Investigating Chromatin Dynamics Following the Loss of BCL-3 in the Context of Irradiation Induced DNA Damage in Colorectal cancer.

Abstract

Colorectal cancer (CRC) poses a significant global health burden, accounting for 11% of all new cancer cases and ranking as the UK’s second leading cause of cancer - related mortality. While early - stage CRC is often managed by surgical resection alone, locally advanced cases typically require pre - operative chemoradiotherapy. Unfortunately, treatment responses vary widely among patients. Recent studies have implicated the oncogene BCL-3 in this variability, with higher expression levels correlating with poorer overall and disease - free survival. Moreover, BCL-3 acts as a co-factor with histone modifying enzymes such as KAT5/TIP60 and HDAC3, suggesting an epigenetic role in therapy resistance. Therefore, this study aimed to investigate BCL-3’s role in chromatin organisation, hypothesising that BCL-3 knockdown increases chromatin accessibility and, as a result, enhances radiosensitivity in CRC cells.

Initial western blot analysis demonstrated that BCL-3 knockdown increased H3K27 acetylation and decreased H3K4 tri-methylation 15 minutes post-irradiation, highlighting BCL-3’s influence on chromatin accessibility. To investigate this further, ATAC-see - a transposase-mediated technique that uses fluorophore-labelled DNA adaptors to directly visualise the 3D spatial organisation of the genome - was employed to quantify dynamic changes in chromatin accessibility following BCL-3 knockdown and irradiation. Successful implementation of ATAC-see first required the optimisation of in-house Tn5 transposase production. Unsuccessful attempts with the pTXB1 - Tn5 plasmid led to the adoption of the VR124 10His-pG-Tn5 plasmid, with successful production confirmed by mass spectrometry.

Although preliminary ATAC-see results were inconclusive regarding BCL-3’s specific mechanisms, this thesis provides foundational insights into BCL-3’s impact on chromatin dynamics and lays the groundwork for future ATAC-see analysis, potentially offering new avenues for personalised treatment strategies in CRC.

Date of Award	25 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Adam Christian Chambers (Supervisor) & Ann C Williams (Supervisor)