Construction of Comprehensive Dosage-Matching Core Histone Mutant Libraries for Saccharomyces cerevisiae

Abstract

contains two genes for each core histone, which are presented as pairs under the control of a divergent promoter, ,, , and , and encode histone H3 and H4 with identical amino acid sequences but under the control of differently regulated promoters. Previous mutagenesis studies were carried out by deleting one pair and mutating the other one. Here, we present the design and construction of three additional libraries covering ,, and respectively. Together with the previously described library of mutants, a systematic and complete collection of mutants for each of the eight core histone genes becomes available. Each designed mutant was incorporated into the genome, generating three more corresponding libraries of yeast strains. We demonstrated that, although, under normal growth conditions, strains with single-copy integrated histone genes lacked phenotypes, in some growth conditions, growth deficiencies were observed. Specifically, we showed that addition of a second copy of the mutant histone gene could rescue the lethality in some previously known mutants that cannot survive with a single copy. This resource enables systematic studies of function of each nucleosome residue in plasmid, single-copy, and double-copy integrated formats.

Synthetic refactor of essential genes decodes functionally constrained sequences in yeast genome

“…The Yeast genome Deletion Project and further functional profiling (Costanzo et al, 2010(Costanzo et al, , 2016Giaever et al, 2002;Kuzmin et al, 2018;Winzeler et al, 1999) have depicted a functional blueprint of yeast genes. Moreover, systematic targeted mutations have detailed the importance of each amino acid for regulating the functions of a protein (Dai et al, 2008;Jiang et al, 2017Jiang et al, , 2019. However, at the nucleotides level, how the synonymous codons, promoters, and terminators are selected to make a functional gene has not been systematically studied, thereby largely limiting our understanding about the genome sequence and our ability to engineer desired functions.…”

Genetic interaction mapping informs integrative structure determination of protein complexes

“…The state of the nucleosome is controlled by histone posttranslational modifications (PTMs) (21)-including acetylation, methylation, phosphorylation, and ubiquitination-that help maintain and regulate chromatin structure and transcription. Our library of point mutations in the core histones H3 and H4 was designed to comprise a comprehensive alanine scan, as well as contextspecific mutations of modifiable residues (e.g., lysine and arginine), such as charge removal or reversal and substitutions mimicking PTMs (22,23). Partial deletions of the N-terminal tails of H3 and H4 were also included, because these regions play important and sometimes redundant roles in chromatin biology (24,25).…”

“…The histone H3 and H4 mutant strain library was constructed essentially as described (22,23). Briefly, the mutants (tail deletions, complete alanine-scan, and context specific point mutations) were generated in the YMS196 background (MATa his3D leu2D ura3D can1:: STE2pr-spHIS5 lyp1::STE3pr-LEU2) (table S1).…”

A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis

“…The budding yeast Saccharomyces cerevisiae has been served as a model organism to explore histone functions, because of several advantages (i) its highly-conserved histone sequences and modification functions with higher eukaryotes, (ii) its only two copies of histone genes in haploid cell stage, (iii) its ability to survive and grow with only one copy of histone genes, and (iv) its availability of powerful genetic manipulation tools. Several strategies have been carried out for systematic histone mutation libraries in S. cerevisiae: (1) transforming an episomal plasmid containing histone mutants with both endogenous copies deleted, (2) mutating one copy of endogenous histones by homologous recombination (HR) with the other copy deleted, (3) mutating both endogenous copies of histones by HR using two different selection markers [9][10][11] . These strategies have been very helpful to study histone functions and modifications, however additional requirements have emerged for new tools.…”

“…These strategies have been very helpful to study histone functions and modifications, however additional requirements have emerged for new tools. Firstly, histone dosage effects have been discovered for some point mutations, thus previous identified crucial histone mutants by deleting one or two copies of endogenous histone may have different phenotypes with both endogenous copies mutated 11,12 . Secondly, multiple histone modifications have the same effect on gene expression or chromatin structure, and methods are needed for generating combinational mutagenesis on histones simultaneously.…”