Global regulation of heterochromatin spreading by Leo1

Abstract

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.

Keywords: Leo1; epigenetics; fission yeast; genome regulation; heterochromatin.

Figure 1.

Leo1 is required to prevent spreading of heterochromatin across an IRC boundary. (a) Schematic showing the position of the IRC1L:ura4⁺ insertion at centromere 1, relative to the outer repeats (otr), innermost repeats (imr), central domain (cnt), tRNA genes (red lines) and IRC elements (red triangles). (b) Assay for silencing at IRC1L:ura4⁺. Plates are non-selective (N/S) or supplemented with 5-FOA (+FOA); growth in the presence of 5-FOA indicates silencing of ura4⁺. (c) RT-qPCR analysis of IRC1L:ura4⁺ transcript levels relative to a control transcript act1⁺, normalized to wild-type. (d,e) ChIP-qPCR analysis of H3K9me2 levels at the IRC1L:ura4⁺locus relative to the act1⁺ gene, normalized to wild-type, in strains grown in the presence of 5-FOA (d), or overexpressing Swi6 (e). Data are averages of three biological replicates and error bars represent 1 s.d.

Figure 2.

Loss of PAF complex components, but not other transcriptional regulators, results in silencing at IRC1L:ura4⁺. (a,b) Assay for silencing at IRC1L:ura4⁺ in cells lacking factors involved in transcription elongation or transcription-coupled chromatin modification (a) or cells lacking PAFc components (b). Plates are non-selective (N/S) or supplemented with 5-FOA (+FOA). (c) RT-qPCR analysis of IRC1L:ura4⁺ transcript levels relative to a control transcript act1⁺, normalized to wild-type. Data are averages of three biological replicates and error bars represent 1 s.d.

Figure 3.

Identification of Leo1 genetic interactors. (a) Schematic of the SGA analysis. Wild-type or leo1Δ query strains (bearing the IRC1L:ura4⁺ reporter and overexpressing swi6⁺) were crossed with the deletion library, and growth of the progeny was measured by colony size and represented as log₂ values of the ratio of growth on selective media (+FOA or –URA) versus non-selective media, normalized to the median ratio (blue, small colonies; yellow, large colonies). (b) Examples of epistatic (upper panel) and suppressing (lower panel) interactions. The indicated mutants are circled. (c) Cluster analysis showing deletion mutants that suppress the leo1Δ phenotype at IRC1L:ura4⁺, or exhibit synthetic (aggravating) or epistatic interactions. The upper panel shows examples of mutants displaying no genetic interaction (neutral). Four replicates are shown for each experiment; blue indicates small colonies and yellow indicates large colonies. (d) IRC1L:ura4⁺ silencing assay to validate SGA analysis results for the indicated strains.

Figure 4.

Loss of Leo1 results in reduced H4K16ac levels at the IRC1 locus. (a) ChIP-qPCR analysis of H4K16ac levels at the endogenous IRC1 element relative to the act1⁺ gene, normalized to wild-type. (b,c) ChIP-qPCR analysis of levels of other transcription-associated chromatin marks, (b) H3K4me3 and (c) H4K12ac, at IRC1 relative act1⁺, normalized to wild-type. (d) ChIP-qPCR analysis of H4K16ac levels at IRC1 in strains lacking Swi6. (e) ChIP-qPCR analysis of Mst1-flag association with IRC1 relative to act1⁺, normalized to wild-type. (f) Assay for silencing at IRC1L:ura4⁺ in strains with or without Swi6 overexpression; plates are non-selective (N/S) or supplemented with 5-FOA (+FOA). (g) ChIP-qPCR analysis of Bdf2-flag association with IRC1 relative to act1⁺, and normalized to wild-type. Data are averages of three biological replicates and error bars represent 1 s.d.

Figure 5.

Tethering the histone acetyltransferase Mst1 is sufficient to suppress heterochromatin spreading in leo1Δ cells. (a) Schematic of the IRC1L:ura4:TetO-ade6⁺ locus, which contains an ade6⁺ reporter gene flanked by four TetO binding sites for recruitment of TetR-Mst1. (b) ChIP-qPCR analysis of H3K9me2 levels at the IRC1L:ura4:TetO-ade6⁺ locus relative to the act1⁺ gene, normalized to wild-type. (c) ChIP-qPCR analysis of TetR-Mst1 levels at the IRC1L:ura4:TetO-ade6⁺ locus relative to the act1⁺ gene. Data are averages of three biological replicates and error bars represent 1 s.d.

Figure 6.

Leo1 regulates heterochromatin spreading independently of boundary elements. (a) Assay for silencing at the ade6⁺:L5-ura4⁺ ectopic silencer locus. The schematic shows the arrangement of the locus comprising the L5 sequence (a 2.6 kb fragment of otr sequence) adjacent to a ura4⁺ gene inserted at the euchromatic ade6⁺ locus. Plates are non-selective (N/S), lacking uracil (−URA) or supplemented with limiting amounts adenine (LOW ADE). Silencing of ura4⁺results in loss of growth on –URA; silencing of ade6⁺ results in red rather than white colonies on LOW ADE. (b) RT-qPCR analysis of ura4⁺and ade6⁺transcript levels relative to a control transcript act1+, normalized to wild-type. (c) ChIP-qPCR analysis of H3K9me2 levels at ura4⁺and ade6⁺relative to act1⁺, normalized to wild-type. Data are averages of three biological replicates and error bars represent 1 s.d.

Figure 7.

Leo1 functions as a global regulator of heterochromatin spreading. (a,b) Genome browser views showing ChIP-seq analysis of H3K9me2 levels in wild-type (blue) and leo1Δ (green) cells in log2 scale. leo1Δ/wt ratios are shown in black in linear scale. In each case, genome annotation is shown below; in the schematic in (a) red lines indicate the positions of relevant tRNA genes. The positions analysed by ChIP-qPCR are indicated in purple. (c,d) ChIP-qPCR analysis of H3K9me2 levels at the indicated loci relative to total H3, normalized to wild-type. (e) RT-qPCR analysis of SPAC186.05c⁺ transcript levels relative to a control transcript act1⁺, normalized to wild-type. (f) ChIP-qPCR analysis of H4K16ac levels at the SPAC186.05c⁺ locus relative to total H4, normalized to wild-type. ChIP-seq data represents the average of two biological replicates; other data are averages of three biological replicates and error bars represent 1 s.d.