Luyties Olivia, Sanford Lynn, Rodino Jessica, Nagel Michael, Jones Taylor, Rimel Jenna K, Ebmeier Christopher C, Palacio Megan, Shelby Grace S, Cozzolino Kira, Brennan Finn, Hartzog Axel, Saucedo Mirzam B, Watts Lotte P, Spencer Sabrina, Kugel Jennifer F, Dowell Robin D, Taatjes Dylan J
Department of Biochemistry, University of Colorado, Boulder, CO 80303, USA.
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80303, USA; BioFrontiers Institute, University of Colorado, Boulder, CO 80303, USA.
Cell Rep. 2025 Jul 22;44(7):115904. doi: 10.1016/j.celrep.2025.115904. Epub 2025 Jun 25.
CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases prevent reliable CDK7 analysis with nuclear extracts, we reconstitute RNAPII transcription with purified factors. We show that CDK7 inhibition slows and/or pauses RNAPII promoter-proximal transcription and suppresses re-initiation, and these effects are Mediator and TFIID dependent. Similarly in human cells, CDK7 inhibition reduces transcriptional output by suppressing RNAPII initiation and/or re-initiation. Moreover, widespread 3' end readthrough transcription occurs in CDK7-inhibited cells; mechanistically, this results from rapid nuclear depletion of RNAPII elongation and termination factors (e.g., DSIF, Integrator, NELF, SPT6, PPP1R10/PNUTS, and SCAF8), including high-confidence CDK7 kinase targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5' ends and 3' ends and reveal that nuclear abundance of elongation and termination factors is kinase dependent. Because 3'-readthrough transcription is commonly induced during stress, our results further suggest that regulated suppression of CDK7 activity enables this transcriptional response.
细胞周期蛋白依赖性激酶7（CDK7）通过尚未完全明确的机制调控RNA聚合酶II（RNAPII）的起始、延伸和终止。由于污染性激酶会干扰利用核提取物对CDK7进行可靠分析，我们利用纯化的因子重建了RNAPII转录过程。我们发现，抑制CDK7会减缓并/或暂停RNAPII在启动子近端的转录，并抑制重新起始，而且这些效应依赖于中介体复合物（Mediator）和TATA框结合蛋白相关因子IID（TFIID）。同样，在人类细胞中，抑制CDK7会通过抑制RNAPII的起始和/或重新起始来降低转录输出。此外，在受CDK7抑制的细胞中会广泛出现3'端通读转录；从机制上来说，这是由于RNAPII延伸和终止因子（如DSIF、整合因子、负性延伸因子NELF、SPT6、蛋白磷酸酶1调节亚基10/核仁磷酸蛋白（PPP1R10/PNUTS）和剪接因子相关因子8（SCAF8））在细胞核内迅速耗竭所致，其中包括高可信度的CDK7激酶作用靶点。总体而言这些结果明确了CDK7如何在基因的5'端和3'端调控RNAPII的功能，并揭示了延伸和终止因子在细胞核内的丰度是依赖于激酶的。由于应激过程中通常会诱导产生3'端通读转录，我们的结果进一步表明，对CDK7活性进行调控性抑制能够引发这种转录反应。
