Applying a novel PROTAC technology in stem cells to elucidate the regulatory role of transcription factors in cartilage ageing

The correct spatiotemporal expression of transcription factors (TFs) in key tissues is essential for skeletal development and homeostasis. There is a well-established link between upregulation of catabolic TFs in ageing chondrocytes (the single cell type in articular cartilage) and cartilage breakdown, however little is known about the trans-regulatory networks orchestrated by these proteins.
PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional degraders consisting of a ligand that binds to a protein of interest (POI), linked to an E3-ligase recruiting moiety (Fig.1a). The recruited E3-ligase catalyses POI ubiquitylation (Fig.1b), targeting it for proteasomal degradation (Fig.1c). A recent publication described TRAnscription Factor TArgetting Chimeras (TRAFTACs). Based upon the Cas9 system, TRAFTACs exploit the intrinsic capacity of TFs to bind to a specific DNA sequence. TRAFTAC chimeras are a single-stranded CRISPR-RNA bonded to a double-stranded DNA motif for the TF of interest (TOI) and are co-expressed with a catalytically dead Cas9-HaloTag (dCas9-HT) protein (Fig.1d). The complex recruits the TOI (via the TRAFTAC) along with a HaloPROTAC (via the HaloTag, Fig.1e), leading to ubiquitylation (Fig.1f), and targeted degradation of the TOI. In pre-clinical investigations, TRAFTACs offer huge advantages over alternative methods for protein depletion including CRISPR and siRNA, which rely on endogenous protein turnover, thus are inefficient when targeting abundant/stable proteins.
TRAFTACs present a novel tool for investigating the role of TFs in musculoskeletal ageing. The primary supervisor has worked in collaboration with the Crews Lab (Yale) to establish and optimise the required cell line (Fig.1g). The primary aim of this project is to employ TRAFTACs to understand age-associated chondrocyte regulatory networks using a multi-omics approach.

Through a series of complementary work packages, the student will:
1. Engineer, express, and optimise TRAFTACs in the dCas9-HT immortalised chondrocytes. Confirm targeted degradation of TFs by western blotting and reporter assays.
2. Create an inducible adipose-derived stem cell dCas9-HT line. Following chondrogenic differentiation, transfect cells with TRAFTACs and culture in 3D (Fig.1h) to mimic the joint environment. Conduct histological analyses, RNA-seq, and proteomic experiments for trans-regulatory network analyses.
3. Define the proteomic signatures of human developmental and aged primary articular chondrocytes to identify the age-associated phenotypic shift. Apply TRAFTACs to aged primary cells, with the aim of improving the age-associated phenotype.

This project will investigate the ageing processes mediated by TFs in the human skeleton with the overarching goal of understanding the trans-regulatory networks that contribute to a decline in human cartilage health in older age. This research will contribute to our knowledge on the discrepancy between human lifespan and musculoskeletal healthspan. This will ultimately be consolidated through comparing primary tissues from aged patients to human foetal samples to investigate how developmental factors can impact upon health in later life. We aim to generate new knowledge to advance regenerative biology and improve the quality of life for the ageing population.