Figure 6
P-Ser83-HP1γ status alters cell proliferation and cell cycle-related gene networks. (A) P-Ser83-HP1γ plays a role in cell proliferation. Cell proliferation was measured in the presence of control (EV), wild type HP1γ, the nonphosphorylatable (S83A)- or phosphomimetic (S83D)-HP1γ mutants by EdU incorporation, using both FACS and microscopy. Wild type HP1γ demonstrated only a slight increase in EdU incorporation compared to EV. However, while mutation of S83A-HP1γ decreased the levels of EdU, the S83D-HP1γ mutant had a significant increase in levels of EdU incorporation over control cells. Western blot controlling expression of His-tagged wild type and mutant HP1γ proteins is shown (top, inlay). A representative immunofluorescence image (40 × magnification) of EdU-positive cells (green) is shown below each respective experimental condition. Cells were counterstained with DAPI to detect total number of cells present in a field. *P values <0.05. (B) Genome-wide expression analysis of HP1γ highlights consequences of Ser83 phosphorylation. Hierarchical clustering of significant targets (P value <0.05) from Affymetrix Human Gene 1.0 ST microarray demonstrates the close relationship between EV and the nonphosphorylatable S83A-HP1γ mutant. Large clusters of genes show deregulation in the presence of either the nonphosphorylatable (S83A)- or phosphomimetic (S83D)-HP1γ mutants. (C) P-Ser83-HP1γ status influences the expression of G2/M-related genes. Gene Ontology (GO) ANOVA reveals significant differential expression of genes by both wild type and mutant HP1γ in functional groupings related to mitosis and cell division, again indicating that the presence of an active phosphorylation site at Ser83 is necessary for proper mitotic function as a sizeable number of targets are deregulated in the presence of the HP1γ mutants with altered phosphorylation abilities. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2´-deoxyuridine; EV, empty vector; FACS, fluorescence-activated cell sorting; GO, Gene Ontology; P-Ser83-HP1γ, phosphorylation of HP1γ at serine 83; Ser83, serine 83.