Abstract 5417: Phospho-p27 as a novel regulator of transcriptional dynamics during quiescence exit and early G1 progression

In addition to its role as a CDK inhibitor, p27 functions as a transcriptional co-regulator of c-Jun and STAT3 in cancer cells, driving tumor metastasis and stem cell expansion. Here, we investigated how p27 regulates cell cycle-dependent transcriptional dynamics. The transition from quiescence to G1 re-entry and progression involves tightly regulated transcriptional changes. RNA-seq analysis of G0-synchronized and released NIH3T3 cells revealed distinct transcriptional landscapes across the cell cycle, with T0 (quiescence) showing the most divergent transcriptome compared to other phases. This highlights the extensive transcriptional reprogramming required for cell cycle entry and progression. Our data suggest that C-terminally phosphorylated p27 promotes cell cycle progression through p27-dependent transcriptional regulation. While total p27 is maximal in G0, phosphorylated p27 is minimal to non-existent at this stage but increases in early G1 via AKT activation. Two transcription factor partners, c-Jun and STAT3, increase upon G0 exit, are phosphorylated in early G1, and form complexes with p27, peaking in early G1. We identified novel categories of p27 target genes co-regulated with c-Jun and/or STAT3 in early G1. Differential expression (DEG) analysis between T3 and T0 revealed enrichment of pathways associated with protein translation, underscoring preparation for cell cycle progression. ChIP-seq showed that p27pT197 chromatin binding increases in early G1, with 21% of sites localized to promoter regions, suggesting p27pT197 regulates transcription. Enhanced c-Jun co-binding and H3K27ac enrichment at these sites support a model where phospho-p27 collaborates with immediate early TFs to recruit or stabilize the transcriptional machinery and activate genes required for quiescence exit. Motif analysis identified enrichment of EMT transcription factors, including Snail1, Zeb1, and Zeb2, at p27pT197 binding sites, suggesting roles in early G1 progression. We are evaluating whether G0-specific p27-activated genes, repressed in early G1, are required for G0 arrest. We identified early G1-specific p27pT197 target genes linked to pathways governing mitogenic kinases, EMT, stem cell renewal, and cytoskeletal dynamics. Ongoing work aims to determine if these targets are required for G1 progression. These findings suggest that C-terminal phosphorylation of p27 acts as a molecular switch, enabling transcriptional reprogramming required for quiescence exit and early G1 progression. This work provides new insights into cell cycle regulation. Citation Format: Amir Bagheri, Seyedeh Fatemeh Razavipour, JiaXiang Tao, Madison Sharp, Joyce Slingerland. Phospho-p27 as a novel regulator of transcriptional dynamics during quiescence exit and early G1 progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5417.