Direct assessment of histone function using histone replacement

Abstract

Histones serve many purposes in eukaryotic cells in the regulation of diverse genomic processes, including transcription, replication, DNA repair, and chromatin organization. As such, experimental systems to assess histone function are fundamental resources toward elucidating the regulation of activities occurring on chromatin. One set of important tools for investigating histone function are histone replacement systems, in which endogenous histone expression can be partially or completely replaced with a mutant histone. Histone replacement systems allow systematic screens of histone regulatory functions and the direct assessment of functions for histone residues. In this review, we describe existing histone replacement systems in model organisms, the benefits and limitations of these systems, and opportunities for future research with histone replacement strategies.

Keywords: Arabidopsis; Drosophila; chromatin; epigenetics; unicellular eukaryotes.

Figure 1:. Functions for histone replacement mutants.

Mutated histones can 1) prevent the post-translational modification of histone residue(s), 2) inhibit recognition by a chromatin-binding protein, 3) partially or completely emulate a histone variant, and/or 4) mimic a constitutively modified state (e.g. lysine (K) to glutamine (Q) mutation to mimic the constitutively acetylated state of lysine residues). Importantly, as mutations in histones have been linked to human cancers (“oncohistones”) and developmental disorders, histone replacement systems have broad applications for studying disease mechanisms in vivo. Additionally, due to the high conservation of histone proteins across eukaryotes, histone replacement systems established in model organisms remain relevant for translational applications towards human disease research. So far, histone replacement systems have been implemented in several model organisms: the single-celled eukaryotes S. cerevisiae, S. pombe, T. thermophila, and T. brucei as well as the multicellular eukaryotes D. melanogaster and A. thaliana.

Figure 2:

Three types of histone replacement systems. (Top) Expression of a mutant histone in a wild-type background produces chromatin with minor insertion of mutant histones. (Middle) Partial histone replacement (some endogenous histone genes are mutated) produces chromatin with significant insertion of mutant histones. (Bottom) Complete histone replacement (all endogenous histone genes are mutated) produces chromatin that exclusively contains mutant histones. One major technical challenge with the generation of complete histone replacement systems is that the diploid genome of most organisms typically contains more than two copies encoding each histone.

Figure 3:. Strategy to generate histone H3 and H4 replacement mutants in S. cerevisiae.

First, synthetic H3 and H4 genes (containing the same protein sequences as endogenous H3 and H4 but different codons to minimize undesired homology-directed repair) are shuffled in to replace endogenous H3 and H4 in situ using CRISPR/Cas9. Next, synthetic H3 and H4 are targeted by CRISPR/Cas9 and donor templates containing endogenous H3 and H4 genes with the desired mutations are provided for repair, resulting in histone replacement mutants.

Figure 4:. Strategy for in situ histone cluster replacement in D. melanogaster.

The histone cluster deletion (His^D) line is generated through a multistep process. First, the attP-FRT cassette is knocked in using CRISPR/Cas9-mediated HR on either side of the replication-dependent histone gene cluster (right side contains attP-FRT duplication). Next, these two fly lines are crossed and flippase activity is induced to generate the His^D line through HR. Varying numbers of histone gene units (His-GUs) are then introduced into the His^D background in situ through the attP/attB integration system to generate histone replacement lines. Viable flies are recovered with the introduction of 8 His-GUs and flies with 12 or 20 His-GUs show similar histone mRNA and protein levels to wild-type flies.

Figure 5:. Strategy to generate H4 replacement mutants in A. thaliana.

Multiple guide RNAs (gRNAs) are used to simultaneously target all except one of the endogenous H4 genes for homozygous mutation and generate the H4 septuple mutant background. Sequence homology between H4 nucleotide sequences is taken advantage of to target multiple H4 genes with the same gRNA. H4 replacement plasmids, containing a replacement H4 gene and a gRNA targeting the remaining endogenous H4 gene are individually transformed into the H4 septuple mutant background. Resulting H4 replacement plants display a complete replacement of endogenous H4 with replacement H4 after selecting plants with homozygous or biallelic mutations in the remaining endogenous H4 gene.