Heterochromatin protein 1gamma epigenetically regulates cell differentiation and exhibits potential as a therapeutic target for various types of cancers

Abstract

Heterochromatin protein 1 (HP1) is a chromosomal protein that participates in both chromatin packaging and gene silencing. Three HP1 isoforms (alpha, beta, and gamma) occur in mammals, but their functional differences are still incompletely understood. In this study, we found that HP1gamma levels are decreased during adipocyte differentiation, whereas HP1alpha and beta levels are expressed constitutively during adipogenesis in cultured preadipocyte cells. In addition, ectopic overexpression of HP1gamma inhibited adipogenesis. Furthermore, we did not detect any HP1gamma protein in the differentiated cells of various normal human tissues. These results suggest that the loss of HP1gamma is required for cell differentiation to occur. On the other hand, the methylation levels of lysine 20 (K20) on histone H4 showed a significant correlation with HP1gamma expression in both these preadipocyte cells and normal tissue samples. However, all cancer tissues examined were positive for HP1gamma but were often negative for trimethylated histone H4 K20. Thus, a dissociation of the correlation between HP1gamma expression and histone H4 K20 trimethylation may reflect the malfunction of epigenetic control. Finally, suppression of HP1gamma expression restrained cell growth in various cancer-derived cell lines, suggesting that HP1gamma may be an effective target for gene therapy against various human cancers. Taken together, our results demonstrate the novel function of HP1gamma in the epigenetic regulation of both cell differentiation and cancer development.

Figure 1

Loss of HP1γ is required for adipocyte differentiation. A: Western blot analysis of HP1α, β, and γ in 3T3L1 and human preadipocyte cells during adipogenesis. GAPDH was also examined as a loading control. B: Western blot analysis of HP1γ in 3T3L1 cells (wt) and 3T3L1-derived cells with a mifepristone-inducible HP1γ expression system (tr). The cells were treated with or without mifepristone and/or the differentiation reagents (3-isobutyl-1-methylxanthine, dexamethasone, and insulin). GAPDH was used as a loading control. C: Oil red staining of 3T3L1 cells (wild) and 3T3L1-derived cells with a mifepristone-inducible HP1γ-full length, -ΔCSD, or -ΔCD expression system (transfectant) during adipogenesis. The cells were treated with mifepristone and/or the differentiation reagents. Lipid duplets in adipose are visualized as red stains.

Figure 2

Global histone modifications in various states of 3T3L1 cells. A: Western blot analysis of global histone modifications in 3T3L1 cells at several time points after induction of adipogenesis. We examined the following histone modification sites: acetylated H3 K9 (AceH3K9), acetylated H3 K18 (AceH3K18), dimethylated H3 K4 (DiMetH3K4), dimethylated H3 K9 (DiMetH3K9), dimethylated H3 R17 (DiMetH3R17), acetylated H4 K8 (AceH4K8), acetylated H4 K12 (AceH4K12), acetylated H4 K16 (AceH4K16), and trimethylated H4 K20 (TriMetH4K20). HP1γ and GAPDH are also examined. B: Western blot analysis of global histone modifications in 3T3L1-derived cells with a mifepristone-inducible HP1γ expression system at several time points after mifepristone treatment. C: Western blot analysis of global histone modifications in 3T3L1 cells transfected with HP1γ siRNA (HP1γ) or negative control siRNA (Cont). UT, untransfected 3T3 cells.

Figure 3

HP1γ and trimethylated histone H4 K20 disappear in terminally differentiated cells in a variety of tissues. A–C: Mature fat; D–F: esophagus; G–I: skin; J–L: colon. A, D, G, and J: H&E-stained sections; B, E, H, and K: immunohistochemical analysis of HP1γ; C, F, I, and L: trimethylated histone H4 K20. Positivity is visualized as a brown stain. Scale bars = 50 μm.

Figure 4

HP1γ is highly expressed in human malignant tumors. H&E-stained sections and nuclear staining of malignant tumor cells by immunohistochemistry with antibodies against HP1γ and trimethylated histone H4 K20. Esophageal cancer (cases 1, 2, and 3), cervical cancer (cases 4, 5, and 6), colon cancer (cases 7, 8, and 9), breast cancer (cases 10, 11, and 12), lung cancer (cases 13, 14, and 15), and myxoid liposarcoma (case 16, 17, and 18). Positivity is visualized as a brown stain. Scale bars, 100 μm. Magnification of each inset is 10× to the main panel.

Figure 5

Repression of HP1γ expression inhibits tumor cell growth. The cell numbers of various cell lines at 72 hours after siRNA transfection are shown. UT, untransfected control. A: All three active siRNAs specific for human HP1γ (named H-3, H-7, and H-9, respectively) inhibit the growth of human colon cancer cell line, DLD-1. B: Three active siRNA specific for mouse HP1γ (named cbx1, cbx2, and cbx3, respectively), however, do not inhibit the cell growth of mouse normal preadipocyte 3T3L1. C: The active siRNA specific for human HP1γ, H-7, inhibits the growth of human tumor cell lines derived from various cancers of colon (HCT116, HT29), cervix (HeLa, SiHa), stomach (MKN1, MKN28), and lung (NCI-H23), and from myxoid liposarcoma (402/91, 2645/94). The expression of HP1γ at 72 hours after siRNA transfection of each cell line was evaluated by Western blot analysis. GAPDH was used as a loading control.