Analysis of Bcl6 in a genetically tractable human B cell line

Abstract

B cell lymphoma 6 (Bcl6) is a proto-oncogene functioning as a transcriptional repressor at the germinal centre (GC) stage of B cell development, regulating proliferation, differentiation and survival. Bcl6 is commonly dysregulated in GCderived lymphomas and hence a promising therapeutic target. Although a number of agents targeting Bcl6 are being developed, there is currently no good model available for functional analysis and hence drug target validation. To address this issue, I have established a conditional BCL6 knockout cell line for in-depth functional analyses of Bcl6. I have used homologous recombination to disrupt one BCL6 allele in the human Burkitt’s lymphoma cell line DG75 with a zeocin resistance cassette and have shown this line to be capable of supporting tetracycline (tet)-regulated Bcl6 expression from an integrated expression construct. The zeocin cassette used to disrupt the first BCL6 allele was subsequently deleted by Cre recombination. This allowed me to reuse the same targeting construct to disrupt the second BCL6allele. I subsequently set out to characterise the resulting conditional knockout cell line and found that depletion of Bcl6 leads to less proliferation, characterised by a prolonged G0/G1cell cycle phase. I went on to further characterise the effect of Bcl6 depletion in my system by cDNA microarray analysis, which revealed interesting insights into functions of Bcl6 such as the modulation of B cell receptor signalling and calcium signalling. The system I have developed is the first precisely regulated genetic model for studying Bcl6 function and will be of great benefit for research aiming to develop Bcl6 targeted drug therapy.