Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains

Abstract

Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving DNA sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of the fission yeast Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their diverse activities. Such complexity has so far precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional in vitro reconstitution approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in budding yeast Saccharomyces cerevisiae cells. The mechanism conferring memory upon these cells is remarkably simple and requires only two proteins, one that recognizes histone H3 lysine 9 methylation (H3K9me) and catalyzes the deacetylation of histone H4 lysine 16 (H4K16), and another that recognizes deacetylated H4K16 and catalyzes H3K9me. Together, these bilingual "read-write" proteins form an interdependent positive feedback loop that is sufficient for the transmission of DNA sequence-independent silent information over multiple generations.

Keywords: HP1; SIR complex; epigenetic inheritance; heterochromatin; histone deacetylation.

Fig. 1.

Establishment of H3K9me-dependent gene repression in S. cerevisiae cells. (A) Cartoon schematic of how HMR was replaced by a tetO_10X-ADE2 reporter via homologous recombination and how DNA sequence–specific recruitment of TetR-SET—consisting of bacterial TetR fused to the catalytic SET domain of human SUV39H2—results in continuous local H3K9me (red spheres). TetR-SET is depicted as a dimer bound to a single tet operator. H3K9me ceases in the presence of anhydrotetracycline (aTc), which untethers TetR-SET from the tet operator array. (B) ChIP-seq profile of H3K9me2 at the tetO_10X-ADE2 reporter in cells producing TetR-SET. Data ranges (in counts per million) are indicated in brackets. The region visualized encompasses ~50 kb of the right arm of chromosome III. The H3K9me2 profile shown in red encompasses 1,150 bp upstream and 1,825 bp downstream of tetO_10X. The input DNA profile is shown in gray. The position of tetO_10X is marked by a white square, and DNA features including select genes are indicated by arrows. (C) ChIP-qPCR analysis of H3K9me2 levels at the tetO_10X-ADE2 reporter in cells producing TetR or TetR-SET. Primers (half-headed arrows) bind to unique DNA sequences (black rectangle) adjacent to tetO_10X (white square). TetR-SET-producing cells were cultured in the presence or absence of aTc for 6 h. Values represent the averages and SD of three biological replicates. The level of H3K9me2 at the reporter locus in aTc-treated cells approached the limit of detection. (D) Cartoon depiction of Sir2^Sp-HP1—consisting of full-length S. pombe Sir2 fused to the chromodomain (CD), hinge region, and chromo shadow domain (CSD) of S. pombe Chp2—bound as a dimer to a dinucleosome containing two methylated H3K9 residues (red spheres). Arrows are drawn to indicate Sir2^Sp-mediated deacetylation of H4K16 and other lysines. (E) RT-qPCR analysis of ADE2 mRNA levels in cells producing TetR-SET and either HP1, Sir2^Sp-HP1, or a Sir2^Sp-HP1 variant lacking histone deacetylase activity (Sir2^Sp N247A-HP1). Values represent ADE2 levels (normalized to ACT1) in the indicated cells relative to ADE2 levels in reporter gene-containing cells producing TetR alone. Averages and SD of three biological replicates are shown. Significance was assessed by a two-tailed Student’s t-test (P ≤ 0.01). (F) Phenotypes of tetO_10X-ADE2 reporter-containing cells producing TetR-SET and either HP1, Sir2^Sp-HP1, Sir2^Sp N247A-HP1, a Sir2^Sp-HP1 variant containing a mutant chromodomain incapable of methyllysine recognition (Sir2^Sp-HP1 W199A), or a monomeric Sir2^Sp-HP1 variant lacking its CSD (Sir2^Sp -HP1ΔCSD). HP1, Sir2^Sp-HP1, and Sir2^Sp-HP1 variants were produced from pRS315, a low-copy plasmid encoding LEU2. Cells were spotted on leucine-lacking medium containing limiting adenine (10 μg/mL; Ade¹⁰). White dashed lines indicate sites of cropping between spots of cells grown and photographed on the same plate.

Fig. 2.

An H3K9me read–write protein allows for distinction between local gene repression and regional gene silencing in S. cerevisiae cells. (A) Schematic of the modified reporter and phenotypes of reporter-containing cells producing either TetR-SET and Sir2^Sp-HP1 alone (row one), or TetR-SET and Sir2^Sp -HP1 in combination with Suv39^χ, a Suv39^χ variant containing a mutant chromodomain incapable of methyllysine recognition (Suv39^χ W31G), or a Suv39^χ variant lacking its chromodomain (Suv39^cΔCD) (rows two through four). Suv39^χ and Suv39^χ variants were encoded on pRS315. Cells were spotted on leucine-lacking medium containing the indicated compounds. White dashed lines indicate sites of cropping between spots of cells grown and photographed on the same plate. (B) Cartoon depiction of how Suv39^χ—consisting of the CD and hinge region of S. pombe Clr4 fused to the catalytic SET domain of human SUV39H2—enables H3K9me spreading. Two Suv39^χ monomers that “read” a nucleosome containing two methylated H3K9 residues (red spheres) and “write” H3K9me on adjacent nucleosomes are illustrated. (C) ChIP-qPCR analysis of H3K9me2 levels at the URA^Kl-tetO_10X-ADE2 reporter and sites located ~3 kb away from tetO_10X. Primer binding sites are indicated by half-headed arrows. Cells produce TetR-SET, Sir2^Sp -HP1, and either Suv39^χ (pink) or Suv39^χ W31G (white). Suv39^χ-producing cells were cultured in the presence (gray) or absence (pink) of aTc for 6 h. Values represent the averages and SD of three biological replicates.

Fig. 3.

An interdependent positive feedback loop maintains DNA sequence–independent gene silencing in S. cerevisiae cells. (A) Phenotypes of URA3^Kl-tetO_10X-ADE2 reporter-containing cells producing TetR-SET, Sir2^Sp -HP1, and either Suv39^χ (row one) or Sir3-SET. Sir3-SET was produced in sir3Δ cells (row two) or sir2Δ sir3Δ sir4Δ (sir⁻) cells (row three). Suv39^χ and Sir3-SET were encoded on pRS315. Cells were spotted on leucine-lacking medium containing the indicated compounds. White dashed lines indicate sites of cropping between spots of cells grown and photographed on the same plate. (B) Phenotypes of URA3^Kl-tetO_10X-ADE2 reporter-containing sir⁻ cells producing TetR-SET and Sir2^Sp-HP1 alone, or TetR-SET and Sir2^Sp -HP1 in combination with either Sir3, Sir3-SET, a Sir3-SET variant harboring a mutation that prevents Sir3 acetylation and disrupts bromo adjacent homology domain structure (Sir3 A2G-SET), or a monomeric Sir3-SET variant lacking its wH domain (Sir3ΔwH-SET). Sir3, Sir3-SET, and Sir3-SET variants were encoded on pRS315. Cells were spotted on leucine-lacking medium containing the indicated compounds. White dashed lines indicate sites of cropping between spots of cells grown and photographed on the same plate. (C) Cartoon depiction of the interdependent positive feedback loop formed by a pair of bilingual “read–write” proteins: Sir2^Sp-HP1 and Sir3-SET. Sir3-SET is shown bound as a dimer to a dinucleosome containing two unmodified H4K16 residues. The Sir3 wH domain mediates Sir3-SET dimerization, and the Sir3 bromo adjacent homology (BAH) domain mediates the recognition of unmodified H4K16 by Sir3-SET. Sir3-SET methylates nearby H3K9 residues of the dinucleosome to which it is bound (as illustrated) as well as H3K9 residues of flanking nucleosomes (not illustrated). Two newly methylated H3K9 residues (red spheres), in turn, recruit the Sir2^Sp-HP1 dimer (Fig. 1D). Sir2^Sp-HP1 deacetylates nearby H4K16 residues (and other lysines) of the dinucleosome to which it is bound (as illustrated) in addition to H4K16 (and other lysines) of flanking nucleosomes (not illustrated). Two newly deacetylated H4K16 residues, in turn, recruit the Sir3-SET dimer. Arrows are drawn to indicate either Sir3-SET-mediated methylation of H3K9 or Sir2^Sp-HP1-mediated deacetylation of H4K16 and other lysines. (D) ChIP-qPCR analysis of H3K9me2 levels at the URA^Kl-tetO_10X-ADE2 reporter and sites located ~3 kb away from tetO_10X. Primer binding sites are indicated by half-headed arrows. Cells are sir⁻ and produce TetR-SET, Sir2^Sp-HP1, and Sir3-SET. Cells were cultured for 6 h in the presence (gray) or absence (white) of aTc. Values represent the averages and SD of three biological replicates. (E) ChIP-seq profiles of H3K9me2 and Sir3-SET in URA3^Kl-tetO_10X-ADE2 reporter-containing sir⁻ cells producing TetR-SET and Sir2^Sp-HP1. Sir3-SET contains a FLAG_3X epitope tag. Data ranges (in counts per million) are indicated in brackets. The whole genome is visualized, with telomere junctions for all 16 chromosomes indicated on the hash-marked line. H3K9me2 profiles are shown in red, the Sir3-SET profile is shown in black, and input DNA profiles are shown in gray. The H3K9me2 profile of reporter-containing sir3Δ cells producing TetR-SET, Sir2^Sp -HP1, and Suv39^χ is shown for comparison. Black and gray arrowheads point to the reporter locus and rDNA repeats, respectively. (F) ChIP-seq profiles of H3K9me2 and Sir3-SET in sir⁻ cells containing the URA3^Kl-tetO_10X-ADE2 reporter in place of HFL1. In addition to Sir3-SET, which contains a FLAG_3X epitope tag, cells also produce TetR-SET and Sir2^Sp-HP1. Data ranges (in counts per million) are indicated in brackets. The region visualized encompasses ~30 kb of chromosome XI centered about tetO_10X. The position of tetO_10X is indicated by a white rectangle, and select genes are indicated by arrows. MRS4, which is naturally positioned adjacent to HFL1, is highlighted in pink. (G) RT-qPCR analysis of MRS4 mRNA levels in sir⁻ cells containing the URA3^Kl-tetO_10X-ADE2 reporter in place of either HMR or HFL1. Cells produce TetR-SET, Sir2^Sp-HP1, and Sir3-SET. Values represent MRS4 levels (normalized to ACT1) in cells carrying the reporter in place of the indicated locus relative to MRS4 levels in reporter-containing cells producing TetR alone. Averages and SD of three biological replicates are shown. Significance was assessed by a two-tailed Student’s t-test (P ≤ 0.001).

Fig. 4.

Reporter gene silencing is heritable but intrinsically unstable. (A) Phenotypes of sir⁻ cells producing Sir2^Sp -HP1 and Sir3-SET in the presence or absence of TetR-SET-encoding DNA. Three strains lacking TetR-SET (Δ #1–3) isolated on and maintained in FOA-containing medium are shown. Cells were grown in the presence of FOA and spotted on medium containing FOA or Ade¹⁰. (B) Silent information can propagate over multiple generations. Colonies—established by a Δ #1, Δ #2, or Δ #3 FOA-resistant founder cell—grown without selection either contain some fraction of FOA-resistant cells (red letter R) or contain FOA-sensitive cells only (black letter S). FOA-resistant daughter cells can be traced from round to round and emerge only from FOA-resistant mother cells, as evidenced by the Δ #1 lineage. Cells are unlikely to acquire resistance to FOA spontaneously, and once resistance to FOA is lost, it becomes irretrievable, as exemplified by the Δ #3 lineage. (C) For 20 Round 1 colonies grown on adenine-replete medium lacking FOA, the fraction of FOA-resistant (FOA^R), pigment-accumulating cells per colony was determined (pink), and the probability (P) of silent information loss per generation was calculated (gray) (Materials and Methods). The final calculated probability of silent information loss per generation represents the average and SD of P for all 20 colonies.