Introduction
Most cancer cells undergo periods of genotoxic stress due to their inherent genetic instability, and as a consequence of chemotherapy. In addition to the essential functions of DNA repair and checkpoint control in responding to genotoxic stress, recent work also implicated innate immune responses. Together, DNA damage signalling and innate immune signalling determine the cellular outcome of genotoxic stress, and trigger inflammation, cell death and senescence, which can determine cancer development and therapy effects (Fig. 1). However, how the innate immune system responds to chromosomal aberrations, and the consequences for the cell, are poorly understood.
The major innate immune sensor of pathogen DNA is the cyclic GMP-AMP synthase cGAS, which triggers inflammation, senescence
and apoptosis upon activation by pathogen DNA (Fig. 2a). Under unperturbed conditions, self-DNA is shielded from activating cGAS, but this is negated in response to genotoxic stress, making cGAS a major regulator of cancer development and therapy responses.
We previously asked the question, How is cGAS prevented from being activated by self-DNA? A major feature distinguishing self-DNA from the DNA of most pathogens is its organisation into chromatin, with the nucleosome, formed by ~150 bp of DNA wrapped around histone protein octamers at its centre. Our recent work showed that it is indeed this organisation into chromatin, which marks
self-DNA (Fig. 2b), thereby providing a mechanism for how cGAS activation is prevented during normal growth, with important implications for how self-sensing occurs during genotoxic stress.
Our current work is aimed at understanding how self-DNA triggers immune responses during genotoxic stress, and how this locks cells into the important fates of inflammation, cell death and senescence.
Chromatin Functions in Innate Immune Responses
Human cGAS uses three surfaces to interact with DNA, which are all required for activation. Additionally, DNA mediates cGAS dimerisation, which is also required for activity. Through cell biology, reconstitution biochemistry and structural biology, we understand the mechanism of chromatin inhibition of cGAS in molecular detail (Fig. 3a). All these DNA binding sites, as well as dimerisation, are blocked when cGAS is bound by nucleosomes. Most dramatically, DNA binding site B of cGAS is repurposed to bind the acidic patch of histones H2A–H2B within the context of the nucleosome.
The cGAS:nucleosome structure revealed cGAS protomers to be sandwiched in between nucleosomes, which can lead to the formation of long cGAS:nucleosome stacks (Fig. 3a and b). This has important implications and raises a number of critical questions: Does cGAS binding affect chromatin structure and function, and – conversely – does chromatin structure affect cGAS binding and activity? Are other innate immune processes also regulated by chromatin? We are currently tackling these questions using a multi-disciplinary approach combining tissue culture, biochemistry and bioinformatics.
A Cell-Free System to Understand Innate Immune Responses
It is unknown how genotoxic stress relieves the silencing of innate immune self-sensing. Studying this question is difficult in cellular model systems due to complex regulation through processes such as cell cycle stage, chromatin organisation and signalling feedback.
In collaboration with Hasan Yardimci from the Francis Crick institute, we are bridging this gap by using a novel cell-free approach. This is based on cytosolic preparations from eggs of the African clawed frog Xenopus laevis, known as Xenopus egg extracts (Fig. 4). These extracts accurately recapitulate complex intracellular biology (such as chromatin organisation, nuclear assembly, DNA repair, DNA replication, and mitotic spindle assembly) in a biochemically accessible manner. We have adapted these extracts for studying cGAS activation, and are currently using them to determine cGAS regulation, and to reconstitute other innate immune processes.
Cellular Decision-Making Processes in the Response to Genotoxic Stress
How does innate immune signalling ultimately lead to cellular consequences such as inflammatory signalling, cell death or growth arrest? How does innate immune signalling drive cells into these fates? And how can these processes be manipulated? We are using a combinatorial approach based on genetic screening, automated imaging and large-scale gene expression analysis, as well as proteomics, to address these questions.
Laboratory of Genome Stability and Innate Immunity