University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, OH, USA.
Institute of Biochemistry, University of Giessen, 35392 Giessen, Germany.
Nucleic Acids Res. 2022 Dec 9;50(22):13083-13099. doi: 10.1093/nar/gkac1137.
The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.
Notch 通路在相邻细胞之间传递信号，引发下游转录程序。Notch 是细胞命运特化、增殖和凋亡的主要调节剂，因此异常信号会导致多种人类疾病，包括发育障碍和癌症。该通路通过转录因子 CSL（哺乳动物中的 RBPJ）发出信号，CSL 与 Notch 受体的细胞内域和共激活子 Mastermind 形成激活复合物。CSL 还可以通过与几种不同的核心抑制蛋白形成复合物来发挥转录抑制作用，如哺乳动物中的 FHL1 或 SHARP 和果蝇中的 Hairless。最近，我们鉴定出 L3MBTL3 是一种真正的 RBPJ 结合核心抑制蛋白，它可以募集具有抑制性的赖氨酸去甲基酶 LSD1/KDM1A 到 Notch 靶基因上。在这里，我们定义了 L3MBTL3 与 RBPJ 的相互作用域，并报告了 RBPJ-L3MBTL3-DNA 复合物的 2.06 Å 晶体结构。该结构揭示了 L3MBTL3 通过与其他 RBPJ 结合伙伴相比不寻常的结合基序与 RBPJ 相互作用，我们使用一系列基于结构的突变体对其进行了全面分析。我们还表明，这些破坏突变会影响细胞中的 RBPJ 和 L3MBTL3 功能，为 Notch 介导的转录调控提供了进一步的见解。
