Competition between Heterochromatic Loci Allows the Abundance of the Silencing Protein, Sir4, to Regulate <i>de novo</i> Assembly of Heterochromatin

<div><p>Changes in the locations and boundaries of heterochromatin are critical during development, and <i>de novo</i> assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. <i>De novo</i> assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of <i>DOT1</i>, the histone H3 lysine 79 (K79) methyltransferase, and <i>SET1</i>, the histone H3 lysine 4 (K4) methyltransferase, speed <i>de novo</i> assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of <i>de novo</i> establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. <i>yku70Δ</i> and <i>ubp10Δ</i> cells also speed <i>de novo</i> assembly, and like <i>dot1</i>Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed <i>de novo</i> establishment by liberating Sir4 from telomeres. Deleting <i>RIF1</i> and <i>RIF2</i>, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced <i>de novo</i> establishment in <i>yku70</i>Δ and <i>ubp10</i>Δ cells, but not in <i>dot1Δ</i> cells, suggesting that <i>YKU70</i> and <i>UBP10</i> regulate Sir4 availability by modulating subtelomeric silencing, while <i>DOT1</i> functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate <i>de novo</i> assembly of heterochromatin.</p>