Single-cell profiling reveals disruption of cell-cycle homeostasis following JDP2 depletion in colorectal epithelial cells

Intestinal epithelial cells (IECs) undergo rapid and continuous renewal to maintain gut epithelial homeostasis and barrier integrity at the interface with luminal microbiota, dietary antigens, and enteric pathogens. IEC proliferation is tightly regulated by a complex machinery of cell cycle regulators and effectors, and genetic or transcriptional dysregulation of cell-cycle regulatory pathways is a hallmark of colorectal cancer development and progression. Previously, JDP2 (Jun dimerization protein 2) has been shown to be involved in cell cycle control and is associated with an array of cancers. JDP2 functions as a transcription factor or epigenetic regulator depending on cellular context, disease, and cancer types. However, the role of JDP2 in CRC, cell cycle homeostasis of colon cancer cells, and their fate remains poorly defined. In this study, we found that patients with higher JDP2 expression have significantly worse survival than patients with lower JDP2 expression. Our functional assays showed that JDP2 depletion increased EdU incorporation during S phase and PHH3 abundance, which is consistent with increased proliferative and mitotic activity. Our single-cell RNA sequencing and transcriptomic profiling further revealed reprogramming of cell-cycle-associated transcriptional states following JDP2 depletion. Differential-expression and pathway analysis identified changes in mitotic and cell-cycle regulatory genes, including programs involving cyclins, cyclin-dependent kinases, chromosome segregation, and mitotic progression. We also found that lower residual JDP2 expression was associated with escalated G2/M representation and a change toward later states along a G1 - S - G2/M-associated transcriptional trajectory. Furthermore, low-JDP2 cells showed significantly greater late-state occupancy than High-JDP2 cells (33.6% versus 24.0%; paired P=0.048). Among the JDP2-siRNA-treated cells, analysis of residual JDP2-expressing cells also revealed heterogeneous associations of JDP2 with cell-cycle, survival, apoptotic, and stress-response programs, consistent with JDP2's context-dependent transcriptional functions. Together, these data identify JDP2 as an important component of intestinal epithelial cell-cycle homeostasis and transcriptional landscape and suggest that JDP2 depletion perturbs the balance of proliferative cell states.