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

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.

process were carried out with equipment and protocols recommended by Affymetrix. The Ambion WT Expression kit, combined with the GeneChip® WT Terminal Labeling and Controls Kit (Affymetrix) was used to generate amplified and biotinylated sense strand DNA targets from the entire expressed genome. The software used for data analysis was Affymetrix® GeneChip® Command Console® (AGCC 3.1, Affymetrix®) and Expression Console™ (EC 1.1,Affymetrix®). Detailed information about the GeneChip technology and Rat Gene 1.1 ST used in this study can be found on the Affymetrix web page. dChip (www.dchip.org) (Li and Wong, 2001) and Partek Genomics Suite (www.partek.com) were used for the bioinformatic analysis. Data was deposited in the gene expression omnibus GEO dataset (GSE158658).

Estimation of exon retention
Efficiency of exon splicing was calculated by standard RT-PCR. Primers were designed at the boundaries of each Lef1 exon (Supplementary Table 4a) and PCR was performed employing cDNA obtained from Q-and A-NSPCs as template. The % exon retention was calculated using IS*100/(IS + ES) where IS (inclusion signal) corresponds to the intensity of the PCR band in gel electrophoresis that includes the exon and ES (exclusion signal) is the intensity of the band that excludes the exon. Values indicate the percentage of transcripts that contain the exon compared to the total transcript population.

RT-qPCR
RNA was extracted from proliferating and quiescent cells collected at 4 DIV using Illustra RNAspin Mini Kit (GE Healthcare). cDNA was obtained by reverse-transcription (RT) employing PrimeScript RT Reagent Kit (Takara). Gene expression was determined by quantitative PCR (qPCR) or standard PCR using TB Green Premix EX Taq (2×)(Takara) and the corresponding forward and reverse primer for each gene are listed in Supplementary  Table 4b. Sdha was used as the internal reference for normalization. Data were analysed according to the 2 −ΔΔCt method (Livak and Schmittgen, 2001

Protein dephosphorylation assay
Proteins were dephosphorylated before SDS-PAGE. Q-and A-NSPCs cells were lysed in the presence of protease inhibitors and crude extracts were treated with 1U of alkaline phosphatase (Thermo scientific, #EF0654) at 37ºC for one hour. To stop reaction EDTA 0.5M was added.

Wnt reporter assays
For the 7xTCF-eGFP-SV40-mCherry (p7GC) assay, NSPCs were plated in 24-well plates and were transduced with the p7GC lentiviral vector, produced as described in Supplemental experimental procedures. Next, NSPCs were washed and grown in proliferation or quiescence medium for 4 DIV. Gene expression of the eGFP WNT reporter gene was normalised to mCherry expression by RT-qPCR or alternatively, eGFP+ and mCherry+ cells were counted in a fluorescence inverted microscope LEICA DM IL LED with a 20X objective and images were acquired. For the TOPFlash Luciferase assay, Neuro2A cells were co-transfected with M50 Super 8x TOPFlash (plasmid #12456, Addgene), Renilla luciferase, pEGFP-Lef1FL and/or pEGFP-Lef1∆E6 (Jesse et al. 2010) plasmids using Polyethylenimine (PEI, 1mg/mL). EGFP (plasmid #6084-1, Clontech) and beta-catenin-CA plasmids were used as a control and co-factor respectively. Firefly and Renilla luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega) at 48 hr post-transfection.
To test for enrichment in enhancers versus flanking sequences, bootstrap confidence intervals of the ratio between motifs per kb inside the enhancers and in the 2.5kb flanking regions were calculated for each enhancer group separately. In each bootstrap replicate, the location of motifs with a score of 80% or higher was randomized taking into account spatial constraints due to masked and border regions. Out of the randomized motifs, one was selected at random per enhancer, since we were only considering the highest-scoring motif in each sequence, and the length-adjusted ratio between motifs inside enhancers and in the flanking regions was calculated for each replicate. A motif is considered to be enriched in enhancers when the observed inside/outside ratio falls above the 0.975 quantile of the simulated ratios. 100 bootstrap replicates were used. [Bootstrapped intervals for each motif and enhancer type can be found in Supplementary Table 6].
To test for differential enrichment of motifs between enhancer types, only motifs with the highest scoring within each enhancer were considered, and their positions were randomized 1000 times between enhancer regions of the three groups, taking into account spatial constraints. Confidence intervals for the proportion of motifs falling in each group were calculated, and a motif was considered to be differentially enriched in an enhancer group if the proportion of motifs in the enhancer groups was above or below the confidence interval.
[Bootstrapped intervals for each motif can be found in Supplementary Table 6].
In order to test for preferential LEF motif localization, we used the CentriMo tool of the MEME suite (Timothy, Bailey & Machanick, 2012), using default parameters. To search for motifs enriched in the vicinity of LEF putative binding sites, we retrieved the sequences of the LEF1 motif matches plus 15 flanking base pairs and used them as input for the DREME tool of the MEME suite (Timothy & Bailey, 2011). Enriched motifs were then fed to the TOMTOM program (Gupta et al., 2007) in order to find matches between enriched motifs and known PWMs.

Motif presence in genes differentially regulated in quiescent vs proliferative cells
We used RNA-seq and ChIP-seq data from (Martynoga et al., 2013) to test whether LEF1 motif was overrepresented in genes that were differentially regulated between quiescent and proliferative states. We considered that genes were up or down regulated if they had a pvalue < 0.01 in (Martynoga et al., 2013) Table 2. Briefly, each gene is assigned the closest enhancer and significance of the association is assessed through a permutation test. By crossing these data with our previous motif search, we built a 3x2 contingency matrix with up-regulated, down-regulated and not differentially regulated genes vs presence and absence of LEF1 motif (Jaspar PF0013.1, score greater or equal to 85%) and used Chi-squared test.