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

Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo 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 DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo 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 de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 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 de novo assembly of heterochromatin.

异染色质（heterochromatin）的位置与边界变化在发育进程中发挥关键作用，而出芽酵母中沉默染色质的从头组装（de novo assembly）是研究异染色质新位点组装机制的经典模型。细胞周期（cell cycle）的G1期无法发生从头组装，完整的沉默染色质组装与转录抑制（transcriptional repression）需要经历1至2次细胞分裂。组蛋白H3赖氨酸79（K79）甲基转移酶（methyltransferase）DOT1与组蛋白H3赖氨酸4（K4）甲基转移酶SET1的突变可加速从头组装过程。上述观测结果支持了“组蛋白的调控性去甲基化（demethylation）可能是细胞调控异染色质建立的一种机制”这一模型。本研究发现，沉默染色质组装必需的蛋白质沉默信息调节因子4（Sir4）的丰度在G1期阻滞过程中显著下降，因此我们验证了改变Sir4的表达水平是否也会改变异染色质从头建立的速度。将Sir4的表达水平减半会延缓异染色质的建立过程，而提升Sir4的表达水平则会加速该过程。yku70Δ与ubp10Δ细胞同样可加速从头组装过程，且与dot1Δ细胞一样存在亚端粒沉默缺陷，这表明这些突变体可能通过将Sir4从端粒（telomeres）中释放，间接加速异染色质的从头建立。敲除RIF1与RIF2可抑制上述突变体的亚端粒沉默缺陷，该操作能够挽救yku70Δ与ubp10Δ细胞中提前发生的从头组装过程，但对dot1Δ细胞无效。这提示YKU70与UBP10通过调控亚端粒沉默来调节Sir4的可获得性，而DOT1则直接参与调控异染色质的建立过程。本研究的数据支持如下模型：组蛋白H3 K79的去甲基化以及Sir4丰度与可获得性的变化，构成了调控异染色质从头组装的两个限速步骤。