Figure 3.
Regulation of CRBN splicing and CC-90009 response by the ILF2 and ILF3 complex. (A-C) Assessment of the effect of ILF3 knockout on CC-90009 response by a flow cytometry-based CRISPR competition assay. U937 cells stably expressing Cas9 were infected with lentiviral vectors coexpressing RFP and a nontargeting sgRNA (sgNT-1), an sgRNA targeting a noncoding region (sgNC-1), or an sgRNA targeting ILF3 (sgILF3-2 or sgILF3-4). The expression of RFP is driven by an EF1a-HTLV hybrid promoter, whereas the expression of sgRNAs was under the control of a doxycycline-inducible H1/TO promoter. Three days after sgRNA induction with 1 μg/mL doxycycline, the cells were mixed at a 1:1 ratio with U937 Cas9 cells infected with a lentiviral vector constitutively expressing GFP and a nontargeting sgRNA (sgNT-1) and treated with DMSO or 10 μM CC-90009. The change of RFP+/GFP+ ratio was monitored by flow cytometry every 2 days thereafter. (A) Schematic design of the flow cytometry-based CRISPR competition assay. (B) Immunoblot analysis of U937-Cas9 cells inducibly expressing sgNT-1, sgNC-1, sgILF3-2, or sgILF3-4. Cells were treated with doxycycline (DOX) for 6 days. (C) The RFP+/GFP+ ratios of U937-Cas9 cells coexpressing RFP and sgNT-1, sgNC-1, sgILF3-2, or sgILF3-4 mixed with cells coexpressing GFP and sgNT-1 at each indicated timepoint were normalized to the RFP+/GFP+ ratio of the cell mixtures on day 0. (D) Immunoblot analysis of U937-Cas9 cells stably expressing sgNT-1 or sgILF3-2 under the control of the H1/TO promoter. Cells were treated with or without DOX for 4 days, followed by incubation with DMSO or an increasing concentration of CC-90009 for 6 hours. (E) RNA sequencing analysis of U937-Cas9 cells with inducible expression sgNT-1 or sgILF3-2 for 7 days. Evidence of differential splicing was observed in a total of 967 unique genes by up- and/or downregulated exon usage with ILF3 knockout in U937 cells, reaching a corrected significance level (FDR) <0.05. At the gene level, 791 genes were found to be significantly (FDR <0.05) up- or downregulated with ILF3 knockout. Top: Venn diagram showing the overlap of genes with significant differential exon usage (DEU; LHS) and genes with differential expression at the gene-level (DEG; RHS). Bottom: pathway enrichment analysis of DEU and DEG genes. The color and size of the dots represent adjusted significance level and gene ratio respectively. Gene ratio refers to the number of input genes annotated to an individual pathway as a ratio of all input genes annotated to any Reactome pathway. (F) DEU analysis revealed significant differential splicing of individual CRBN exons (red bars) with knockout of ILF3. An exon, which annotates to the truncated transcript, CRBN.213 (exon bin no.13), is significantly elevated (FDR, 0.02) with ILF3 knockout relative to NT controls. Conversely, exons downstream of this isoform are significantly underrepresented (FDR, 0.05; exon bin no. 14) in the ILF3 knockout cells relative to parental. (G) Quantitative PCR analysis of the expression levels of CRBN transcripts as indicated in U937-Cas9 cells with inducible expression sgNT-1 or sgILF3-2 for 7 days. Data in panel G are shown as mean ± standard deviation (SD), n = 4 technical replicates. Result shown in all figure panels is representative of 3 biological replicates.

Regulation of CRBN splicing and CC-90009 response by the ILF2 and ILF3 complex. (A-C) Assessment of the effect of ILF3 knockout on CC-90009 response by a flow cytometry-based CRISPR competition assay. U937 cells stably expressing Cas9 were infected with lentiviral vectors coexpressing RFP and a nontargeting sgRNA (sgNT-1), an sgRNA targeting a noncoding region (sgNC-1), or an sgRNA targeting ILF3 (sgILF3-2 or sgILF3-4). The expression of RFP is driven by an EF1a-HTLV hybrid promoter, whereas the expression of sgRNAs was under the control of a doxycycline-inducible H1/TO promoter. Three days after sgRNA induction with 1 μg/mL doxycycline, the cells were mixed at a 1:1 ratio with U937 Cas9 cells infected with a lentiviral vector constitutively expressing GFP and a nontargeting sgRNA (sgNT-1) and treated with DMSO or 10 μM CC-90009. The change of RFP+/GFP+ ratio was monitored by flow cytometry every 2 days thereafter. (A) Schematic design of the flow cytometry-based CRISPR competition assay. (B) Immunoblot analysis of U937-Cas9 cells inducibly expressing sgNT-1, sgNC-1, sgILF3-2, or sgILF3-4. Cells were treated with doxycycline (DOX) for 6 days. (C) The RFP+/GFP+ ratios of U937-Cas9 cells coexpressing RFP and sgNT-1, sgNC-1, sgILF3-2, or sgILF3-4 mixed with cells coexpressing GFP and sgNT-1 at each indicated timepoint were normalized to the RFP+/GFP+ ratio of the cell mixtures on day 0. (D) Immunoblot analysis of U937-Cas9 cells stably expressing sgNT-1 or sgILF3-2 under the control of the H1/TO promoter. Cells were treated with or without DOX for 4 days, followed by incubation with DMSO or an increasing concentration of CC-90009 for 6 hours. (E) RNA sequencing analysis of U937-Cas9 cells with inducible expression sgNT-1 or sgILF3-2 for 7 days. Evidence of differential splicing was observed in a total of 967 unique genes by up- and/or downregulated exon usage with ILF3 knockout in U937 cells, reaching a corrected significance level (FDR) <0.05. At the gene level, 791 genes were found to be significantly (FDR <0.05) up- or downregulated with ILF3 knockout. Top: Venn diagram showing the overlap of genes with significant differential exon usage (DEU; LHS) and genes with differential expression at the gene-level (DEG; RHS). Bottom: pathway enrichment analysis of DEU and DEG genes. The color and size of the dots represent adjusted significance level and gene ratio respectively. Gene ratio refers to the number of input genes annotated to an individual pathway as a ratio of all input genes annotated to any Reactome pathway. (F) DEU analysis revealed significant differential splicing of individual CRBN exons (red bars) with knockout of ILF3. An exon, which annotates to the truncated transcript, CRBN.213 (exon bin no.13), is significantly elevated (FDR, 0.02) with ILF3 knockout relative to NT controls. Conversely, exons downstream of this isoform are significantly underrepresented (FDR, 0.05; exon bin no. 14) in the ILF3 knockout cells relative to parental. (G) Quantitative PCR analysis of the expression levels of CRBN transcripts as indicated in U937-Cas9 cells with inducible expression sgNT-1 or sgILF3-2 for 7 days. Data in panel G are shown as mean ± standard deviation (SD), n = 4 technical replicates. Result shown in all figure panels is representative of 3 biological replicates.

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