A core nucleosome surface crucial for transcriptional silencing

Abstract

Transcriptional silencing in yeast provides a genetically tractable system for analyzing the formation and maintenance of heterochromatin, a transcriptionally repressive chromatin structure found in all organisms. The nucleosome constitutes the central structure of chromatin and comprises two chains each of histones H2A, H2B, H3 and H4. The structure of the nucleosome consists of a central globular core surrounded by outwardly protruding amino-terminal histone tails. We show that a specific surface of the assembled nucleosome core is required for silencing in yeast. This surface is located at a H3/H4 histone-fold motif and contains amino-acid side chains located on the nucleosome disk surface and on an adjacent surface that interacts with DNA. The side chains, identified from mutants in which all three forms of silencing (rDNA, telomere and silent mating locus silencing) are eliminated, are centered around Lys79 of histone H3, a residue methylated by the yeast Dot1 protein. Moreover, mutations in the genes encoding H3 (HHT1 and HHT2) and H4 (HHF1 and HHF2) mapping to spatially adjacent amino-acid residues affected the three forms of silencing distinctly, suggesting that specific interactions mediate each form of silencing. Several of the mutations that we identified resemble those in a cluster of previously identified mutations affecting a distinct histone-fold motif elsewhere in the nucleosome core. These two clusters relieve distinct forms of transcriptional repression (silencing versus repression resulting from lack of Swi/Snf chromatin remodeling activity).

Histone H3 Lysine 36 Methylation Antagonizes Silencing in Saccharomyces cerevisiae Independently of the Rpd3S Histone Deacetylase Complex

“…Consistent with these results, loss of methylation at H3-K79 causes increase of Sir2 and Sir3 occupancy at Y9 subtelomeric elements (van Leeuwen et al 2002). A previous screen for histone mutations identified several residues surrounding H3-K79, including H3-K79 itself, as important for silencing (Park et al 2002). This study identifies a nonoverlapping set of residues near H3-K79 that are important for anti-silencing.…”

“…This reporter construct has been found to be sensitive to mutations that cause the spread of silencing (R. M. Raisner and H. D. Madhani, unpublished results). The strain further contained deletions of the two gene pairs encoding for H3 and H4 (HHT1-HHF1 and HHT2-HHF2) complemented by a LYS2-marked centromeric plasmid containing the HHT1-HHF1 locus and an ADE2 reporter integrated at the chromosome VR telomere (TELVR) (Park et al 2002). We mutagenized the HHT2 gene by PCR amplification and introduced it into this strain as a TRP1-marked centromeric plasmid containing the HHT2-HHF2 gene pair using a gap repair method.…”

“…Strain YM2330 was generated from strain JPY16 described in Park et al (2002) by a plasmid shuffle to replace pDM9 (URA3 CEN HHT1-HHF1) with pJP11 (LYS2 CEN HHT1-HHF1) and integration of a PCR product containing the Candida albicans URA3 gene plus 50 bp of promoter 200 bp to the right of the 39 end of the tRNA gene at HMRa. SET2, EAF3, RCO1, SIR2, and DOT1 genes were then replaced in this strain with MX markers to generate strains YM2332, YM2333, YM2334, YM2331, and YM2450, respectively.…”

Application of mass spectrometry to the identification and quantification of histone post‐translational modifications

“…K79 and K77 lie in the L2 loop of H4. K79 was identified in a large scale genetic screen that isolated point mutations in histones H3 and H4 that disrupt silent chromatin structure in yeast [Park et al, 2002]. Many of these mutations cluster in a region on the surface of the nucleosome that includes histone H3 K79 van Leeuwen et al, 2002].…”

Histone Regulation in the CNS: Basic Principles of Epigenetic Plasticity

“…Although specific core amino acids located at the H3/H4 histone-fold motif have been shown to be important for aspects of transcriptional silencing in yeast (this core is centered around lysine 79 of H3 and may be important to nucleosomal stability) (Park et al, 2002), these data indicate a high level of functional redundancy in histone modifications that must be considered. With the advent of ChIP-seq, we are now beginning to gain a clearer picture of the relative distribution of histone marks throughout the mammalian epigenome, both basally and following periods of cellular activity.…”