Research

Mechanisms of cellular senescence

Cellular senescence is a state of stable cell cycle arrest with active metabolism. Similar to apoptosis, senescence can be a failsafe program against a variety of cellular insults. In contrast to apoptosis, in which cytotoxic signals converge to a common mechanism, senescence is typically a delayed stress response involving multiple effector mechanisms. These effector mechanisms include epigenetic regulation, the DNA damage response, and the senescence-associated secretion phenotype. The relative contribution of these effectors varies depending on the trigger and cell type, and it is possible that the combination and balance of these effectors determines the quality of the senescence phenotype. Thus, to understand the senescence program, it is important to identify new effector mechanisms and examine how they associate with each other, and also to identify which effector mechanisms could be potential targets for cancer therapy.

Identification of senescence-associated chromatin factors

To further understand the molecular basis underlying the irreversibility of senescence arrest, we have been using a biochemical approach to analyse the alteration of the cell's chromatin protein profile during senescence. Using SDS-PAGE to visualize the protein composition of each chromatin preparation, we have identified HMGA proteins as new components of senescence (Narita et al., Cell 2006; 126: 503). Considering the identification of HMGA proteins in this system as a proof of concept, we are currently taking a more systematic approach for a thorough analysis of the chromatin protein profile in senescent cells. So far, we have several candidate proteins that specifically associate with chromatin to form senescent cells. Now we are in the process of verifying and analysing them in the context of senescence.

Genome-wide analysis of heterochromatin components in SAHFs

Certain types of cells undergo distinct alterations in chromatin structure during senescence, called senescence-associated heterochromatic foci (SAHF). SAHF have been widely used as a marker of senescence, and more importantly, several new components of senescence machinery have been successfully identified using SAHF as a readout. Thus, it is important to understand SAHF structure in more detail and how SAHF are actually formed. Interestingly, we have shown that SAHF are indistinguishable from the inactive X (Xi) chromosome, one of the best studied heterochromatin models, and other groups recently suggested that each individual SAHF might represent each chromosome territory. In contrast to Xi, SAHF formation can be dynamically regulated in normal human diploid fibroblasts (HDFs), thus providing a unique tool to study not only senescence, but also chromatin biology. To further characterise SAHF in detail, we are currently investigating a dynamic redistribution of the specific histone modifications and their adaptor proteins using confocal microscopy, with a new panel of highly specific monoclonal antibodies against histone marks (in collaboration with Dr. Hiroshi Kimura, Osaka University, Japan). In addition, we are currently analysing the genome-wide redistribution of these chromatin components by chromatin-IP coupled with deep sequencing (ChIP-seq).

From mRNA to proteins

Oncogene induced senescence (OIS) is a very dynamic process where cells typically undergo an initial burst of cell proliferation ('mitotic phase'), followed by the induction of pro-senescent factors, including p16 and HMGA2 ('transition phase'). Eventually, the senescent phenotype dominates ('senescence phase'). During the transition phase, oncogenic and pro‑senescence activities work against each other, and senescence usually prevails in normal cells. How cells can achieve such a drastic phenotype remodelling is unclear. A new area of interest in my group is in another layer of gene expression control, namely protein metabolism, during the senescence and transition phases. We reason that global epigenetic alteration should be coupled with efficient protein turnover as a part of the execution of epigenetic 'blue prints', in such an emergent context (Figure 1). Consistent with this idea, we have identified that autophagy, a bulk protein degradation program, facilitates synthesis of IL6/8, which are pro-senescence secretory proteins (Young et al., Genes Dev. 2009; 23: 798). This new functional link between senescence and autophagy will be further characterised in the future.

Concept of stress-responsive gene regulation (Narita report 2010; figure 1)
Figure 1
Concept of stress-responsive gene regulation.

Identification of senescence-associated p53 function

A tumour suppressive transcription factor,p53, plays a critical role in many stress responsive phenotypes, including DNA damage checkpoints, apoptosis, and senescence. Although ample data have supported a role for p53 in senescence, the precise mechanism is not clear. To address this issue, we are currently using HDFs, where we can induce different phenotypes depending on environmental stimuli or other conditions (Figure 2). By adding either retroviral- or lentiviral-mediated stable RNAi to HDFs, we are comparing the impact of p53 knockdown on the gene expression profile in each condition, which represents a phenotypespecific p53 function. We have finished the array experiments, and are now attempting to build a comprehensive picture of p53's functions. So far, in a primary analysis, we have identified several genes whose products are upregulated in a p53-dependent manner during senescence, but not in the other stress responsive contexts (e.g. apoptosis). We are currently undertaking functional verification of one of the genes.

Model system for understanding a comprehensive picture of p53 functions (Narita report 2010; figure 2)
Figure 2
Model system for understanding a comprehensive picture of p53 functions. HDFs are genetically defined cells and we can induce indicated phenotypes by different triggers or at different time points. In each phenotype, p53 can be stably and acutely down-regulated by retroviral or lentiviral RNAi.