Abstract
Histone H3 mutations in residues that cluster in a discrete region on the nucleosome surface around lysine 79 of H3 affect H3-K79 methylation, impair transcriptional silencing in subtelomeric chromatin, and reveal distinct contributions of histone H3 to various DNA-damage response and repair pathways. These residues might act by recruitment of silencing and DNA-damage response factors. Alternatively, their location on the nucleosome surface suggests a possible involvement in nucleosome positioning, stability and nucleosome interactions. Here, we show that the yeast H3 mutants hht2-T80A, hht2-K79E, hht2-L70S, and hht2-E73D show normal nucleosome positioning and stability in minichromosomes. However, loss of silencing in a subtelomeric URA3 gene correlates with a shift of the promoter nucleosome, while nucleosome positions and stability in the coding region are maintained. Moreover, the H3 mutants show normal repair of UV lesions by photolyase and nucleotide excision repair in minichromosomes and slightly enhanced repair in the subtelomeric region. Thus, these results support a role of those residues in the recruitment of silencing proteins and argue against a general role in nucleosome organization.
Highlights
Chromatin structure serves as a central regulator for DNAassociated cellular processes in eukaryotic cells, including transcription, replication, and repair
Histone H3 mutants maintain nucleosome positioning and stability in minichromosomes To investigate whether histone H3 mutations in close proximity to methylatable H3-K79 affect chromatin structure, we used yeast strains in which both genomic loci coding for histones H3 and H4 (HHT1-HHF1 and HHT2-HHF2) were disrupted and replaced with either an HHT2 wild-type or an hht2 mutant allele of H3 on a centromeric plasmid [26]
The relative expression levels of wildtype H3 and the H3 mutants were very similar [41]. To test whether these H3 mutations affect the stability of nucleosomes, nucleosome positioning, and nucleosome-nucleosome contacts, the strains were transformed with circular minichromosomes (YRpFT35 and YRpFT38; Figure 1 B) that were shown to have distinct chromatin structures in strains containing the wild type set of histone genes (S288c) [10]
Summary
Chromatin structure serves as a central regulator for DNAassociated cellular processes in eukaryotic cells, including transcription, replication, and repair. Eukaryotic genomes are folded in arrays of nucleosome cores connected by linker DNA and further condensed into compact fibers and additional levels of higher order structures. While nucleosome cores are formed by intranucleosomal histone-histone and histone-DNA contacts, the close proximity of nucleosomes in arrays and higher order structures may require internucleosomal interactions of histones and DNA as well as a contribution of nonhistone chromosomal proteins [1,2]. The tails may interact with the DNA or histones of adjacent nucleosomes, thereby contributing to higher order structures [4,5]. The histone fold domains show an irregular surface with a distinct charge distribution that has been implicated in nucleosome-nucleosome contacts to promote chromatin higher order structure formation [3,4,6]
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