Interactions With Histone H3 & Tools to Study Them

Abstract

Histones are an integral part of chromatin and thereby influence its structure, dynamics, and functions. The effects of histone variants, posttranslational modifications, and binding proteins is therefore of great interest. From the moment that they are deposited on chromatin, nucleosomal histones undergo dynamic changes in function of the cell cycle, and as DNA is transcribed and replicated. In the process, histones are not only modified and bound by various proteins, but also shuffled, evicted, or replaced. Technologies and tools to study such dynamic events continue to evolve and better our understanding of chromatin and of histone proteins proper. Here, we provide an overview of H3.1 and H3.3 histone dynamics throughout the cell cycle, while highlighting some of the tools used to study their protein-protein interactions. We specifically discuss how histones are chaperoned, modified, and bound by various proteins at different stages of the cell cycle. Established and select emerging technologies that furthered (or have a high potential of furthering) our understanding of the dynamic histone-protein interactions are emphasized. This includes experimental tools to investigate spatiotemporal changes on chromatin, the role of histone chaperones, histone posttranslational modifications, and histone-binding effector proteins.

ATRX proximal protein associations boast roles beyond histone deposition

“…We theorized that a proximity-based protein labeling methodology in live cells would capture ATRX protein-protein associations that are not represented when using biochemical means. We therefore made use of proximity-dependent biotinylation (BioID) [ 42 , 43 ] to identify proteins that directly or indirectly associate with the ATRX protein ( Fig 1A ). ATRX was fused to a FLAG-tagged BirA* biotin ligase at either the N- or C-terminus, and expressed from isogenic, tetracycline-inducible HEK293 Flp-In T-REx cells (herein denoted as HEK293 Flp-In).…”

“…Comprehensive biochemical characterizations of ATRX protein-protein interactions have been invaluable, but are limited to a few stable interactions (i.e., DAXX, the MRN complex) that remain associated after harsh extractions [ 30 , 31 , 41 ]. We therefore screened ATRX protein-protein associations using proximity-dependent biotinylation (BioID) [ 42 , 43 ], which tags proteins (prey) that are proximal to a protein of interest (bait) in live cells. Our experiments confirmed robust associations between ATRX and DAXX, as well as with the MRN complex.…”

Activation of TC10-Like Transcription by Lysine Demethylase KDM4B in Colorectal Cancer Cells

“…Transcriptional events in mammalian cells are programmed by the coordinated actions between sequence-specific transcription factors and epigenetic co-factors. The epigenetic machinery, including histone and DNA modifying enzymes ( Audia and Campbell, 2016 ), chromatin remodeling proteins ( Kaur et al, 2019 ; Centore et al, 2020 ), histone variants ( Cavalieri, 2020 ; Scott and Campos, 2020 ), and non-coding regulatory RNAs ( Fazi and Fatica, 2019 ; Zhan et al, 2020 ), plays a key role in the pathogenesis of CRC development and progression ( Lao and Grady, 2011 ). Generally speaking, promoters of actively transcribed genes are marked by high levels of acetylated histones ( Eberharter and Becker, 2002 ) and methylated H3K4 ( Shilatifard, 2012 ).…”

A common classification framework for histone sequence alterations in tumours: an expert consensus proposal

“…When pairs of these dimers are bound to DNA, they form a histone octamer, which corresponds to a nucleosome. A chain of nucleosomes is wrapped in a spiral called a solenoid, in which the nucleosomes are further stabilised by the linker histone H1 to support the chromatin structure [7,8].…”