CCNT1 Knockout HeLa Polyclonal Cells are a CRISPR/Cas9-edited cell population with targeted disruption of the CCNT1 gene, providing a loss-of-function model for Cyclin T1, the regulatory subunit of P-TEFb. This polyclonal population abrogates CCNT1 protein expression and avoids clonal selection artifacts, enabling robust investigation of CCNT1-dependent transcriptional regulation and its roles in disease-relevant pathways.
The host HeLa cell line, derived from cervical adenocarcinoma, is HPV18-positive and exhibits epithelial morphology. HeLa cells are widely used in cancer and virology research due to their susceptibility to viral infection, including HIV-1. Their permissiveness to HIV-1 makes them particularly suitable for studying Tat-mediated transactivation, which requires CCNT1. HeLa cells endogenously express transcription elongation factors, providing a physiologically relevant system for P-TEFb functional analysis.
The CCNT1 protein forms the active P-TEFb kinase complex with CDK9, which phosphorylates serine-2 residues in the RNA polymerase II CTD, promoting promoter-proximal pausing release and productive elongation. CCNT1 is activated by HIV Tat, NF-??B, and BRD4, while it is inhibited through sequestration by HEXIM1 and the 7SK snRNP. It interacts directly with CDK9, Tat, BRD4, AFF4, HEXIM1, and 7SK RNA, and mediates phosphorylation of downstream targets SPT5 and DSIF, thereby facilitating both HIV LTR-driven viral transcription and cellular gene expression programs. This positions CCNT1 at the nexus of pathways including transcriptional elongation, HIV life cycle, CDK9 signaling, and p53 regulation.
In HeLa cells, CCNT1 knockout disrupts P-TEFb function, leading to impaired RNA Pol II CTD phosphorylation and reduced transcriptional elongation. This directly abolishes HIV Tat-dependent transactivation of the LTR, enabling precise dissection of viral gene expression and identification of host dependency factors critical for HIV replication. Additionally, the model permits investigation of P-TEFb’s roles in cancer-specific transcription programs and enables evaluation of P-TEFb inhibitors on cell proliferation and survival in a cervical cancer background.
Suitable applications include Western blotting for CCNT1, RT-qPCR for HIV LTR-driven transcripts, detection of RNA Pol II CTD phosphorylation, co-immunoprecipitation with CDK9, and ChIP-qPCR for RNA Pol II occupancy. HIV LTR reporter assays directly measure Tat transactivation efficiency. The cells also support validation of small-molecule P-TEFb inhibitors and studies of transcription elongation dependencies in cancer. For further technical details, contact Ascent Research.
