Table7_The transcription factor LEF1 interacts with NFIX and switches isoforms during adult hippocampal neural stem cell quiescence.xlsx

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical β-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active β-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.

成年哺乳动物组织中的干细胞会长时间处于一种可逆的静息状态，该状态被称为静息态（quiescence）。近年来的研究极大地加深了我们对干细胞静息态背后表观遗传（epigenetic）与转录调控图谱的认知。然而，主动维持静息态程序的转录因子调控代码（transcription factor code）仍未得到明确阐释。同样，在干细胞静息态中影响转录因子的可变剪接（alternative splicing）事件也长期被忽视。本研究发现，经典依赖β-连环蛋白（β-catenin）的Wnt信号通路核心调控因子——转录因子T细胞因子/淋巴增强结合因子1（LEF1），在静息态神经干细胞中发生可变剪接并发生亚型转换。研究团队观察到，活性β-连环蛋白与其结合因子LEF1在静息态海马神经干细胞及前体细胞（quiescent neural stem and progenitor cell, Q-NSPC）培养物中出现富集。相应地，静息态海马神经干细胞及前体细胞表现出增强的TCF/LEF1介导的转录活性，以及基础Wnt信号活性，该活性可在依赖Wnt的实验中为培养细胞赋予功能优势。在分子机制层面，本研究发现Lef1基因的表达受到精细调控。Lef1转录的协同上调与可变剪接外显子6（E6）的保留，共同导致全长蛋白亚型（LEF1-FL）的积累，该亚型在静息态中稳定性增强。从出生后海马体中前瞻性分离得到的GLAST阳性细胞，在体内静息态建立的过程中同样发生了E6保留。值得注意的是，LEF1结合基序在静态海马神经干细胞及前体细胞中上调基因的静息态相关增强子（enhancer）中显著富集，且静息态相关核因子I X（NFIX）转录因子结合基序紧邻LEF1结合位点。本研究进一步证实LEF1可与NFIX发生相互作用，并鉴定出潜在的LEF1/NFIX共同靶基因。综上，本研究结果揭示了LEF1在静息态神经干细胞及前体细胞基因调控中此前未被发现的功能，并阐明可变剪接作为转录后调控机制，参与了干细胞激活向静息态的转变过程。