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稳态至关重要。老化软骨细胞（关节软骨中的单细胞类型）中分解代谢TF的上调与软骨分解之间存在明确的联系，但对这些蛋白质协调的反式调节网络知之甚少。PROTAC（PROteolysis Targeting Chimeras）是一种异双功能降解剂，由一个与目标蛋白（POI）结合的配体组成，该配体与一个E3连接酶募集部分连接（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）。最近的出版物描述了TRANscription Factor Targetting Chimeras（TRAFTAC）。基于Cas9系统，TRAFTAC利用TF的内在能力结合特定的DNA序列。TRAFTAC嵌合体是与目标TF（TOI）的双链DNA基序结合的单链CRISPR-RNA，并与催化死亡的Cas9-HaloTag（dCas 9-HT）蛋白共表达（图1d）。该复合物沿着与HaloPROTAC（通过HaloTag，图1 e）一起募集TOI（通过TRAFTAC），导致泛素化（图1f）和TOI的靶向降解。在临床前研究中，TRAFTAC比CRISPR和SiRNA等替代蛋白质去除方法具有巨大优势，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在肌肉骨骼衰老中的作用提供了一种新工具。主要主管与Crews Lab（耶鲁大学）合作，建立并优化所需的细胞系（图1g）。本项目的主要目的是利用TRAFTACs来了解与年龄相关的软骨细胞调控网络，使用多组学方法。在dCas 9-HT永生化软骨细胞中工程化、表达和优化TRAFTAC。通过蛋白质印迹和报告基因测定确认TF的靶向降解。2.创建诱导型脂肪来源的干细胞dCas 9-HT系。软骨形成分化后，用TRAFTAC转染细胞并在3D中培养（图1h）以模拟关节环境。进行组织学分析、RNA-seq和蛋白质组学实验以进行反式调控网络分析。3.定义人类发育和老化的原代关节软骨细胞的蛋白质组特征，以确定年龄相关的表型转变。将TRAFTAC应用于老化的原代细胞，目的是改善与年龄相关的表型。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反式调节网络。这项研究将有助于我们了解人类寿命和肌肉骨骼健康之间的差异。这最终将通过比较老年患者的初级组织与人类胎儿样本来研究发育因素如何影响晚年的健康来巩固。我们的目标是产生新的知识，以促进再生生物学和提高老龄人口的生活质量。

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.