N-Termini of Fungal CSL Transcription Factors Are Disordered, Enriched in Regulatory Motifs and Inhibit DNA Binding in Fission Yeast

BackgroundCSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins.
Methodology/Principal FindingsWe identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay.
Conclusions/SignificanceThis study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.

研究背景：
CSL（CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1）转录因子是Notch受体信号通路的效应分子，该通路对后生动物（metazoan）的发育具有关键作用。后生动物来源的CSL蛋白（M类）同样能够以不依赖Notch的方式行使功能。近期，研究人员在真菌中鉴定出两类新型CSL蛋白，分别命名为F1类与F2类。由于真菌体内不存在Notch信号通路，因此真菌CSL蛋白的功能仍不明确。在裂殖酵母中，CSL旁系同源蛋白Cbf11与Cbf12在细胞黏附以及细胞与细胞核分裂的协调过程中发挥拮抗作用。真菌与无脊椎动物的CSL家族成员普遍具有异常延长的N端延伸序列。本研究旨在探究CSL蛋白此类延长N端序列的功能意义。

研究方法与主要发现：
本研究从7个真菌物种中鉴定出15个全新的CSL家族成员，并对包含34个真菌CSL蛋白序列与11个后生动物CSL蛋白序列的整合数据集开展生物信息学分析。结果显示，真菌CSL蛋白较长且非保守的N端尾部大概率处于无序构象，且富含磷酸化位点与PEST基序。以F2类蛋白Cbf12为对象进行案例研究，我们通过实验证实该蛋白存在蛋白水解加工过程，并且在电泳迁移率变动分析（electrophoretic mobility shift assay）中，其N端序列会抑制Cbf12依赖的DNA结合活性。

研究结论与意义：
本研究揭示了真菌CSL蛋白延长N端尾部的特征，此类特征可能对调控DNA结合能力与CSL蛋白功能至关重要。我们提出，裂殖酵母通过Cbf12的N端区域调控DNA结合的方式，是平衡F1类与F2类旁系蛋白活性的重要手段。这种调控模式可能也存在于其他携带CSL蛋白的真菌中，其中部分真菌与人类疾病及生物技术应用密切相关。