Figure 7
PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. (a) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) (n=3, **P<0.01). (b) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. (c) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. (d) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n=114). (e, f) ING5 expression levels negatively correlate with survival of the Proneural subtype (n=24) and the Classical subtype (n=30) of GBM patients. (g) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n=61). (h) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.
