Abstract SY02-03: Chromatin architecture and gene regulation in oncogene-induced senescence.
Authors: Narita Masashi 
Published: August 2014

Cellular senescence was originally a term describing the ‘irreversible’ state of cell cycle arrest induced by the replicative exhaustion of human diploid fibroblasts (HDFs) in culture1. In marked contrast to the readily reversible quiescent state, senescent cells typically exhibit distinct morphological changes, including enlarged cells and nuclei, prominent nucleoli, and cytoplasmic vacuoles. While the ‘replicative exhaustion’ was later attributed to the limits imposed by critically short telomeres, which provoke a persistent DNA damage response (1), a similar phenotype can also occur acutely upon oncogenic stress (oncogene-induced senescence, OIS) as well as in response to other cytotoxic stresses (2).

While it is widely accepted that both of the p53 and p16/Rb tumour suppressor pathways are involved in senescence, the precise mechanisms underling the phenotype are still elusive. The senescence phenotype can be heterogeneous and, depending on the stress or cell type, the ‘quality’ of the phenotype varies. A number of markers of senescence have been described, but none of them are definitive. Nevertheless, combinations of these markers have been useful to extend the concept of the senescence phenotype, not only within cultured cell models but also to in vivo systems. Furthermore, it is important that several ‘effector mechanisms’ that can modulate senescence have been identified, including senescence associated heterochromatic foci (SAHFs) and epigenetic gene regulation, the DNA damage response, senescence associated secretory phenotype (SASP) and autophagy (3). These multiple effector mechanisms seem to collectively define the phenotype, generating a wide spectrum of phenotypes.

We and others have previously shown that HDFs exhibit dramatic heterochromatin (HC) alteration during oncogenic Ras-induced senescence (i.e. SAHFs) in a p16/Rb dependent manner and have proposed that SAHF formation is associated with the stability of the phenotype (4). Senescent HDFs with high p16 (non-reversible by p53 depletion) show more prominent SAHFs compared to p16-low HDFs (reversible by p53 depletion), indicating a correlation between SAHF formation and the stability of senescence arrest (5,6). Furthermore, non-histone chromosomal architectural proteins, HMGA1 and HMGA2, which have been implicated in cancer, are essential structural components of SAHFs, and disruption of SAHFs by depletion of HMGA1 makes it easier for cells to bypass senescence (7).

More detailed characterization of SAHFs using highly specific monoclonal antibodies against modified histones, has revealed that SAHFs are distinct non-overlapping multi-layer structures, in which H3K9me3 (a constitutive HC mark) is enriched in the ‘core’ of SAHFs, surrounded by a layer of H3K27me3 (a facultative HC mark), which separates the core from the transcriptionally active H3K4/36me3 regions (8). This multi-layered structure suggests that SAHF formation involves at least two events: co-association of similar types of chromatin and segregation of distinct chromatin types into the layers. Surprisingly, despite this dramatic alteration of microscopic pattern of heterochromatic marks during SAHF formation, global linear epigenomic profiles of these repressive histone marks are largely unaltered, although regional levels of the repressive marks can alter in a subset of gene bodies during the process. These data suggest that SAHF formation involves ‘spatial reorganization’ of pre-existing HC. This is in contrast to the ‘spreading’ of HC occurring during ES cell differentiation (9). Thus SAHF formation involves at least two ‘modules’: accumulation of architectural components of HC, and spatial rearrangement of HC. Interestingly, a recent study showed that loss of nuclear Lamin B1, which forms fibrillar network at nuclear lamina, is downregulated during senescence, and that knockdown of Lamin B1 induces senescence in human fibroblasts (10,11). As constitutive HC is often associated with the nuclear envelope, the depletion of Lamin B1 might disrupt the HC anchoring to nuclear lamina and facilitate its spatial repositioning during senescence. We are currently testing this hypothesis using both cell biological and epigenomic approaches (12).

Finally our preliminary analyses of time series microarray experiments during Ras-induced senescence indicate that levels of both up- and down-regulated genes are mostly static once senescence is established. Thus it is tempting to speculate that such a layer structure of SAHFs might promote the maintenance of the efficient silencing as well as constitutive expression of genes at the cost of dynamic gene regulation.

1. Shay, J. W. & Wright, W. E. Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol 1, 72-76 (2000).

2. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593-602 (1997).

3. Kuilman, T., Michaloglou, C., Mooi, W. J. & Peeper, D. S. The essence of senescence. Genes Dev 24, 2463-2479 (2010).

4. Narita, M. Cellular senescence and chromatin organisation. Br J Cancer 96, 686-691 (2007).

5. Narita, M. et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113, 703-716 (2003).

6. Beauséjour, C. M. et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 22, 4212-4222 (2003).

7. Narita, M. et al. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation. Cell 126, 503-514 (2006).

8. Chandra, T. et al. Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation. Mol Cell (2012).doi:10.1016/j.molcel.2012.06.010

9. Hawkins, R. D. et al. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 6, 479-491 (2010).

10. Shimi, T. et al. The role of nuclear lamin B1 in cell proliferation and senescence. Genes Dev 25, 2579-2593 (2011).

11. Freund, A., Laberge, R. M., Demaria, M. & Campisi, J. Lamin B1 loss is a senescence-associated biomarker. Mol Biol Cell 23, 2066-2075 (2012).

12. Haferkamp, S. et al. The relative contributions of the p53 and pRb pathways in oncogene-induced melanocyte senescence. Aging (Albany NY) 1, 542-556 (2009).

Citation Format: Masashi Narita. Chromatin architecture and gene regulation in oncogene-induced senescence. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY02-03. doi:10.1158/1538-7445.AM2013-SY02-03