We have previously reported that CK is downregulated in sene
We have previously reported that CK2 is downregulated in senescent human lung fibroblast IMR-90 cells, aged rat tissues, and aging nematodes [17,18]. CK2 downregulation induces cellular senescence via the PI3K-AKT-mTOR-reactive oxygen species-p53-p21Cip1/WAF1 pathway in human cells [, , ]. We have also shown that kin-10 (the Caenorhabditis elegans ortholog of CK2β) knockdown induces age-related biomarkers including reduced longevity and retardation of locomotion in C. elegans . In this study, we examined the physiological significance of CK2 in the context of chromatin reorganization during cellular senescence. Our results indicate, for the first time, that CK2 downregulation stimulates SUV39h1 expression and decreases G9a expression, and this event triggers H3K9 tri-methylation and SAHF formation.
Materials and methods
Discussion We have previously shown that CK2 downregulation induces premature senescence in IMR-90, HCT116, and MCF-7 cells [17,, , ], and enhances expression of age-related biomarkers and shortens lifespan in nematodes . Because SAHF are formed to irreversibly block PF-6405761 progression during senescence [, , , , ], we examined the role of CK2 downregulation in SAHF formation. The present study demonstrated that CK2 downregulation mediates both induction of H3K9me3 and reduction of H3K9me2 (Fig. 1, Fig. 2, Fig. 3A, Supplemental Figs. 1, 2) and represses the expression of cyclin D1 (Fig. 1, Fig. 2B) in human cells and nematodes. In addition, CK2 downregulation accelerates HP1γ binding to H3K9me3 in DAPI-dense heterochromatic foci (Fig. 1A, Supplemental Fig. 1) in human cells. Conversely, CK2 upregulation or treatment with spermidine (a putative CK2 activator) reduce H3K9me3, induce H3K9me2 (Fig. 2, Fig. 3B), and accelerate expression of cyclin D1 (Fig. 1, Fig. 2D). It has been previously reported that whereas silent euchromatin regions are enriched for dimethylated H3K9, heterochromatic regions are highly trimethylated on H3K9 . H3K9me3 is a stable hallmark in DAPI-dense heterochromatin regions, and HP1γ selectively binds to H3K9me3 in SAHF [, , , , ]. Therefore, the present study collectively indicates that CK2 downregulation induces H3K9me3 and SAHF formation, thereby suppressing the expression of cell cycle progression genes such as the gene encoding cyclin D1. SUV39h1 is a main trimethylase of H3K9, and G9a, GLP, and SETDB1 are dimethylases of H3K9. H3K9me2 is a primary substrate for trimethylation by SUV39h1 [, , , ]. Thus, it is expected that CK2 downregulation would enhance expression of SUV39h1 as well as G9a, GLP, and SETDB1 for H3K9me3. Consistently, the present study showed that SUV39h1 expression is increased by CK2 downregulation and decreased by CK2 upregulation. Unexpectedly, however, CK2 downregulation rather reduces expression of G9a, GLP, and SETDB1 and CK2 upregulation stimulates expression of these dimethylases (Fig. 3, Supplemental Fig. 2). Moreover, this study indicated that both SUV39h1 inhibition and G9a overexpression successfully counteract CK2 inhibition-mediated increases of SA-β-gal activity, H3K9me3, and SASF formation (Fig. 4, Supplemental Fig. 4). Based on these data, we propose that CK2 downregulation induces H3K9me3 and SAHF formation through both stimulating SUV39h1 expression and inhibiting G9a expression. The present study found that G9a overexpression apparently suppressed CK2 downregulation-mediated induction of p53, SUV39h1, and H3K9me3 (Fig. 4C, Supplemental Fig. 4). Our previous study has demonstrated that p53 is essential for CK2 downregulation-mediated senescence . In addition, p53 is important for CK2 downregulation-mediated stabilization of SUV39h1 (J.W. Park and Y.S. Bae, manuscript in preparation). Therefore, our previous and present studies suggest that CK2 downregulation-mediated G9a reduction increases the amount of p53 protein, which results in induction of SUV39h1. How does G9a reduce levels of p53? Since G9a specifically methylates p53 at Lys 373 and acetylated p53 is more stable than the methylated form [25,26], we hypothesize that G9a downregulation-mediated demethylation of p53 Lys 373 may increase p53 stability in CK2-downregulated cells. Future studies are needed to investigate how CK2 regulates the expression of G9a and how G9a modulates p53 level.