GSEA found that the genes Brefeldin A buy in the REST 24 gene signature were indeed down regulated in a statistically significant manner in a subset of gliomas. A second gene list examining 30 REST target genes not present in the 24 gene signature that were induced at least 2 fold in at least 2 of three cell lines tested in Wagoner et al was also strongly under expressed in the same subset of gliomas. This suggests that enhanced REST is not limited to a small set of REST target genes. Finally, GSEA was performed based on a geneset comprised of over 800 REST target genes identified in Jurkat T cells as REST targets by ChIP Seq after removal of the 24 gene signature. This analysis confirms the statistically significant down regulation of REST target genes in gliomas with respect Inhibitors,Modulators,Libraries to non neoplastic tissue, sug gesting an increase in REST function in the tumors.
Intriguingly, the increased REST function observed in gli omas was not uniform across all tumors, with some tumors expressing REST target genes near the levels observed in non neoplastic tissue. To determine whether the intertu moral variation of REST function is significant, we ranked tumors by expression of the 24 gene signature and then divided gliomas into groups of high and Inhibitors,Modulators,Libraries low expression of REST signature genes. Tumors with low level expression of genes in the REST signature were termed REST enhanced malignancies, and those with expression levels of REST target genes at or near that of normal, non neoplastic tissue were termed near normal tumors.
Using the above three independent REST gene lists, GSEA found statistically significant decreases Inhibitors,Modulators,Libraries in REST Inhibitors,Modulators,Libraries target gene mRNA levels, suggesting that a significant population of these high grade gliomas have heightened REST function. Given that many REST target genes are highly expressed in mature neurons, one possible explanation for the ele vated REST function observed in REM tumors could Inhibitors,Modulators,Libraries be that those tumors have low levels of neuronal involvement or neuronal contamination. To determine if higher levels of neurons are present in near normal glioma tumor sam ples with respect to REM tumors, we first had to identify genes selectively expressed in neurons that are not likely REST target genes. These genes were selected from a gene expression dataset comparing fluorescently sorted neurons, astrocytes, and glia from the murine CNS.
First, we identified those genes that are most highly and selectively expressed in neurons. Then we product information filtered out any genes that had been identified as a potential REST target in published ChIP ChIP or ChIP Seq experiments, or contained a consensus 21bp REST binding element. Figure 4A validates the resulting 6 genes that are not REST targets as neuron specific. Evaluation of neuronal non REST target genes found that there was no concerted up regulation of all neu ronal non REST markers in either REM or near normal tumors.