Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: a potential mechanism for position-effect variegation

Abstract

Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for association of the Sir silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein association and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This positive feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein association and leads to further methylation, suggests a model for position-effect variegation.

Figure 1

H3-K79 methylation is low at heterochromatic loci. (A) Relative levels of H3-K79 methylation at regions (A–L) at the indicated positions (drawn to scale) with respect to the telomeres and linked genes on chromosome VI. ORF-free regions on chromosomes I and VIII serve as controls and were arbitrarily defined as having H3-K79 levels of 1.0. (B) H3-K79 methylation in wild-type, dot1, and H3-K79 mutant strains at the indicated regions from the telomere of chromosome VI; note difference in scale. (C) H3-K79 methylation at the indicated regions of HMRa, HMRE, and rDNA loci, along with the ORF-free region controls.

Figure 2

H3-K79 methylation occurs at comparable levels at many nonheterochromatic loci, including those that are transcriptionally repressed and contain deacetylated histones. (A) Relative levels of H3-K79 methylation (black bars) and Myc-tagged histone H4 (gray bars) at the indicated positions of active and control loci. (B) H3-K79 methylation at the indicated genomic regions of inactive or repressed genes.

Figure 3

Hypomethylation of H3-K79 at the telomeric and HM, but not rDNA, loci depends on Sir proteins and Sir2 histone deacetylase activity. H3-K79 methylation at the indicated regions (see Fig. 1) in wild-type and sir mutant strains for telomeric (A), HM (regions 1, 3, 4, and 6) (B), and rDNA (C) loci.

Figure 4

Interplay between H4-K16 acetylation and H3-K79 methylation. (A) H3-K79 methylation at the indicated telomeric regions (see Fig. 1) in wild-type (white bars) and H4-K16Q mutant (gray bars) strains. (B) H4 acetylation (lysines 5, 8, 12, and 16) at the indicated telomeric regions in wild-type (white bars), dot1 (gray bars), and H3-K79 (black bars) mutant strains.

Figure 5

Overexpression of Sir3 concomitantly extends the telomeric domain of Sir3 and H3-K79 hypomethylation. (A) H3-K79 methylation at the indicated telomeric and control positions (see Fig. 1) in wild-type (white bars) and Sir3-overexpressed (black bars) strains. (B) Relative levels of Sir3 in wild-type (white bars) and Sir3-overexpressed (black bars) strains.

Figure 6

H3-K79 methylation localizes to active gene segments and correlates with H3 acetylation at IgH and TCRβ loci. H3-K79 methylation (fold-enrichment in the immunoprecipitated samples relative to the input) at V, D, J, C (constant), and E (enhancer) regions of the IgH and TCRβ loci, and at CAD, a ubiquitously expressed gene involved in pyrimidine biosynthesis. The gene segments are arranged in 5′ to 3′ orientation as they appear at the endogenous IgH (VH15, VH81X, DQ52, JH1, JH4, Eμ, and Cγ3) and TCRβ (Vβ10, Dβ1, Jβ1.2, Jβ1.5, Cβ1, Eβ, and Vβ14).

Figure 7

Model for maintaining stable on–off states. Positive-feedback loops for the on and off states involve the preferential recognition of chromatin with hypomethylated H3-K79 by Sir proteins and inhibition of Dot1-mediated methylation of H3-K79 by bound Sir proteins. Stochastic events that affect either Sir protein association or H3-K79 methylation can lead to switching between the two states.