Identification of epigenetic modifiers involved in neural stem cell function

Abstract

It has become clear that epigenetic modifications of chromatin play a crucial role in regulating gene expression and cell fate decisions in stem cells; however, epigenetic pathways in neural stem cells (NSCs) have not been well characterised. In order to identify new epigenetic factors involved in NSC function, I conducted a pooled competitive RNAi screen in cultured primary embryonic NSCs using a bespoke, high coverage shRNA library that specifically targets epigenetic factors. With this approach, I discovered a novel NSC epigenetic modifier, Prdm8. When Prdm8 expression was reduced in NSCs they displayed a competitive advantage over control cells. Further studies determined that this advantage was mediated by increased cellular proliferation.

In a separate targeted individual competition screen of polycomb group complex factors, I also discovered that reduced expression of the Ying Yang 1 gene, Yy1, decreased NSC competitiveness. Further investigation revealed that Yy1 knockdown resulted in a cell cycle defect; specifically, NSCs with reduced Yy1 expression accumulated in the G1 phase of the cell cycle and exhibited increased expression of the cyclin-dependent kinase inhibitor, p21, without co-induction of p53. Correspondingly, Yy1 overexpression led to decreased levels of p21. RNA sequencing of NSCs with reduced Yy1 expression revealed other differentially expressed cell cycle regulatory genes, including Cend1 and Ccna1. Cend1 has a previously published role in driving neural progenitors out of the cell cycle, and in promoting neuronal differentiation and thus the mechanism behind increased Cend1 expression in Yy1 knockdown NSCs is of particular interest. Chromatin immunoprecipitation followed by next generation sequencing using antibodies targeting H3K4me3 and H3K27me3 in control and Yy1 knockdown NSCs revealed the absence of genome-wide alterations or individual peak alterations indicating that the Yy1 phenotype may be correlated with alterations in another histone mark(s).

In summary, I have shown that Yy1 and Prdm8 are novel epigenetic factors critical to NSC function. Future experiments will further elucidate their functions in vivo and in human brain cancer as NSCs share many characteristics with cancer stem cells.