Establishing the importance of DNA helicases and G-quadruplex homeostasis for the maintenance of proteome integrity with age
Lead Research Organisation:
University College London
Department Name: Genetics Evolution and Environment
Abstract
The accumulation of misfolded, mislocalised and aggregated proteins (also known as proteostasis collapse) is a highly conserved driver of age-related tissue dysfunction in worms, flies, mice and humans. Our work in the small nematode worm C. elegans has demonstrated that, contrary to long-held assumptions, age-related proteostasis collapse does not simply arise from the accumulation of molecular damage, but instead, emerges from the transcriptional remodelling of the Proteostasis Network (PN; the complete collection of mechanisms that cooperate to maintain proteome integrity) early in life. Given that similar observations have been made in flies and human brain tissues, maintaining the expression of PN genes with age may be a potent way to maintain proteome integrity and prolong healthy tissue function. However, at present, it remains unclear precisely why the expression of PN genes changes with age and how this could be prevented.
One currently unexplored and provocative possibility is that alterations in the structure of the genome itself may underlie changes in the expression of PN genes during adulthood. Non-helical DNA secondary structures known as G-quadruplexes (G4s) are highly prevalent across the genome and have emerged as important regulators of gene expression. While dynamic G4 formation can promote transcription factor binding and chromatin accessibility, the stabilisation of G4s can impede RNA polymerase II and repress transcription. As such, maintaining G4 homeostasis is crucial for normal cell function.
G4 formation and stability is regulated by the action of DNA helicases, which bind to and actively resolve DNA secondary structures. We recently discovered that the expression of two prominent G4 resolving DNA helicases, wrn-1/WRN and him-6/BLM, declines prior to the loss of proteostasis capacity during early C. elegans adulthood, and that G4 structures are associated with candidate PN genes known to be down-regulated prior to protoestasis collapse. In addition, we find that reduced WRN-1 and HIM-6 activity leads to the repression of PN genes and accelerates the loss of proteostasis capacity in muscle tissues. These observations raise the intriguing possibility that reduced DNA helicase activity and increased G4 stability may be among the earliest events governing the loss of proteostasis capacity with age, and that by maintaining WRN/BLM activity or G4 homeostasis in aged cells, it may be possible to protect the ageing proteome.
Here, we propose to explore this possibility by determining the precise relationship between DNA helicase activity, G4 homeostasis, proteostasis collapse and long-term tissue health. We will use a combination of tissue-specific proteostasis sensors and fluorescent reporters/probes to establish the specific tissues in which compromised WRN-1/HIM-6 activity and impaired G4 homeostasis underlies proteostasis collapse. In addition, we will combine genomics, mass-spectrometry-based proteomics and genetic screening approaches to identify the precise genes and proteins that are functionally relevant to age-related proteostasis collapse and tissue dysfunction downstream of reduced WRN-1/HIM-6 activity and compromised G4 homeostasis. Finally, we will genetically engineer animals to preserve wrn-1 and him-6 expression with age and use small molecule "helicase mimetics" to suppress G4 stabilisation in order to prevent transcriptional remodelling of the PN early in adulthood, maintain proteostasis capacity throughout life and promote healthy tissue function with age.
We expect that this work will establish a new relationship between DNA secondary structure and the long-term health of the proteome, thereby acting as a foundation for future studies aimed at developing small molecule "helicase mimetics" that can be directed to specific genomic loci in order to discretely maintain the expression of select PN genes, maintain proteome integrity and promote healthy tissue function.
One currently unexplored and provocative possibility is that alterations in the structure of the genome itself may underlie changes in the expression of PN genes during adulthood. Non-helical DNA secondary structures known as G-quadruplexes (G4s) are highly prevalent across the genome and have emerged as important regulators of gene expression. While dynamic G4 formation can promote transcription factor binding and chromatin accessibility, the stabilisation of G4s can impede RNA polymerase II and repress transcription. As such, maintaining G4 homeostasis is crucial for normal cell function.
G4 formation and stability is regulated by the action of DNA helicases, which bind to and actively resolve DNA secondary structures. We recently discovered that the expression of two prominent G4 resolving DNA helicases, wrn-1/WRN and him-6/BLM, declines prior to the loss of proteostasis capacity during early C. elegans adulthood, and that G4 structures are associated with candidate PN genes known to be down-regulated prior to protoestasis collapse. In addition, we find that reduced WRN-1 and HIM-6 activity leads to the repression of PN genes and accelerates the loss of proteostasis capacity in muscle tissues. These observations raise the intriguing possibility that reduced DNA helicase activity and increased G4 stability may be among the earliest events governing the loss of proteostasis capacity with age, and that by maintaining WRN/BLM activity or G4 homeostasis in aged cells, it may be possible to protect the ageing proteome.
Here, we propose to explore this possibility by determining the precise relationship between DNA helicase activity, G4 homeostasis, proteostasis collapse and long-term tissue health. We will use a combination of tissue-specific proteostasis sensors and fluorescent reporters/probes to establish the specific tissues in which compromised WRN-1/HIM-6 activity and impaired G4 homeostasis underlies proteostasis collapse. In addition, we will combine genomics, mass-spectrometry-based proteomics and genetic screening approaches to identify the precise genes and proteins that are functionally relevant to age-related proteostasis collapse and tissue dysfunction downstream of reduced WRN-1/HIM-6 activity and compromised G4 homeostasis. Finally, we will genetically engineer animals to preserve wrn-1 and him-6 expression with age and use small molecule "helicase mimetics" to suppress G4 stabilisation in order to prevent transcriptional remodelling of the PN early in adulthood, maintain proteostasis capacity throughout life and promote healthy tissue function with age.
We expect that this work will establish a new relationship between DNA secondary structure and the long-term health of the proteome, thereby acting as a foundation for future studies aimed at developing small molecule "helicase mimetics" that can be directed to specific genomic loci in order to discretely maintain the expression of select PN genes, maintain proteome integrity and promote healthy tissue function.
Technical Summary
The accumulation of aberrant protein species (proteostasis collapse) is a conserved driver of age-related tissue dysfunction that emerges from transcriptional remodelling of the Proteostasis Network (PN) during early adulthood. At present, the underlying basis for changes in the expression of PN genes and the loss of proteostasis capacity with age, remain poorly understood, thereby hampering attempts to promote healthy tissue function in aged individuals. Recently, we have discovered that the expression of two DNA helicases, wrn-1/WRN and him-6/BLM, declines prior to the onset of proteostasis collapse, and that this correlates with the formation of DNA G-quadruplex (G4) structures and transcriptional repression at PN genes. Furthermore, we find that reducing WRN-1 or HIM-6 activity through RNAi, accelerates the transcriptional repression of PN genes and the loss of proteostasis capacity during adulthood. These observations strongly suggest that reduced WRN-1/HIM-6 activity and loss of G4 homeostasis early in life promotes transcriptional remodelling of the PN and leaves cells vulnerable to age-related proteostasis collapse. Here, we will use a multi-disciplinary approach to explore this possibility. We will establish precisely how reduced WRN-1/HIM-6 activity and increased G4 stability impact proteostasis capacity in different tissues with age and use genetic and chemical approaches to maintain DNA helicase activity and suppress G4 stabilisation in adulthood, thereby protecting the ageing proteome and prolonging healthy tissue function. This work will re-shape our understanding of the origins of age-related proteostasis collapse and act as a precursor for future work aimed at designing small molecules that can discretely maintain the expression of key PN genes in aged cells, thereby preserving proteome integrity and prolonging healthy tissue function with age.
