The CCNT1 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited human cell population designed to disrupt the CCNT1 gene, encoding Cyclin T1, in the HT29 colorectal adenocarcinoma cell line. This polyclonal knockout model enables loss-of-function studies of the P-TEFb regulatory subunit without the constraints of single-cell clonal selection, providing a robust tool for investigating Cyclin T1-dependent transcriptional regulation.
The HT29 cell line is an established adherent epithelial model derived from a primary colorectal adenocarcinoma of a 44-year-old female. This line harbors several oncogenic mutations characteristic of aggressive colorectal cancer, including an APC truncation, a TP53 mutation, and the activating BRAF V600E alteration, and is microsatellite stable. These genetic features make HT29 a widely employed platform for studying colorectal cancer signaling, drug responses, and tumor biology.
CCNT1 (Cyclin T1) functions as the regulatory subunit of the P-TEFb complex, where it partners with the cyclin-dependent kinase CDK9. P-TEFb drives transcription elongation by phosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II at Ser2, along with the negative elongation factors DSIF and NELF, thereby releasing promoter-proximal paused polymerase into productive synthesis. The activity and assembly of P-TEFb are modulated by an intricate network of factors: the bromodomain protein BRD4 and the super elongation complex component AFF4 recruit P-TEFb to chromatin, while the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP), comprising HEXIM1 and 7SK RNA, sequesters the complex. CCNT1 is also a critical host cofactor for the HIV-1 Tat protein, enabling viral transactivation. Upstream signals from c-Myc and NF-??B promote CCNT1 expression, and key downstream transcriptional targets include the proto-oncogene MYC, the AP-1 transcription factor subunit FOSL1, and the anti-apoptotic factor MCL1. Additionally, elongation factors ELL2, SUPT5H, and SUPT6H interact with the P-TEFb complex to control processivity.
In the context of the HT29 colorectal cancer model, loss of CCNT1 disrupts P-TEFb-dependent transcript elongation, leading to diminished expression of proliferation and cell-survival gene programs driven by MYC, FOSL1, and MCL1. Given the high proliferative drive imposed by the BRAF V600E oncogene and the compromised tumor suppressor landscape, CCNT1 knockout in this background provides a physiologically relevant system to dissect the contribution of transcriptional elongation to colorectal tumor maintenance. This model thus enables the interrogation of cyclin-dependent kinase-mediated transcription addiction in colon cancer, a pathway increasingly recognized as a therapeutic vulnerability.
These polyclonal knockout cells are optimized for applications such as mechanistic studies of transcription elongation control, HIV-1 latency and Tat-dependent reactivation, and colorectal cancer proliferation pathways. The model is suitable for screening CDK9 inhibitors like flavopiridol and dinaciclib, and for identifying P-TEFb-dependent gene programs through RNA-seq and ChIP-qPCR of RNA polymerase II occupancy. Validated assays include western blotting for Cyclin T1, CDK9, and Ser2-phosphorylated RNA Pol II CTD; RT-qPCR for MYC, FOSL1, and MCL1; flow cytometry; co-immunoprecipitation of the P-TEFb complex; and HIV-1 LTR-Tat luciferase reporter assays. For further details and technical specifications, please contact Ascent Research.
