Chromatin immunoprecipitation assay for analyzing histone H3-mediated regulation of copper homeostasis.

Histone tails undergo various post-translational modifications that aid in recruiting effector proteins involved in diverse transcriptional responses. Our previous work explored the role of histone H3 N-terminal residues and their modifications on the transcriptional regulation of the CUP1 metallothionein gene during cellular copper stress. To pinpoint critical histone residues, we used complementary methods, including growth assays and gene expression kinetics. We screened a library of synthetic histone H3 and H4 mutants for copper response defects by assessing their sensitivity to copper stress and quantifying CUP1 transcript levels via real-time PCR. We further investigated the mechanisms of impaired CUP1 transcription by assessing the recruitment of Ace1, a copper-sensing transcription factor that activates CUP1 in response to high intracellular copper, and of TATA-binding protein (TBP), using chromatin immunoprecipitation (ChIP) assays. Our results showed that mutations in the H3 N-terminal tail impair the recruitment of Ace1 and TBP to the CUP1 promoter, causing defects in its induction. Collectively, these findings highlight the significance of histone residues and their modifications in regulating metal homeostasis. This chapter outlines the methods used to study histone-mediated regulation of copper homeostasis, with a focus on the chromatin immunoprecipitation assay.