Methods to Study Histone Chaperone Function in Nucleosome Assembly and Chromatin Transcription

Abstract

Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

14–3‐3γ prevents centrosome duplication by inhibiting NPM1 function

“…The different His-tagged NPM1 proteins were purified by Ni-NTA-based affinity purification method as per protocol described previously (Gadad et al, 2010;Senapati et al, 2015;Swaminathan et al, 2005). Briefly, E. coli BL-21 (DE3) competent cells were transformed with the respective plasmids, cultured at 37°C, 180 rpm and induced with 0.5 mM BOSE Et al.…”

“…The nucleosome assembly activity of wild-type nucleophosmin (NPM1 WT), the phospho-deficient mutant (NPM1 S48A) and phospho-mimic mutant (NPM1 S48E) was assessed by the in vitro histone transfer or supercoiling assay as described previously, with a few modifications (Senapati et al, 2015). Briefly, 200 ng of supercoiled G 5 ML plasmid was relaxed using Drosophila Topoisomerase I (dTopoI) in 1X assembly buffer (2X buffer composition: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl and 0.1 mg/ml BSA (Sigma)) for 40 min at 37°C.…”

Interactions With Histone H3 & Tools to Study Them

“…There are, of course, several other specialized techniques to quantify and qualify nucleosome assembly, such as electron microscopy (EM) and single-molecule techniques (see Duzdevich and Greene, 2013;Schwartzman and Tanay, 2015;Senapati et al, 2015).…”

Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

“…L18Q NPM1 was less efficient in pulling down histone H3 as compared to WT NPM1 ( Figure 4D). We next compared the histone chaperone activity of WT NPM1, L18Q NPM1 and Y17T-C21F NPM1 using a histone transfer assay (Senapati et al 2015) with increasing protein amounts. Both the mutants displayed a reduced ability to deposit histones measured by the extent of supercoiling of plasmid DNA as compared to WT NPM1 ( Figure 4E, compare lanes 8, 11 versus 7 and 4F) at both protein concentrations tested ( Figure 4E, compare lanes 10, 12 versus 9 and 4F).…”

“…The results show that oligomerization of NPM1 protein is critical for its histone chaperone activity. We then tested the transcriptional activation property of the histone chaperone-deficient mutants of NPM1 using an acetylation-dependent chromatin transcription assay as indicated in ( Figure 4G) (Senapati et al 2015). The mutant proteins L18Q NPM1 and Y17T-C21F NPM1 were tested in two different concentrations in the transcription assay and compared with WT NPM1.…”