Understanding and targeting chromatin-surveillance mechanisms in human ageing

Human cells are highly organised and regulated, and any change in this organisation can affect the entire body. It has become evident that one crucial part of the cell is a structure called the nucleus, which generally looks like a sphere-shaped compartment that contains the well-packaged genetic code: DNA. This is why the nucleus is considered to act as a control-center for the cell. The nucleus and its DNA content are formidably organized. One of the elements that contribute to this organization is a mesh-like network composed of filaments made up of a protein called lamin-A/C. Interestingly, this mesh is disrupted in cells from patients that suffer premature ageing as well as in cells from elderly individuals. When the lamin-A/C mesh is disrupted, the nuclei become misshapen and as a result cells become generally unhealthy and grow very slowly, or even stop dividing. This suggests that defects in the lamin-A/C mesh contribute to ageing, but how this happens is not completely understood at the moment.

We have discovered a trick that a cell uses to sense problems with the organization of it nucleus and DNA. This trick involves the marking and stimulation of a protein enzyme called KAT5. Because of our discovery, we thought that KAT5 may become marked and stimulated in ageing cells, where it may contribute to making ageing cells unhealthy and causing their nucleus to become misshapen. Indeed, our initial results support this idea because we found that KAT5 becomes stimulated in ageing cells. Importantly, we also found that when we stop KAT5 stimulation (using an available medicine) in ageing cells, their nucleus becomes normal and they start dividing again. Therefore, our initial work has revealed a new explanation for how ageing cells become unhealthy, and has established a novel strategy that can render these aged cells healthy again.

This proposed work describes experiments to help us increase our understanding of how marked (stimulated) KAT5 makes cells unhealthy and determine exactly how stopping the marking of KAT5 and its stimulation help in correcting the problems that ageing cells face. In order to achieve these, we set four specific aims:

1. We will use a complementary (and a more definitive) strategy to stop KAT5 stimulation and examine the effect of using this strategy on turning ageing cells into healthy ones again.

2. We will determine how exactly KAT5 becomes marked and stimulated in ageing cells.

3. We will examine the direct consequences of stimulating KAT5 on making ageing cells unhealthy.

4. We will identify the reason why stopping KAT5 stimulation helps ageing cells recover.

We believe that this work could improve our fundamental understanding of the ageing process, and will provide us with new insights into how stimulated KAT5 contributes to these events. Moreover, this work will also open up new perspectives for therapeutic intervention in premature ageing diseases, which are also likely to be useful in reducing normal ageing-associated frailty. Our work has the potential to improve health and well-being in a population with increased life expectancy; which of course in the long term may have great impact on public health.

A common cellular feature between progeroid laminopathies and physiological ageing is chromatin disorganization. Yet, the mechanistic understanding and therapeutic reversal of these chromatin perturbation's onset remain challenging. Having recently discovered the c-Abl-mediated KAT5 phosphorylation (KAT5-pY44) as a mechanism for chromatin surveillance; here, we hypothesized that this pathway operates in cell-models of ageing. Thus, we detected KAT5-pY44 in progeria and lamin-A/C-depleted cells, and found that drug-based inhibition of KAT5-pY44 reverses their chromatin disorganization and alleviates their overall cellular defects. Hence, our central hypothesis is that KAT5-pY44 senses initial chromatin perturbations in ageing cells, thereby contributes to the onset of further defects in chromatin organization resulting in overall cellular unfitness. This proposal seeks to mechanistically determine how KAT5-pY44 ensues upon lamin-A/C loss-of-function and to establish the molecular bases for the protective effect of inhibiting KAT5-pY44. We will use cell-based genetic complementation systems and pharmacological inhibitions in combination with quantitative cellular and molecular biology approaches to determine:
-The direct contribution of inhibiting KAT5-Y44 phosphorylation to the cellular amelioration of lamin-A/C-depleted cells.
-How KAT5-Y44 becomes phosphorylated in progeria and lamin-A/C-depleted cells.
-The direct effects of KAT5-pY44 in progeria and lamin-A/C-depleted cells.
-How inhibiting KAT5-pY44 mechanistically increases the fitness of lamin-A/C-defective cells.
This study will provide new mechanistic insight into the causal contribution of chromatin disturbances to the overall defects in ageing cells, and will shed light on this challenging research area. Also, it will open potential therapeutic opportunity for laminopathies with possible impact on improving life-quality during normal ageing, which may in the longer term influence public health.

This proposal addresses the MRC's strategic objective to "understanding tissue disease and degeneration and advancing knowledge in the biology of ageing...". Accordingly, the proposed work unravels a novel role for KAT5-mediated chromatin surveillance in experimental models of human ageing associated with defective nuclear lamina/architecture. This research will increase our understanding of the regulation of chromatin organization and surveillance in relation to the overall functions within the nucleus, and thus will stimulate further work in these fields. Consequently, we strongly believe that the results arising from our research will have a significant impact in a number of different areas:
-It will impact on researchers working on other human diseases where chromatin disturbances are observed, including cancer and neuro-degeneration. These researchers will benefit from this work upon its dissemination by using similar experimental approaches as well as novel reagents (e.g. antibody).
-It will impact on academics working in the fields of nuclear architecture, chromatin organization, cell cycle, and DNA damage; as our results will mechanistically shed light on how nuclear lamina defects can affect chromatin organization, checkpoint signaling, and cell proliferation.
-It will impact on laminopathy-related fields of study as well as on researchers studying cellular and organism ageing.
-The results arising from our research will provide insights into how to rescue nuclear defects in progeria/laminopathies-associated ageing and could thus not only help us understand these diseases but may also suggest new ways of treating these incurable diseases in the longer term.
-Premature ageing diseases are associated with the similar nuclear defects as those arising during normal ageing. Our research might thus provide important information on how to improve the global cellular fitness in both premature and normal ageing.
-Our findings may be of great value to the global health system in suggesting possible ways to improve the health and quality of life of the elderly.
-Consequently, our work may in the longer term directly impact on the elderly population.
-Our work will be of interest for non-scientific audiances since it will help explain the mechanisms of ageing and age-related cellular pathologies, something that is appreciated by society.
-Our research might open new perspectives on how to delay age-associated phenotypes and signs of ageing, and could thus be of great interest to the pharma, biotech and cosmetics industries which might wish to exploit our findings to benefit human health and quality-of-life, and for commercial benefit for UK-based organizations.
-Indeed, our study suggests targeting KAT5-pY44 to alleviate cellular ageing using existing small molecule therapeutics (c-Abl inhibitors). Further, there is potential for inhibiting the pathological KAT5-pY44 through rational drug design to target its chromodomain. The research outlined in this proposal will provide the rationale for such a novel therapeutic strategy, and thus will be of direct use to industry. The Elizabeth Blackwell Institute and the Research Commercialization team in the Research and Enterprise Development Office will facilitate in developing discussions with key industrial partners.
-The Postdoctoral Researcher working on this project will gain key skills in cutting-edge research techniques, scientific communication, and supervision. The Researcher will play a central role in the reporting of the work, both by presentation at scientific meetings and publications. The experience obtained by the postdoctoral researcher will be equally applicable to a career in academia, industry, and other science-related endeavors. Indeed, the Investigator previously supervised an Amgen Scholar and as an MPhil student, who is currently reading for a PhD at CRUK Cambridge Research Institute, as well as a Research Technician who now works at Illumina.