefly, the cells are simultaneously lyzed and reversibly cross-linked to stabilize protein-DNA interactions. Chromatin is prepared from isolated nuclei and sheared by sonication. Hsp90-bound loci are immunoprecipitated and the corresponding DNA fragments are purified and sequenced. The described approach revealed that Hsp90 binds close to the transcriptional start site of around one-third of all Drosophila coding genes and characterized the role of the chaperone at chromatin.
Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/metabolism , Drosophila Proteins/metabolism , Heat-Shock Proteins/metabolism , Sequence Analysis, DNA/methods , Animals , Cell Line , DNA/metabolism , Drosophila melanogaster/metabolismABSTRACT
Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.