Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

The retinoblastoma (RB)/p16(INK4a) pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16(INK4a)-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1 beta (HP1 beta)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated beta-galactosidase marker. Notably, formation of RB/HP1 beta foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1 beta/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.     

Cellular senescence and chromatin structure

Cellular senescence is characterized by stable cell cycle arrest that is triggered by various forms of stress stimuli. Senescent cells show a series of morphological and physiological alterations including a flat and enlarged morphology, an increase in acidic β-galactosidase activity, chromatin condensation, and changes in gene expression pattern. These features are not observed in proliferating cells or quiescent cells in vitro. Using these senescence markers, cellular senescence has been shown to occur in benign or premalignant lesions but not in malignant lesions and to act as a tumor-suppressing mechanism in vivo. The onset and maintenance of the senescent state are regulated by two tumor suppressor proteins, p53 and Rb, which mediate senescence signals through p38 mitogen-activated protein kinase and cyclin-dependent kinase inhibitors. Alterations of chromatin structure are believed to contribute to the irreversible nature of the senescent state. Senescent cells form characteristic heterochromatin structure called senescence-associated heterochromatic foci (SAHFs), which may repress the expression of proliferation-promoting genes, such as E2F target genes. Recent studies have provided molecular insights into the structure and the mechanism of SAHF formation. In this paper, we review the role of cellular senescence in tumor suppression in vivo and the molecular mechanism of stable growth arrest in senescent cells, focusing on the special form of heterochromatin, SAHFs.     

Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21WAF1, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.     

ING1a expression increases during replicative senescence and induces a senescent phenotype

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.