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

关键组织中转录因子（tf）的正确时空表达对骨骼发育和体内平衡至关重要。在老化的软骨细胞（关节软骨中的单细胞类型）中，分解代谢tf的上调与软骨破裂之间存在着明确的联系，然而，对这些蛋白质精心策划的反调节网络知之甚少。蛋白水解靶向嵌合体（PROteolysis TArgeting Chimeras, PROTACs）是一种异双功能降解物，由一个与目标蛋白（POI）结合的配体组成，该配体与e3连接酶招募片段相连（图1a）。募集的e3连接酶催化POI泛素化（图1b），靶向其进行蛋白酶体降解（图1c）。最近的一份出版物描述了转录因子靶向嵌合体（TRAFTACs）。基于Cas9系统，traftac利用tf结合特定DNA序列的内在能力。TRAFTAC嵌合体是一种单链CRISPR-RNA，与目标TF （TOI）的双链DNA基元结合，并与催化死亡的Cas9-HaloTag （dCas9-HT）蛋白共表达（图1d）。该复合物招募TOI（通过TRAFTAC）和HaloPROTAC（通过HaloTag，图1e），导致泛素化（图1f），并有针对性地降解TOI。在临床前研究中，traftac与CRISPR和siRNA等其他蛋白质消耗方法相比具有巨大优势，这些方法依赖于内源性蛋白质周转，因此在靶向丰富/稳定的蛋白质时效率低下。traftac为研究tf在肌肉骨骼老化中的作用提供了一种新的工具。主要主管与耶鲁大学克鲁斯实验室合作建立和优化所需的细胞系（图1g）。该项目的主要目的是利用多组学方法利用traftac来了解与年龄相关的软骨细胞调节网络。通过一系列的补充工作包，学生将：在dCas9-HT永生化软骨细胞中设计、表达和优化traftac。通过western blotting和报告基因试验确认tf的靶向降解。2. 建立可诱导的脂肪来源干细胞dCas9-HT系。软骨分化后，用traftac转染细胞并在3D环境中培养（图1）以模拟关节环境。进行组织分析，RNA-seq和蛋白质组学实验进行跨调控网络分析。3. 定义人类发育和衰老原发性关节软骨细胞的蛋白质组学特征，以确定年龄相关的表型转移。将traftac应用于衰老的原代细胞，目的是改善年龄相关的表型。该项目将研究人类骨骼中由tf介导的衰老过程，其总体目标是了解导致老年人软骨健康下降的跨调节网络。这项研究将有助于我们了解人类寿命与肌肉骨骼健康寿命之间的差异。通过比较老年患者的原发组织与人类胎儿样本，研究发育因素如何影响晚年的健康，最终将巩固这一点。我们的目标是产生新的知识，以推进再生生物学和提高生活质量的人口老龄化。

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.