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. 2022 Feb 28;11:e74338. doi: 10.7554/eLife.74338

Figure 6. Transcriptional consequences of loss of Fbw7 in neural stem cells.

(A) Clustering analysis separates differentially expressed protein-coding genes in NSCs into two groups. Heatmap shows the intensity of expression of each gene (y-axis) for three replicates per cell type (x-axis). Three replicates were from two independently engineered cell samples. (B) TFs, pathways, and GO terms enriched in upregulated genes in Fbw7−/− NSCs. (C) Sites with increased (red) and decreased (blue) Jun in Fbw7−/− NSCs compared to WT. (D) Nondifferential and differential Jun peaks located within each gene feature. (E) Fold change of CIITA and MHC Class II genes in Fbw7−/− NSCs compared to WT. FDR values are given at the top of each bar. n=3. (F) Quantitative RT-PCR analysis of MHC Class II (HLA-DRA, HLA-DRB, HLA-DPA, and HLA-DPB) expression in Fbw7−/− NSCs. Mean fold change in Fbw7−/− cells with respect to WT cells. Error bars=SEM, n=3. (G) Genome browser view of Myc, Jun, and H3K27ac occupancy on CIITA regulatory regions in WT and Fbw7−/− NSCs. Black scale bar=8 kb. See Figure 6—figure supplements 1–3 and Figure 6—source data 1–4, Figure 6—figure supplement 1—source data 1, Figure 6—figure supplement 3—source data 1, Figure 6—figure supplement 5—source data 1. GO, gene ontology; TF, transcription factor; WT, wild-type.

Figure 6—source data 1. Differential expression analysis of U5-NSC RNA-Seq.
This Excel file contains the differential analysis output of U5-NSC RNA-Seq data from control (Ctrl23, Ctrl4.1, and Ctrl4.2) and Fbw7−/− (Fb23, Fb4.1, and Fb4.2) cells. DE=0, not differentially expressed (DE) in the mutant compared to WT; DE=1, DE in the mutant compared to WT. NSC, neural stem cell; WT, wild-type.
Figure 6—source data 2. Enrichr output for U5-NSC differentially expressed genes.
This Excel file includes the TRANSFAC & JASPAR, MSigDB, and GO term outputs enriched in differential (upregulated or downregulated) genes in Fbw7−/− NSCs. GO, gene ontology; NSC, neural stem cell.
Figure 6—source data 3. Jun differential sites in U5-NSCs.
(U5F=Fbw7−/− and U5W=WT). NSC, neural stem cell; WT, wild-type.
elife-74338-fig6-data3.xlsx (827.2KB, xlsx)
Figure 6—source data 4. Quantitative RT-PCR analysis of MHC Class II genes in NSCs.
NSC, neural stem cell.

Figure 6.

Figure 6—figure supplement 1. Validation of U5-NSC Fbw7−/− generation and CUT&RUN Jun signal.

Figure 6—figure supplement 1.

(A) Western blot showing Fbw7; samples 1–4: U5 NSCs with sgRNA targeting Fbw7 exons 3, 4, and 9, and all three exons in one pool; samples 5–6: U5 NSCs with control sgRNA 1× and 3×; and sample 7: Hct116 WT. (B) Sequence logo of AP-1 motif enriched in Jun peaks in U5 NSCs (E value=1.2e−146). CUT&RUN, cleavage under target and release using nuclease; NSC, neural stem cell; WT, wild-type.
Figure 6—figure supplement 1—source data 1. Confirming loss of Fbw7 in U5-NSC Fbw7−/− cells.
This folder contains the original western blots for Figure 6—figure supplement 1A. Western blots that confirm the loss of Fbw7 in two others separately performed nucleofection reactions are also included.
Figure 6—figure supplement 2. Proliferation of Fbw7 mutant U5 NSCs.

Figure 6—figure supplement 2.

(A) Cell proliferation was monitored with an IncuCyte for 81 hr. Data are shown as mean ± SEM. Proliferation was measured at each time point normalized to percent confluence at time 0. Doubling time, WT 61.3 hr and Fbw7−/− 88.82 hr. (B) Flow cytometry analysis of DNA replication in Fbw7 mutant cells. Density plots of EdU versus DNA content (DAPI) in NSCs. Single cells were identified by gating events on DAPI-H/DAPI-A. Cells were labeled with EdU prior to harvest and were later fixed and immuno-stained for flow cytometry. NSC, neural stem cell; WT, wild-type.
Figure 6—figure supplement 3. Percentage of peaks with decreased Jun in U5 NSC Fbw7−/− within different gene regions.

Figure 6—figure supplement 3.

NSC, neural stem cell.
Figure 6—figure supplement 3—source data 1. Original gels for Figure 6—figure supplement 3.
Figure 6—figure supplement 4. CIITA isoform III amplified using isoform specific primers in U5 NSCs.

Figure 6—figure supplement 4.

NSC, neural stem cell.
Figure 6—figure supplement 5. MHC Class II protein expression in Hct116 cells.

Figure 6—figure supplement 5.

(A) Flow cytometry analysis of HLA-DR in Fbw7 mutant NSCs. Density plots of side scatter (SSC) versus PE HLA-DR. (B) Western blot detecting HLA-DR/DP/DQ in NSCs. Protein intensity of HLA band was measured using ImageJ in comparison to the loading control. HLA protein is ~3-fold higher in Fbw7−/− cells in comparison to WT. NSC, neural stem cell; WT, wild-type.
Figure 6—figure supplement 5—source data 1. Original western blots for Figure 6—figure supplement 5.
Figure 6—figure supplement 6. Comparison of differentially expressed (DE) genes in Hct116 and NSCs.

Figure 6—figure supplement 6.

(A) Hierarchical clustering of DE protein-coding genes in Hct116. Expression level of the same genes in NSCs is also mapped. Genes that are not captured in NSC are in gray. Log2FC of DE genes specific to Hct116 cells was higher than the DE genes shared by both Hct116 and NSCs (Wilcoxon test: p=5.33e−16). (B) Hierarchical clustering of DE protein-coding genes in NSC. Expression level of the same genes in Hct116 cells is also mapped. Log2FC of DE genes specific to NSCs was higher than the DE genes shared by both Hct116 and NSCs (Wilcoxon test: p=3.727e−10). NSC, neural stem cell.