Journal: Nature

Article Title: OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage

doi: 10.1038/s41586-022-04842-7

Figure Lengend Snippet: (a) Two-class comparison of gene dependencies of four SCLC-P versus 986 other cancer cell lines. The average essentiality of each gene from SCLC-P is subtracted to its mean in other cell lines. This difference is moderated with an empirical Bayes method using the adaptive shrinkage method described in CRNA package - ashr. (b) Scatter plot of C11orf53, COLCA2, and POU2F3 dependency scores across a panel of human cancer cell lines. Data obtained from DepMap (21Q2). (c) Control sgRNAs (2 negative controls or one sgCDK1) for the competition-based proliferation assays (related to Fig. 4a), (n = 3). Mean ± s.d. is plotted. (d) BrdU incorporation assays following CRISPR-based targeting of POU2F3, C11orf53, or COLCA2 or negative control in the indicated Cas9+ cell lines. Two technical replicates with two independent sgRNAs for each gene as biological replicates. Adjusted P value was calculated with two-way ANOVA with Tukey’s multiple comparison tests. (e-f) Tumour weights and imaging at the terminal timepoint of the xenograft experiments shown in Fig. 4b. Mean ± s.e.m. is plotted for (d-e). P-values are derived from two-tailed unpaired student’s t-test with Welch’s correction. (g) Controls for NCI-H211 gene complementation assay shown in Fig. 4c. (n = 3). Mean ± s.d. is plotted. (h) Competition-based proliferation assays in NCI-H1048 cells cotransduced with indicated cDNAs and sgRNAs lentivirally to assess functionality of indicated mutants. cDNAs were engineered to be resistant to Cas9/sgRNA-mediated cutting. Mean ± s.d. of normalized GFP percentage is plotted (n = 3). (i) anti-HA western blot of the indicated cDNAs from (h). Data are representative of two biological replicates. Source data for all GFP depletion assays, BrdU assays and tumour weights are provided in Supplementary Table 3–4, gating strategy for BrdU assay is provided in Supplementary Figure 2, sgRNA sequences are provided in Supplementary Table 4, uncropped gels are provided in Supplementary Fig. 1j.

Article Snippet: Plasmid construction and sgRNA cloning The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T, Addgene, 108098) and the lentiviral Cas9 vector (Addgene, 108100).

Techniques: Comparison, Control, BrdU Incorporation Assay, CRISPR, Negative Control, Imaging, Derivative Assay, Two Tailed Test, Western Blot, BrdU Staining

Journal: Nature

Article Title: OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage

doi: 10.1038/s41586-022-04842-7

Figure Lengend Snippet: a, Competition-based proliferation assays in Cas9-expressing SCLC cells after lentiviral expression of the indicated sgRNAs (linked with GFP). Data are mean ± s.d. normalized percentage of GFP (to day 4 after infection) of two to three sgRNAs. n = 3. b, The growth kinetics of Cas9-expressing NCI-H526 and NCI-H1048 xenografts implanted in immunodeficient mice. Cells were lentivirally transduced with the indicated sgRNAs before injection into mice. n = 5, except for C11orf53, for which n = 10. Data are mean ± s.e.m. sgNEG, sgRNA targeting negative controls. P values were calculated using two-tailed unpaired Student’s t-tests with Welch’s correction. c, Competition-based proliferation assays in NCI-H211 cells cotransduced with the indicated cDNAs and sgRNAs lentivirally to assess the functionality of the indicated mutants. cDNAs were engineered to be resistant to Cas9/sgRNA-mediated cutting. Data are mean ± s.d. of GFP percentage (normalized to day 4). Expression of mutants is shown in Extended Data Fig. 6d. d, CRISPR–Cas9-induced deletion of C11orf53 to generate knockout mice. e, The ratio of different genotypes of C11orf53+/− (HET) progeny (C11orf53 F2 mice). f, Representative immunofluorescence staining of the tuft cell marker DCLK1 in the trachea and small intestine. Scale bars, 100 μm. g,h, Quantification of POU2F3-positive and DCLK1-positive cells. Data are mean ± s.e.m. The P values for the comparison of tuft cell frequencies of knockout versus WT (DCLK1/POU2F3) or HET versus WT of POU2F3 staining in small intestine, trachea, urethra, gallbladder and tongue were calculated using two-tailed unpaired Student’s t-tests with Welch’s correction; the P values for comparing tuft cell frequencies of knockout versus WT in colon, thymus and stomach, or HET versus WT of DCLK1 staining in the small intestine and trachea were calculated using ordinary unpaired two-tailed Student’s t-tests. The numbers of animals involved in f–h are provided in Extended Data Fig. 9. Source data for GFP competition and gene complementary assays are provided in Supplementary Table 3 and mouse data are provided in Supplementary Tables 4 and 5. sgRNA sequences and genotyping primers are provided in Supplementary Table 6.

Article Snippet: Plasmid construction and sgRNA cloning The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T, Addgene, 108098) and the lentiviral Cas9 vector (Addgene, 108100).

Techniques: Expressing, Infection, Transduction, Injection, Two Tailed Test, CRISPR, Knock-Out, Immunofluorescence, Staining, Marker, Comparison

Journal: Nature

Article Title: OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage

doi: 10.1038/s41586-022-04842-7

Figure Lengend Snippet: (a) Comparison of POU2F3, C11orf53, and HA-COLCA2 ChIP-seq peak overlap in the indicated cell lines. For each protein, peaks represent the ones that are consistently identified from 2–3 replicate of experiments. Detailed information is in method. (b) Annotation of POU2F3 and C11orf53 overlapping peaks NCI-H211 cells. (c) Position weight matrix of discovered motif enriched on POU2F3/C11orf53 co-binding sites by MEME from NCI-H211 cell line. (d) Western blot analysis of lentivirally overexpressed constructs used for ChIP-qPCR and gene complementation assay in NCI-H211 cells (n = 2). (e) Sequential ChIP-qPCR analysis of overexpressed HA-C11orf53 (NCI-H211) or HA-COLCA2 (NCI-H1048) co-expressed with FLAG-POU2F3 binding sites in NCI-H211 and NCI-H1048 cells respectively with anti-FLAG (1st IP) or protein G beads (1st IP) and anti-HA (2nd) antibodies. The enrichment is adjusted to the input amount. Two biological replicates are performed and represented as individual dots; the bar value represents the average enrichment over input of two biological replicates. (f) Western blot analysis of POU2F3 or C11orf53 in NCI-H211 (Cas9) cells transduced with indicated sgRNAs. Samples were collected 4 days post infection (n = 1). (g) RNA-seq analysis comparing mRNA changes following C11orf53, COLCA2, or POU2F3 knockout compared to control in three SCLC-P cells. RNA was collected five- or six-days post sgRNA infection. Each dot represents the log2fold-change of a single protein-coding genes (read outs > = 10). (h) Western blot analysis of FLAG-POU2F3, HA-C11orf53, and FLAG-GFP expression in murine YT330 SCLC cells. Data is representative of two biological replicates. Uncropped gel is provided in Supplementary Fig. 1i. Source data for gene expression changes upon knockout in different cells are provided in Supplementary Table 2. sgRNA and primer sequences are provided in Supplementary Table 6.

Article Snippet: Plasmid construction and sgRNA cloning The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T, Addgene, 108098) and the lentiviral Cas9 vector (Addgene, 108100).

Techniques: Comparison, ChIP-sequencing, Binding Assay, Western Blot, Construct, ChIP-qPCR, Transduction, Infection, RNA Sequencing, Knock-Out, Control, Expressing, Gene Expression