NDF/GLYR1 Promotes RNA Polymerase II Processivity via Pol II Binding and Nucleosome Destabilization

The Nucleosome Destabilizing Factor (NDF) facilitates transcription through chromatin, but its precise mechanism remains incompletely understood. Here, we identify a critical region (amino acids 140-160) within NDF that specifically interacts with phosphorylated RPB1, the largest subu-nit of elongating RNA Polymerase II (Pol II). Mutations in this region disrupt Pol II interaction and impair Pol II elongation both in vitro and in cells, yet do not affect NDF's ability to destabilize nu-cleosomes, establishing a functional separation between these two activities. Cellular studies re-veal that NDF knockout cells display faster Pol II elongation rates but produce fewer nascent tran-scripts, demonstrating NDF's primary role in maintaining transcriptional processivity through-out gene bodies. Our findings demonstrate that NDF uses distinct mechanisms to ensure productive transcription elongation rather than simply enhancing elongation speed, offering new insights in-to how transcription efficiency is maintained in chromatin. ChIP-seq analysis of NDF in NDF WT, MT2 and MT4 rescued SW480 KO cells; ChIP-seq analysis of Pol II in NDF WT and KO Hela cells across the conditions: DRB transcription arrested, DRB released for 5, 10 and 20 mins respectively.

核小体去稳定因子（Nucleosome Destabilizing Factor, NDF）可介导染色质环境中的转录延伸过程，但其具体作用机制尚未完全阐明。本研究鉴定出NDF内部一段关键区域（氨基酸140-160位），该区域可特异性结合磷酸化RPB1——后者为延伸状态RNA聚合酶II（Pol II）的最大亚基。该区域发生突变后，会破坏其与Pol II的结合能力，同时在体外及细胞内均损害Pol II的延伸活性，但不影响NDF自身的核小体去稳定功能，由此证实这两种活性存在功能上的分离。细胞实验结果显示，NDF基因敲除细胞的Pol II延伸速率更快，但新生转录本的产量更低，这表明NDF的核心功能是维持基因体全程的转录持续合成能力。本研究结果表明，NDF通过独立的多种机制保障有效的转录延伸，而非仅单纯提升延伸速度，这为染色质环境中转录效率的维持机制提供了全新的研究视角。本研究对NDF野生型、MT2及MT4 rescue型SW480基因敲除细胞开展了NDF的染色质免疫共沉淀测序（ChIP-seq）分析；同时针对NDF野生型与敲除型HeLa细胞，在DRB转录阻滞、DRB释放5、10及20分钟的不同条件下，完成了Pol II的ChIP-seq分析。