Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes

Spt6-Spn1 is an essential histone chaperone complex that associates with RNA Polymerase II (RNAPII) and reassembles nucleosomes during gene transcription. While Spt6-Spn1 interaction is important to its histone deposition and transcription functions, a precise understanding of how Spt6-Spn1 association contributes to its functions is still limited. Here, using temperature sensitive alleles of spt6 and spn1 that disrupt their interaction, we show that Spt6-Spn1 association is broadly important for its interaction with the elongating RNAPII complex and nucleosomes. Using micrococcal nuclease (MNase)-based chromatin occupancy profiling, we further find that Spt6-Spn1 interaction is required for maintaining the preferred nucleosome positioning generated from replication-dependent histone deposition at actively transcribed genes. These analyses also reveal an overall shift of nucleosomes towards the 5’ end of genes that correlates with decreased RNAPII levels. While loss of Spt6 and Spn1 function is known to promote cryptic transcription, the nucleosome defects found in spt6 and spn1 mutants that interfere with their interaction were observed broadly across transcribed genes, further highlighting the role of DNA sequence as a mediator of cryptic transcription when nucleosomes positioning is altered. Taken together, these findings reveal Spt6-Spn1 interaction is key to its association with elongating RNAPII and for its ability to precisely organize nucleosomes across transcription units.Spt6-Spn1是一类必需的组蛋白伴侣复合物，可与RNA聚合酶II（RNA Polymerase II，RNAPII）结合，并在基因转录过程中重新组装核小体。尽管Spt6-Spn1相互作用对其组蛋白沉积与转录功能具有重要意义，但目前学界对Spt6-Spn1结合如何调控其功能的精准认知仍较为匮乏。本研究利用可破坏二者相互作用的spt6与spn1温度敏感等位基因，证实Spt6-Spn1结合对其与延伸态RNA聚合酶II复合物及核小体的相互作用具有广泛的必要性。通过基于微球菌核酸酶（micrococcal nuclease，MNase）的染色质占据谱分析，我们进一步发现，Spt6-Spn1相互作用是维持活跃转录基因中依赖复制的组蛋白沉积所形成的偏好性核小体定位的必要条件。此类分析还揭示，核小体整体向基因5'端发生移位，该现象与RNA聚合酶II水平下调呈相关性。尽管已知Spt6与Spn1的功能丧失会促进隐秘转录（cryptic transcription），但在干扰二者相互作用的spt6与spn1突变体中观察到的核小体缺陷广泛分布于所有转录基因中，这进一步凸显了当核小体定位发生改变时，DNA序列作为隐秘转录介导因子的关键作用。综上，本研究结果表明，Spt6-Spn1相互作用是其与延伸态RNA聚合酶II结合，以及精准调控转录单元内核小体排布的核心要素。