RNAseq analysis of isolated anchor cells expressing the lin-39 hox gene

We have previously reported that he hox gene lin-39 directly induces proliferation of various cell types in C. elegans larvae. To further characterize how lin-39 changes mitotic activity we isolated lin-39 overexpressing anchor cells. By passing single cell suspensions extracted from pbx-ACEL>lin39::gfp larvae at the L3 stage through a fluorescent-activated cell sorter (FACS), we were able to obtain a sufficient number of LIN-39::GFP-positive cells (around 20'000 cells with >90% purity) to perform bulk RNAseq analysis and determine their expression profile. So far, we have not been able to isolate sufficient numbers of non-proliferating ACs from the pbx-ACEL>nls::gfp::lacZ control strain to perform RNAseq. Yet, in comparison with the LIN-39::GFP-negative cell population, several genes known to be preferentially expressed in the wild-type AC, such as the hlh-2, fos-1, egl-43 and nhr-67 transcription factors, were strongly enriched in the LIN-39::GFP-positive cells, suggesting that over-expression of lin-39 did not reprogram the fate of the proliferating ACs. However, many cell cycle regulators, such as the G1 and G2/M cyclins, the mcm genes and other components of the pre-RC, which are normally expressed at very low or even undetectable levels in the AC, were enriched in the LIN-39::GFP-positive cells. Hence, over-expression of lin-39 caused a reactivation of cell cycle genes in the AC and bypassed the inhibitory functions of the nhr-67 and egl-43 transcription factors, which normally maintain the AC in its post-mitotic state. Moreover, the lin-12 notch gene was overexpressed in the proliferating ACs, which is consistent with our previous finding that hyper-activation of NOTCH signaling can cause AC proliferation.