Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor’s validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism. Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

转录因子EB（transcription factor EB, TFEB）与结合IGHM增强子3的转录因子（transcription factor binding to IGHM enhancer 3, TFE3）属于MiT/TFE家族转录因子，可结合靶基因调控区域内完全一致的DNA应答元件。二者参与调控溶酶体生物发生、功能、胞吐作用、自噬以及脂质分解代谢。对TFEB与TFE3活性的精准调控，对于细胞衰老、应激反应、能量代谢以及细胞分解代谢等生理过程至关重要。上述转录因子的调控异常与多种疾病密切相关，因此研究者们将这类转录因子视为潜在治疗靶点，探索可调控MiT/TFE活性的药理学手段。然而，其功能的生理复杂性以及缺乏适配的体内研究工具，制约了选择性MiT/TFE调控剂的开发。本研究构建了一种基于报告基因的生物传感器，命名为CLEARoptimized，可用于TFEB与TFE3介导的转录过程的药理学表征。这款创新工具可通过成像与生化技术，检测活细胞及活体小鼠体内的TFEB与TFE3活性。CLEARoptimized的核心序列包含6个协同溶酶体表达与调控（coordinated lysosomal expression and regulation, CLEAR）基序，该基序是通过对128个TFEB靶基因的启动子进行深度生物信息学分析筛选得到的。该生物传感器可驱动荧光素酶与tdTomato报告基因的表达，从而通过光学成像及生化实验，实现细胞与活体动物内TFEB和TFE3活性的定量检测。通过生理及药理学刺激手段，分别在细胞培养体系与报告基因小鼠模型中调控MiT/TFE活性，验证了该生物传感器的有效性。综上，本研究开发了一款创新工具，可在从单个细胞到完整生物体的多个生物学层次上，开展自噬与溶酶体通路调控的相关研究。缩写说明：CLEAR：协同溶酶体表达与调控（coordinated lysosomal expression and regulation）；MAR：基质附着区（matrix attachment regions）；MiT：小眼症相关转录因子（microphthalmia-associated transcription factor）；ROI：感兴趣区（region of interest）；TBS：Tris缓冲盐溶液（tris-buffered saline）；TF：转录因子（transcription factor）；TFE3：结合IGHM增强子3的转录因子（transcription factor binding to IGHM enhancer 3）；TFEB：转录因子EB（transcription factor EB）；TH：酪氨酸羟化酶（tyrosine hydroxylase）；TK：胸苷激酶（thymidine kinase）；TSS：转录起始位点（transcription start site）。