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

转录因子在关键组织中的正确时空表达对骨骼发育和动态平衡至关重要。在老化的软骨细胞(关节软骨中的单细胞类型)中分解代谢因子的上调与软骨破坏之间存在着公认的联系，然而，关于这些蛋白质协调的跨调控网络却知之甚少。蛋白水解靶向嵌合体(PROTACs)是一种异双功能降解物，由一个配体组成，该配体与感兴趣蛋白(POI)结合，并连接到E3-连接酶招募部分(图1a)。招募的E3-连接酶催化POI泛素化(图1b)，目标是蛋白酶体降解(图1c)。最近的一篇文章描述了转录因子靶向嵌合体(TRAFTACs)。在Cas9系统的基础上，TRAFTAC利用转录因子与特定DNA序列结合的固有能力。TRAFTAC嵌合体是一个单链CRISPR-RNA，连接到感兴趣的TF(TOI)的双链DNA基序，并与催化死亡的Cas9-HaloTag(dCas9-HT)蛋白共表达(图1d)。该复合体与HaloPROTAC(通过HaloTag，图1e)一起招募TOI(通过TRAFTAC)，导致泛素化(图1f)，并靶向降解TOI。在临床前研究中，与依赖内源性蛋白质周转的CRISPR和siRNA等替代方法相比，TRAFTAC具有巨大的优势，因此在靶向丰富/稳定的蛋白质时效率低下。TRAFTAC为研究TFS在肌肉骨骼衰老中的作用提供了一种新的工具。首席主管与Crews Lab(耶鲁大学)合作建立和优化了所需的细胞系(图1g)。本项目的主要目的是使用TRAFTAC通过多组学方法了解与年龄相关的软骨细胞调控网络。通过一系列补充的工作包，学生将：1.在dCas9-HT永生化软骨细胞中设计、表达和优化TRAFTAC。Western blotting和报告分析证实TFs的靶向性降解。2.建立可诱导的脂肪来源干细胞dCas9-HT系。在软骨分化后，用TRAFTAC转染细胞并在3D(图1h)中培养以模拟关节环境。进行组织学分析、RNA-seq和蛋白质组学实验以进行反式调节网络分析。3.明确人类发育和衰老的原代关节软骨细胞的蛋白质组特征，以确定与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。该项目将研究转录因子在人类骨骼中介导的衰老过程，主要目标是了解导致老年人类软骨健康下降的跨调控网络。这项研究将有助于我们了解人类寿命和肌肉骨骼健康寿命之间的差异。通过将老年患者的原始组织与人类胎儿样本进行比较，以研究发育因素如何影响晚年的健康，这一点最终将得到巩固。我们的目标是产生新的知识，以促进再生生物学，并改善老龄化人口的生活质量。

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.