Global Reorganization of the Nuclear Landscape in Senescent Cells2015-01-29 00:04:51
Cell Reports; 29 January 2015 ; DOI:10.1016/j.celrep.2014.12.055
Tamir Chandra, Philip Andrew Ewels, Stefan Schoenfelder, Mayra Furlan-Magari, Steven William Wingett, Kristina Kirschner, Jean-Yves Thuret, Simon Andrews, Peter Fraser, Wolf Reik
Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation.
Cellular senescence is an irreversible cell-cycle arrest, originally described for primary cells after long-term cell culture and attributed to telomere attrition (Hayflick and Moorhead, 1961). More recently, cellular senescence has been established as a cellular response to a variety of stresses such as DNA double-strand breaks or oncogene activation (Di Leonardo et al., 1994, Lin et al., 1998 and Serrano et al., 1997).
Oncogene-induced senescence (OIS) is an intrinsic tumor suppressor mechanism, involving activation of the key tumor suppressor pathways p53 and pRB/p16INK4a. Inactivation of one or both of these pathways is found in the majority of cancers. Markers of senescence, such as p16 upregulation, are particularly prevalent in benign lesions and are often lost upon malignancy (Braig et al., 2005, Haugstetter et al., 2010 and Michaloglou et al., 2005). Reactivation of p53 in mouse models of liver cancer leads to senescence with subsequent immune clearance of cancer cells (Xue et al., 2007). A key aspect of the senescence response implicated in the immune clearance is the senescence-associated secretory phenotype (SASP) (Acosta et al., 2008, Coppé et al., 2008 and Kuilman et al., 2008). SASP is characterized through the secretion of cytokines, which are able to induce paracrine senescence in neighboring cells (Acosta et al., 2013). Recent work has implicated cellular senescence in normal developmental processes (Muñoz-Espín et al., 2013 and Storer et al., 2013).
In addition to its role in oncogenesis, a role for senescence in organismal aging has recently been substantiated; the depletion of senescent cells has been shown to relieve symptoms in mouse models of age-related diseases, suggesting that cellular senescence may be a useful model system for organismal aging (Baker et al., 2011 and López-Otín et al., 2013).
Previous work has shown that cellular senescence in human diploid fibroblasts is accompanied by a large-scale spatial rearrangement of chromatin, forming nuclear structures known as senescence-associated heterochromatic foci (SAHF). SAHF are enriched in constitutive heterochromatic markers, such as H3K9me3 and HP1 proteins (Narita et al., 2003). However, SAHF formation does not occur in all senescent cells. The proportion of cells exhibiting SAHF depends on the method of senescence induction, ranging from a few percent in replicative senescence to nearly 90% in c-raf OIS (Jeanblanc et al., 2012). In contrast, other cellular models of organismal aging such as cells from Hutchinson-Gilford progeria syndrome (HGPS) patients show a decrease in heterochromatin and are devoid of SAHF (Scaffidi and Misteli, 2006 and Shumaker et al., 2006). Cellular models of HGPS and cellular senescence of fibroblasts have proven to be relevant models for organismal aging. It is therefore important to understand the seemingly contradictory roles of heterochromatin in cellular aging and SAHF formation.
We have recently shown that SAHF chromosomes show an inversion of euchromatin, facultative heterochromatin (fHC), and constitutive heterochromatin (cHC), with cHC moving to the center of chromosomal territories (see also Figure 4C; Chandra et al., 2012). This inversion is due to a physical reorientation of the chromatin rather than a redistribution of repressive histone marks, questioning a causal role for classical heterochromatic marks, H3K9me3 (cHC) and H3K27me3 (fHC), in the formation of heterochromatin in somatic cells. A key feature of the senescent nucleus and strong correlate with SAHF formation is the loss of lamin B1 (Sadaie et al., 2013 and Shah et al., 2013). Other factors involved in SAHF formation, such as the cell-cycle regulator pRb, high mobility group proteins HMGA1/HMGA2, histone chaperones HIRA and ASF1a, canonical Wnt signaling, chromatin remodelling proteins p400 and BRG1, and linker histone H1, have been identified; however, knowledge of how the chromosome structure is changed is still lacking (Chan et al., 2005, Chicas et al., 2010, Funayama et al., 2006, Narita et al., 2003, Narita et al., 2006, Tu et al., 2013, Ye et al., 2007a, Ye et al., 2007b and Zhang et al., 2005). More importantly, the function of SAHF is controversial. Whereas the role of SAHF was initially reported as being tumor and cell-cycle suppressive (Narita et al., 2003 and Narita et al., 2006), recent work has suggested that SAHF may in fact be proproliferative (Di Micco et al., 2011).
To gain insight into the function of SAHF, we decided to unravel the physical structure of senescent chromatin in unprecedented detail, combining fluorescence in situ hybridization (FISH) with Hi-C to map the physical changes that accompany SAHF formation. We find dramatic changes in both the global interaction network and local neighborhood of genomic regions. Surprisingly, we find distinct global changes in the interactions and compaction of certain classes of lamin-associated domains, defined by continuous genomic fragments of homogenous guanine-cytosine (GC) content (isochores; Bernardi, 2012). Contrary to the current view of enhanced heterochromatinization in SAHF formation, we find a loss of internal structure in constitutive heterochromatic (cHC) regions in cellular senescence. This loss of internal structure is accompanied by spatial clustering of the cHC regions. We further show that HGPS cells behave similarly to senescent cells in their local interaction changes but do not exhibit the spatial clustering of cHC, suggesting a two-step mechanism for SAHF formation. Finally, we investigate embryonic stem cells (ESCs) and senescent cells and find a fundamentally opposing local architecture with somatic cells representing an intermediate state.
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