Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins—Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2–4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2–4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

异染色质是一个沉默的染色质区域，对于维持基因组稳定性和驱动发育过程至关重要。异染色质的复杂结构和动力学使其难以表征。在芽殖酵母中，异染色质组装需要 SIR 蛋白——Sir3（被认为是 SIR 异染色质的主要结构成分）和 Sir2-4 复合体，负责 SIR 蛋白的定向募集和组蛋白 H4 赖氨酸 16 的脱乙酰化。之前，我们发现 Sir3 结合但不压缩核小体阵列。在这里，我们用完整的 SIR 蛋白重建染色质纤维，并使用沉降速度、分子模型和原子力显微镜来表征 SIR 染色质纤维的化学计量和构象。与单独使用 Sir3 的光纤相比，我们的结果表明 SIR 阵列非常紧凑。引人注目的是，SIR 异染色质纤维的浓缩结构需要 H4K16 的完整性以及 Sir3 和 Sir4 之间的相互作用。我们提出了一个模型，其中 Sir3 的二聚体桥接并稳定两个相邻的核小体，而 Sir2-4 异四聚体与与核小体三聚体相关的 Sir3 相互作用，驱动纤维压缩。