In order to discover new epigenetic factors involved in NSC function, we have conducted an in vitro RNAi screen using our bespoke shRNA library that specifically targets epigenetic genes.
Adherent monolayers of NSCs were transduced with a pool of retrovirus containing shRNA targeting vectors against 130 predicted enzymatic epigenetic modifiers (~8 shRNAs per gene), plus controls. A representation of the number of cells harbouring each shRNA was examined over time using next generation sequencing. ShRNAs that affect NSC function were predicted to display altered representation compared to controls as the pooled cells competed in culture. Reassuringly, our screen showed a reduction in representation over time of cells containing proviral shRNA against the known positive NSC regulators Blbp and Bmi1. Furthermore, immortalisation with p53 knockdown resulted in a gain of shRNA representation in the screen.
Importantly, we have identified and individually validated two epigenetic modifiers, Yy1 and Prdm8, with hitherto uncharacterised roles in NSCs using our shRNA screening approach. Yy1 knockdown NSCs exhibit a competitive disadvantage while Prdm8 knockdown cells show a competitive advantage. These screening results were confirmed using single gene knockdown competition assays and with multiple unique shRNA targeting vectors designed against each gene.
We are currently defining the mechanistic role of these novel NSC epigenetic regulators in vitro and in vivo. We have revealed several interesting Yy1 target genes in NSCs by sequencing RNA from Yy1 knockdown and control cells. The top upregulated gene in Yy1 knockdown NSCs is the nucleosome assembly protein 1-like 2, Nap1l2. Nap1l2 is an epigenetic factor that when deleted in NSCs results in increased proliferation and decreased neuronal differentiation, therefore, upregulation of Nap1l2 may explain the competitive disadvantage displayed by Yy1 knockdown cells.
In summary, we have identified Yy1 and Prdm8 as two novel epigenetic factors critical to NSC function using a pooled shRNA screening approach, are characterising the mechanisms underlying their action, and plan to investigate their role in human brain cancer.