Global analysis for functional residues of histone variant Htz1 using the comprehensive point mutant library

Abstract

Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only 60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.

Chromatin and Transcription in Yeast

“…Genome-wide expression analysis of histone mutants has provided broader understanding of the impact of specific histone mutants (e.g., see Wyrick et al 1999;Sabet et al 2004;Dion et al 2005;Parra et al 2006;Parra and Wyrick 2007;Nag et al 2010). Recently, large-scale studies have systematically constructed and analyzed hundreds of mutations in histone genes, providing a comprehensive data set of the histone residues that are required for normal transcription in vivo (Matsubara et al 2007;Dai et al 2008;Nakanishi et al 2008;Seol et al 2008;Kawano et al 2011). A convenient resource for information on histones and histone mutants is the Histone Systematic Mutation Database (http://baderzone.net/v2/histonedb.html) (Huang et al 2009).…”

Key Functional Regions in the Histone Variant H2A.Z C-Terminal Docking Domain

“…Although different functions have been attributed to the C terminus of H2A.Z, the necessity of the docking domain has not been studied extensively with regard to H2A.Z binding to chromatin, cellular functions, and genetic interactions. A recent comprehensive alanine scan of yeast H2A.Z identified relatively few residues that resulted in sensitivity to genotoxic stressors (24). Pointing toward an important contribution of the C terminus for H2A histone family function, a recently published deletion analysis of human H2A identified its C terminus as being required for nucleosome stability, chromatin remodeling, and binding to histone H1 (50).…”

“…Despite our clear evidence for the last 20 amino acids being important for function, the C-terminal truncation approach employed here does not exclude the possibility of residues located before amino acid 114 also contributing to H2A.Z function. As such, an alanine scan of H2A.Z identified mutants with mutations in two residues, I109 and G113, in the C terminus that have mild sensitivity to genotoxic stressors (24), as do some mutants with mutations in the H2A.Z acidic patch region (20).…”

Roles of common subunits within distinct multisubunit complexes

“…Mutation of the H2A-Y58 and -E62 residues in nonfused H2A-and H2B-expressing cells caused cell death (23,24), whereas mutation of the Htz1-Y65 and -E69 residues in nonfused Htz1-and H2B-expressing cells resulted in HU/MMS/benomyl sensitivities (26). Thus, the proximity and functional relationship of H2B-L109 to H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 might explain why H2B-L109 is functionally important in both the H2B-H2A and H2B-Htz1 fusions.…”

“…H2B-L109 physically interacts with the H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 residues, which are exposed to the nucleosome surface (23,24,26). Mutation of the H2A-Y58 and -E62 residues in nonfused H2A-and H2B-expressing cells caused cell death (23,24), whereas mutation of the Htz1-Y65 and -E69 residues in nonfused Htz1-and H2B-expressing cells resulted in HU/MMS/benomyl sensitivities (26).…”