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特性使其难以表征。在发芽酵母中，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复合物，负责SIR蛋白的靶向募集和组蛋白H4赖氨酸16的脱乙酰化。以前，我们发现Sir3结合但不能压缩核小体阵列。在这里，我们用SIR蛋白的完整互补物重构染色质纤维，并使用沉降速度，分子模型和原子力显微镜表征SIR染色质纤维的化学计量和构象。与仅使用Sir3的光纤相反，我们的结果表明SIR阵列非常紧凑。令人惊讶的是，SIR异染色质纤维的压缩结构既需要H4K16的完整性，又需要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了两个相邻的核小体，而Sir2-4异源四聚体与与核小体三聚体相关的Sir3相互作用，从而驱动纤维紧实。