Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination

SPT6 is both, an important histone chaperone and POL2 elongation factor but its primary role on transcription in mammalian cells remains open, as no acute depletion system is available. We used targeted protein degradation to rapidly deplete SPT6 and analyzed defects in POL2 behavior by a multi-omics approach and estimated POL2 processivity, elongation rates and termination and compared it to gene transcription. Our data indicate that SPT6 is a crucial factor for POL2 elongation. Unexpectedly, SPT6 has also a vital role in POL2 termination, as acute depletion induces POL2 read through at most protein coding genes. In contrast, acute depletion did not induce spurious intragenic initiation, while this behavior can be induced by long-term depletion of SPT6 and can therefore be attributed to its function as a histone chaperone. In conclusion, targeted protein degradation of SPT6 allows the kinetic discrimination of its function as a histone chaperone and POL2 elongation factor. 4sU-seq, ChIP-seq (SPT6, Ser2 Pol2 and Total Pol2), DRB-4sU-seq and SLAM-seq in presence or absence of SPT6 ChIP-seq (CSTF2,H3,TFIIF,TBP,pH2AX) in presence or absence of SPT6

SPT6既是一类重要的组蛋白伴侣（histone chaperone），同时也是RNA聚合酶II（POL2）延伸因子，但由于尚无可用的急性降解系统，其在哺乳动物细胞中转录过程中的核心功能仍未明确。我们采用靶向蛋白质降解技术快速降解SPT6，并通过多组学方法分析POL2的行为缺陷，同时量化POL2的持续合成能力、延伸速率与终止效率，并将其与基因转录情况进行对比。我们的数据表明，SPT6是POL2延伸过程中的关键因子。出乎意料的是，SPT6在POL2终止过程中同样发挥至关重要的作用：急性降解SPT6会导致绝大多数蛋白编码基因出现POL2通读现象。与之相反，急性降解SPT6并未引发虚假的基因内起始，而这一现象仅在长期降解SPT6时才会出现，因此可将其归因于SPT6作为组蛋白伴侣的功能。综上，通过靶向蛋白质降解技术降解SPT6，能够实现对其作为组蛋白伴侣与POL2延伸因子的功能进行动力学区分。本研究包含的测序实验包括：存在或缺失SPT6条件下的4sU-seq、ChIP-seq（检测SPT6、Ser2磷酸化POL2与总POL2）、DRB-4sU-seq及SLAM-seq；以及存在或缺失SPT6条件下的ChIP-seq（检测切割刺激因子2（CSTF2）、组蛋白H3（H3）、转录因子IIF（TFIIF）、TATA框结合蛋白（TBP）、磷酸化组蛋白H2AX（pH2AX））。