Essential role of the Crk family-dosage in DiGeorge-like anomaly and metabolic homeostasis

(A) Crk and Crkl protein levels were determined in time course in immunoblots upon induction of Crk;Crkl deficiency. Crk ^f/f;Crkl ^f2/f2;R26^creERT2/+ MEFs were induced for Crk;Crkl deficiency by 4-hydroxytamoxifen (4OHT) treatment for 24 h, whereas a control group was treated with vehicle only (CTRL). Cell lysates were isolated from MEFs at each time point indicated above each lane (0 h was the time right before 4OHT/vehicle addition). (B) Crk;Crkl double deficiency results in slow cell growth and altered population morphology. Pictures were taken posttreatment day 2 for control and day 4 for Crk;Crkl deficiency group. Note that the cell density of Crk;Crkl-deficient MEFs on day 4 is similar to that of control group on day 2, and that population morphology is distinguishable between the 4OHT and control groups. (C) Time lapse images show cell division from a single MEF (identified at 0:00 time) to two daughter cells in each group. In the control, cells migrated away from each other after division and were no longer visible together within the field after 4 h. In contrast, Crk;Crkl-deficient cells (4OHT-treated) stayed attached with one another, forming a two-cell island after division. Movies are available as supplemental materials. (D) Cell–cell contacts were analyzed by immunostaining with anti-Ctnnb1 (β-catenin) antibody. Individual cells are labeled by numbers. β-catenin localization highlights defined zipper-like cell–cell contacts in Crk;Crkl-deficient MEFs (4OHT-treated) compared with control MEFs. Box and whisker plots show quantitative comparisons of β-catenin levels across cell–cell contacts shown in the images above the plots. Diamonds indicate intensity values derived from tracing seven separate regions that encompass cell–cell contacts. Two plots demonstrate that Crk;Crkl-deficient MEFs (4OHT-treated) had greater accumulation of β-catenin at cell–cell contacts compared with control as shown in both average and maximum levels of the β-catenin staining (“average levels” and “peak levels,” respectively). (E) The area of spreading was measured for individual cells after replating on a gelatin-coated surface (72 cells in each group). Note that whereas MEFs lacking either Crk or Crkl showed smaller spread area at 60 and 90 min, Crk;Crkl double deficiency induced greater degrees of spreading defects over time and did not show a significant increase in the spread area between 60 and 90 min, thus suggesting that cell spreading reached a lower plateau compared with their control (and that of either Crk or Crkl deficiency). (F) The cell size was estimated in dissociated Crk;Crkl-deficient MEFs and their control in the G1 phase (4OHT and CTRL, respectively). FSC-H values (forward scatter height) were analyzed and illustrated in a box-and-whisker plot. See Fig S5 for propidium iodide–binding profiles and gating information for the FACS analysis. Each treatment group was subdivided into two subgroups with or without a split on day 2 posttreatment to adjust and maintain low cell density until harvest on day 3 posttreatment.

(A) The heat map shows a list of the top 30 pathways based on comparison analysis of the Crk and/or Crkl single and double deficiency groups in Ingenuity Pathway Analysis (QIAGEN; see Supplemental Data 1). RNA-Seq experiments were performed on RNA isolated from four independent primary MEF populations for each genotype as described in the Materials and Methods section as well as in Table S2 and Supplemental Data 1. Differentially expressed genes (DE genes) were identified by Benjamini–Hochberg adjusted P-values (p.adj) smaller than 0.05 using DESeq2. (B) Deficiencies for Crk and Crkl genes, separately or combined, resulted in overlapping lists of DE genes, categorized into subsets a-g as shown in the Venn’s diagram. Subsets a, b, c, and d are referred to subsets “red,” “orange,” “yellow,” and “green” hereafter (Supplemental Data 2). The number in each subset indicates the number of DE genes in the subset. The number below the gene symbol (Crk, Crkl, or Crk;Crkl) indicates the total number of DE genes identified in the gene deficiency. The numbers in parentheses separated by colon show the numbers of genes up-regulated versus down-regulated. Note that deficiency of either Crk or Crkl was sufficient to disrupt normal expression of the genes in subset “red,” whereas the genes in subset “green” tolerated single gene disruption of either Crk or Crkl. (C) The DE genes in subsets “red,” “orange,” “yellow,” and “green” were analyzed for their enrichment into pathways using KEGG (Kyoto Encyclopedia of Genes and Genomes). The node circles and annotations are color-coded as appeared in panel (B). Nodes are labeled only for the KEGG modules and pathways with Storey’s q-values smaller than 0.0005 (shown as FDRs), whereas node circles are shown for the pathways/modules with a q-value < 0.05. The diameter of node circle is proportional to −log₁₀(q-value).

(A) CE-TOF/MS metabolome analysis identified differential levels of several metabolites in central carbon metabolism in primary MEFs deficient for either Crk or Crkl, or for both Crk and Crkl. The illustration is a compilation of metabolome and RNA-Seq results. Transporters, enzymes, and metabolites affected in Crk;Crkl double deficiency are highlighted by a color shade (shades of blue indicate down-regulation; shades of red, up-regulation). Nodes encircled by magenta lines are affected not only in Crk;Crkl double deficiency but also in single deficiencies of both Crk and Crkl. Orange-colored labels indicate known targets of the transcription factor Hif1a. (B) Quantitative RT-PCR validated the results of RNA-Seq for several glycolysis genes in primary MEFs deficient for Crk and Crkl. Levels of expression were expressed as a fold change. The basal level without induction of Crk;Crkl deficiency set at 1.0 as shown at the red dotted line. Welch’s t test was performed on raw Ct values between 4-hydroxytamoxyfen (4OHT)–treated and CTRL groups (n = 3); P-values were 0.03462, 0.00061, 0.01735, 0.00027, 0.00834, and 0.00013 for Pfkl, Gapdh, Pgk1, Pgam1, Eno1, and Ldha, respectively. (C) Association of RNA polymerase II to several glycolysis genes were decreased in MEFs deficient for Crk and Crkl. ChIP was conducted with anti-RNA polymerase II hosphor-S5 CTD repeats (Pol2) antibody followed by quantitative PCR. Levels of Pol2 association to each gene were expressed as a fold change. The basal level without induction of Crk;Crkl deficiency set at 1.0 as shown at the red dotted line. Welch’s t test was performed on delta Ct values of chromatin IP samples (relative to their respective DNA input used for IP) between 4-hydroxytamoxyfen (4OHT)–treated and CTRL groups (n = 3); P-values were 0.0254, 0.0064, and 0.0177 for Gapdh, Pgk1, and Ldha, respectively. (D) Differential Hif1a protein levels were observed in the nucleus between Crk/Crkl deficiency–induced and CTRL MEFs with or without CoCl₂ to stabilize Hif1 proteins (box plot). Representative images are shown on the left to the box plot. MEFs were incubated with or without 0.5 mM CoCl₂ for 4 h before fixation. Hif1a proteins were detected in the IN Cell Analyzer 2000 upon immunofluorescent staining with anti-HIF1A antibody. The nuclei were identified by DAPI staining. Signals in ∼2,000–2,300 nuclei were quantified for each group for Hif1a nuclear localization. Kruskal–Wallis tests followed by Dunn’s post hoc tests with Bonferroni corrections yielded virtually identical p-levels to that of Brunner–Munzel tests.

(A) Scatterplots indicate ChIP-Seq signals for H3K27Ac and Pol2 (hosphor-S5 CTD repeats) in the x-axis and mRNA levels (data from RNA-Seq) in the y-axis, in which values are shown in log₂ fold change (log₂ FC) as differentials between Crk/Crkl deficiency–induced and uninduced MEFs. Each dot represents a single gene identified in subset “red” (Fig 3B). Red or light-blue dots indicate down-regulated or up-regulated genes based on their expression identified by RNA-Seq, respectively. Glycolytic genes are labeled for their gene symbols. ChIP-Seq signals in these panels are based on peak heights within transcription start site (TSS) ± 2 kb. Spearman’s rank correlation coefficient ρ was calculated for the entire distribution. (B) Boxplots indicate distributions of the ChIP-Seq signal differentials within TSS ± 2 kb for H3K27Ac and Pol2 in either down-regulated or up-regulated categories of subset “red” genes. Whiskers were drawn between the highest and lowest data points within 1.5× interquartile range (IQR) from the upper or lower quartile. Data points outside the 1.5× IQR are indicated as outliers (dots). The P-values were calculated by Mann–Whitney U tests between down-regulated and up-regulated categories. (C) XY plots indicate the average ChIP-Seq signals as reads per genome content (RPGC) in the y-axis and the distance from TSS in the x-axis in Crk/Crkl deficiency-induced and uninduced MEFs (KO and CTRL) in orange and green lines, respectively. Note that when ChIP-Seq signals are compared in CTRL MEFs between the down-regulated and up-regulated gene groups, the down-regulated group shows greater average peak heights in both H3K27Ac and Pol2 ChIP-Seq signals. (D) Boxplots indicate the distribution of the ChIP-Seq signals (RPGC) within TSS ± 2 kb in Crk/Crkl deficiency-induced and uninduced MEFs (KO and CTRL), for genes down-regulated or up-regulated in subset “red.” The y-axis is in a log₁₀ scale of RPGC. ANOVA and Tukey post hoc tests were performed on log₁₀-transformed RPGC values to bring the data distributions closer to Gaussian distributions. See Fig S7 for boxplots using untransformed data and square-root transformed data.

(A) Glucose availability in culture media throttled dose-dependent phosphorylation of ribosomal protein S6 (pS6-S240/244), as well as that of Akt (pAKT-S473), TSC2 (pTSC2-T1462), and p70 S6 kinase (pS6K-T389) as shown in immunoblots. MEFs were cultured with indicated concentrations of glucose for 24 h after a 24-h period of glucose restriction at a concentration of 0.1 mM, the lowest glucose concentration that MEFs can tolerate in the presence of 10% dialyzed FBS (Fig S8). As a point of reference, basal DMEM includes 5 mM glucose, whereas a high-glucose formula includes 25 mM glucose. (B) The glucose metabolism inhibitor 2-deoxy-D-glucose (2DG) induced a cell blebbing phenotype in a dose-dependent manner, and Crk/Crkl deficiency significantly exacerbated the frequency of the phenotype in single cell analysis. The 2DG concentrations are indicated under the x-axis. The frequency of the blebbing phenotype was determined as described in the Materials and Methods section and Fig S9. Large numbers of cells (n) were imaged in a high-content imaging apparatus, and individual cells were analyzed through a series of MATLAB scripts. Beneath the bar graph is a matrix table for P-values by Fisher’s exact tests adjusted for multiple comparisons (FDR). (C) Crk/Crkl deficiency increased Igf1 mRNA levels. The bar graph represents levels of Igf1 messages upon Crk/Crkl deficiency induction in MEFs, relative to that of their controls without deficiency induction. As a control, fold change of Rplp0 (60S ribosomal protein p0) is also shown. Two non-overlapping primer sets 1 and 2 (ps1 and ps2) were used to confirm up-regulation approximately at an order of magnitude greater. Error bars indicate standard deviations (n = 3). Red-dotted line shows the level in control samples set at a relative fold change of 1. (D) Crk/Crkl deficiency resulted in muted response to exogenous Igf1 over a range of doses in phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6. In contrast, Akt phosphorylation was induced by Igf1, suggesting a role of Crk and Crkl in a regulatory mechanism on S6K and S6. MEFs were cultured without serum for the last 3 h of 48 h post deficiency induction, then were stimulated with Igf1 at different concentrations indicated for 15 min. (E) Crk and Crkl played an important role in S6K and S6 phosphorylation on which both Igf1 and fibronectin (FN) cooperate as shown in immunoblots probed with different antibodies. Note that FN alone did not activate Akt (as seen in pAkt-S473), whereas Igf1 did. After 3 h serum starvation, MEFs were re-plated on FN or poly-L-lysine in serum-free medium for 60 min followed by incubation with Igf1 for 15 min before harvest.

(A) Overexpression of C3GF partially blocked Crk/Crkl-deficiency–induced cell size changes. The histograms show distributions of cell sizes as estimated by FSC-H measurements in FACS analysis of ∼6,000–7,000 cells in the G1 phase in each group. To compare the distributions, a boxplot was generated below the histograms. Human RAPGEF1 fused to a farnesylation sequence (C3GF) or empty vector was introduced into MEFs before Crk/Crkl deficiency induction by 4OHT. Two-way ANOVA followed by Tukey post hoc tests were performed for statistical comparisons. (B) Overexpression of C3GF rescued Crk/Crkl deficiency–induced inhibition of cell proliferation. Cell numbers were counted in tissue culture plates for 3 d after plating. Bars indicate standard deviations from triplicate determinations (n = 3). Two-way ANOVA followed by Tukey post hoc tests were performed for statistical comparisons. (C) C3GF restored expression of the glycolytic enzyme genes. Expression of glycolytic genes were determined in real-time/quantitative RT-PCR. Bars indicate standard deviations. Two-way ANOVA followed by Tukey post hoc tests were performed on raw Ct values (n = 3). (D) C3GF blocked Crk/Crkl deficiency from reducing the level of fructose-1,6-bisphosphate (F1,6P2). Bars indicate standard deviations from triplicate determinations (n = 3). Two-way ANOVA followed by Tukey post hoc tests were performed for statistical comparisons. (E) Immunoblots show C3GF-dependent rescues on S6, S6K, and Akt phosphorylation associated with elevated phosphorylation of the focal adhesion protein p130^Cas (Bcar1). (F) C3GF restored phosphorylated Bcar1, phosphotyrosine, and Fak localization at focal adhesions. Representative fluorescent microscopy images are shown. (G) Violin plots show quantitative results of focal adhesions. Focal adhesions were identified by localization of Fak in immunostained MEFs followed by automated image acquisition and analysis as described in the Materials and Methods section (also see Fig S11). The sample size (the number of cells per group) was 1,481, 516, 1,666, and 1,374 in a 2X2 experimental design (Uninduced/Vector Only, 4OHT-Induced/Vector Only, Uninduced/C3GF, and 4OHT-Induced/C3GF groups, respectively) after applying the cutoffs indicated in Fig S11 to minimize the possibility of counting artifacts in staining and segmentation. However, inclusion of all cells without cutoffs did not affect the statistical outcome. Statistical analysis was performed by a global pseudo rank method with Tukey tests adjusted for multiple comparisons using the mctp function in the nparcomp package written in the programming language R (Konietschke et al, 2015). Similar statistical outcome was obtained by two-way ANOVA after log₁₀-transformation (Fig S11D). White circles in each violin plot indicate the position of the median.