Background

Eukaryotic chromatin structure is broadly divided into euchromatin and heterochromatin (Cheung and Lau, 2005), with heterochromatin structure further subdivided depending on the combination of histone post-translational modifications (PTMs). These PTMs alter not only the chromatin conformation but also establish direct regulatory roles in gene expression and protein recruitment (Felsenfeld and Groudine, 2003; Allshire and Madhani, 2017). Myriad combinations of histone PTMs–including acetylation, phosphorylation, methylation, ubiquitination, biotinylation, sumoylation, and proline isomerization, collectively known as the “histone marks”–can be found, particularly on the unstructured N-terminal tail protruding from the nucleosomal core (Guetg and Santoro, 2012). These PTMs regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair (Suganuma and Workman, 2011), through the activities of different “readers” or effector proteins (Musselman et al., 2012). Thus, large efforts have been made to identify the readers for histone modifications.

Studying the interactions between reader proteins and their target histone PTMs using conventional methods (e.g., surface plasmon resonance [SPR] and SPR imaging [SPRi] biosensors) often requires large amounts of substrates or complex, multistep experimental methods, and is complicated by the various method-specific limitations. These concerns preclude the ease and accuracy of quantifying the strength of an interaction (Phizicky and Fields, 1995; Berggard et al., 2007; Rowley and Corces, 2016; Wierer and Mann, 2016). In recent work, we employed bio-layer interferometry (BLI) octet methodology (Kamat and Rafique, 2017; Petersen, 2017) to elucidate the binding between fission yeast Swi6, the counterpart of the human heterochromatin protein 1, and dimethylated histone H3 lysine 9 (H3K9me2) in the presence or absence of a phosphorylation moiety on tyrosine 41 residue on the histone H3 N-terminus (Ren et al., 2018). BLI is an optical technique for real-time measurement of macromolecular interaction. This aim is achieved via the analysis of interference patterns of the white light that is reflected off the biosensor surface. In a typical BLI experiment, the ligand is immobilized on the biosensor tip and then allowed to interact with the analyte (for example, a protein). The binding of the analyte to the immobilized ligand will increase the thickness of the biological layer at the surface of the biosensor tip resulting in a shift in the interference pattern, which is then documented in real time.

Akin to SPRi biosensors, BLI sensors do not entail isotopic labeling. Label-free biosensors offer a significant advantage when studying PTM-reader interactions, as small modification groups–such as a label–could affect the binding affinity of specific reader proteins. The use of label-free biosensors thus avoids any bias generated using labels with a different molecular mass. Although BLI technology has some limitations where small-sized molecules are concerned, its high flexibility and robustness for the simultaneous analysis of multiple (96) independent analyte-ligand pairs offer significant advantages. The disposable biosensors also allow for ad hoc replacement and real-time re-loading of the analytes or ligand substrate arrays (Abdiche et al., 2008). The BLI technology uses a non-fluidic system of dipping the sensors into a well plate instead of delivering the sample liquid to the sensor. This change in sample delivery not only increases the robustness of the assay but also decreases the operating costs (Nirschl et al., 2011).

Here, we present a simple, quick, and highly sensitive method for the detection of protein interactions using a synthesized peptide as bait and a recombinant protein in the BLI octet approach, in the determination of interaction kinetics of a histone PTM (H3K9me2) with its reader protein (Swi6). The protocol below describes detailed procedures for bacterial expression, extraction, and purification of recombinant protein Swi6, followed by the set-up of BLI octet, detection of Swi6 interaction with biotinylated peptide (H3K9me2), and subsequent processing and analysis of the readout data.