Approaches to Study Native Chromatin-Modifying Complex Activities and Functions

Abstract

The modification of histones—the structural components of chromatin—is a central topic in research efforts to understand the mechanisms regulating genome expression and stability. These modifications frequently occur through associations with multisubunit complexes, which contain active enzymes and additional components that orient their specificity and read the histone modifications that comprise epigenetic signatures. To understand the functions of these modifications it is critical to study the enzymes and substrates involved in their native contexts. Here, we describe experimental approaches to purify native chromatin modifiers complexes from mammalian cells and to produce recombinant nucleosomes that are used as substrates to determine the activity of the complex. In addition, we present a novel approach, similar to the yeast anchor-away system, to study the functions of essential chromatin modifiers by quickly inducing their depletion from the nucleus. The step-by-step protocols included will help standardize these approaches in the research community, enabling convincing conclusions about the specificities and functions of these crucial regulators of the eukaryotic genome.

Recruitment of a splicing factor to the nuclear lamina for its inactivation

“…The same approach was subsequently applied in a proof-of-principle study in Drosophila melanogaster to study loss-of-function phenotypes of nuclear proteins 84 . A similar strategy has been applied in mammalian cells, involving abscisic acid-mediated dimerization of phytohormone factor fusion proteins, with the results suggesting that it could be used to probe the function of chromatin-modifying proteins with reduced side effects 85 . However, most of these alternative strategies either require extensive washout to reverse the effects or the effects cannot be efficiently reversed due to a high affinity of the chemical dimerizer to the target protein fusion (e.g., in the case of rapamycin) 86 .…”

“…In our study, over-production of RPL13-ECFP-SNAP led to an accumulation of the anchor fusion construct in nucleoli, presumably because it was not efficiently or completely integrated into ribosomal complexes for nuclear export. While CRISPR/Cas9-based SNAP-tagging of endogenous RPL13 may in principle prevent these effects, homozygous tagging of RPL13 could not be achieved in a past study 85 , possibly because of loss of RPL13 functionality in the ribosome.…”

The TUDOR domain of SMN is an H3K79me1histone mark reader

“…Introduction of C110A and K79C mutations in xH3 were done by site-directed mutagenesis using QuikChange (Stratagene) and the plasmid was sequence-verified. Recombinant histones were purified from E. coli , and modified where indicated, prior to octamer assembly and subsequent refolding of recombinant nucleosome core particle (rNCP) with a 151 base pair 601 Widom DNA as previously described (50,51). Briefly, the histones were purified from inclusion bodies under denaturing conditions on a 5 mL HiTrap SP FF (GE Healthcare) cation exchange column on a Next Generation Chromatograph (NGC, Bio-Rad).…”

Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active gene

“…Recombinant nucleosome core particles (rNCP) have been produced from recombinant histones as previously described (Dyer, Edayathumangalam et al 2004) (Galloy, Lachance et al 2021). Briefly, after preparation of inclusion bodies, the histones were purified under denaturing conditions on a 5 ml HiTrap SP FF (Cytiva) cation exchange column.…”

“…Octamers were refolded by mixing the four histones in equimolar ratios, followed by dialysis into 2M NaCl, 10 mM Tris pH7.5, 1 mM EDTA, and then purified on a Superdex 200 HiLoad 16/600 size exclusion column (Cytiva). Recombinant nucleosome core particles (rNCPs) were reconstituted as described (Galloy, Lachance et al 2021) (Dyer, Edayathumangalam et al 2004). The 601 Widom DNA sequence with a 72 bp linker used to wrap the mononucleosomes was a gift from B. Bartholomew.…”