Unlocking the chromatin code by deciphering protein– DNA interactions

Abstract

Characterizing the composition of protein complexes bound to different genomic loci is essential for advancing our mechanistic understanding of transcriptional regulation. In their recent study, Krijgsveld and colleagues (Rafiee et al, 2016) report ChIP‐SICAP, a powerful tool for deciphering the chromatin proteome by combining chromatin immunoprecipitation, selective isolation of chromatin‐associated proteins and mass spectrometry.

Decoding Cinnabarinic Acid–Specific Stanniocalcin 2 Induction by Aryl Hydrocarbon Receptor

“…2). Surprisingly, we did not detect H4 K5 acetylation with high confidence possibly due to the limitation of signal detection by MS, low abundance or loss during sample preparation (Bensaddek and Lamond, 2016). Nevertheless, xChIP-Western blotting successfully confirmed presence of H4 K5ac at AhR-bound chromatin exclusively upon CA treatment (Fig.…”

Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

“…Following extensive washing, the cross-link is reversed and proteins are trypsin digested and identified by mass spectrometry. 108 The effectiveness of ChIP-SICAP was demonstrated by characterizing the chromatin-bound network around Oct4, Sox2, and Nanog in mouse ESCs, the so-called OSN system, leading to the discovery of Trim24 as a component of the pluripotency network. ChIP-SICAP uniquely benefits from the double purification of protein−DNA complexes, accomplished by subsequent ChIP of the protein of interest and pull-down of biotinylated DNA allowing the exclusive capture of protein complexes bound to DNA.…”

“…DNA fragments are then biotinylated and chromatin is retrieved along with interacting proteins on streptavidin beads. Following extensive washing, the cross-link is reversed and proteins are trypsin digested and identified by mass spectrometry …”