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Research Article
Transparent Process
Open Access

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

View ORCID ProfileAkira Imamoto  Correspondence email, Sewon Ki, Leiming Li, Kazunari Iwamoto, Venkat Maruthamuthu, View ORCID ProfileJohn Devany, View ORCID ProfileOcean Lu, Tomomi Kanazawa, Suxiang Zhang, Takuji Yamada, Akiyoshi Hirayama, Shinji Fukuda, Yutaka Suzuki, View ORCID ProfileMariko Okada  Correspondence email
Akira Imamoto
1The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA
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  • For correspondence: aimamoto@uchicago.edu
Sewon Ki
2RIKEN Integrative Medical Sciences, Tsurumi, Yokohama, Kanagawa, Japan
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Leiming Li
1The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA
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Kazunari Iwamoto
3Institute for Protein Research, Osaka University, Suita, Osaka, Japan
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Venkat Maruthamuthu
4Department of Mechanical and Aerospace Engineering, Old Dominion University, Norfolk, VA, USA
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John Devany
5Department of Physics, The University of Chicago, Chicago, IL, USA
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Ocean Lu
1The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA
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Tomomi Kanazawa
3Institute for Protein Research, Osaka University, Suita, Osaka, Japan
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Suxiang Zhang
3Institute for Protein Research, Osaka University, Suita, Osaka, Japan
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Takuji Yamada
6Department of Life Science and Technology, Tokyo Institute of Technology, Meguro, Tokyo, Japan
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Akiyoshi Hirayama
7Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan
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Shinji Fukuda
7Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan
8Intestinal Microbiota Project, Kanagawa Institute of Industrial Science and Technology, Kawasaki, Kanagawa, Japan
9Transborder Medical Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan
10PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
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Yutaka Suzuki
11Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan
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Mariko Okada
2RIKEN Integrative Medical Sciences, Tsurumi, Yokohama, Kanagawa, Japan
3Institute for Protein Research, Osaka University, Suita, Osaka, Japan
12Center for Drug Design and Research, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan
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  • For correspondence: mokada@protein.osaka-u.ac.jp
Published 10 February 2020. DOI: 10.26508/lsa.201900635
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  • Figure S1.
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    Figure S1. Gene targeting at the Crk locus.

    (A) A map of the knockout-ready Crk conditional allele (Crk f). The targeting construct included two homology arms at the 5′ and 3′ sides (gray), flanking Crk exon 1 (magenta) and two strips of exogenous sequences 1 and 2 (94 and 139 bp, respectively; shown in orange). These exogenous sequences included recombinase-target sequences such as loxP and FRT (blue). An FneoF cassette (FRT-PGKneobpA-FRT) was initially inserted in the targeting construct at the FRT site, which was later removed in the germ line by a genetic cross with an FLPeR strain. Forward and reverse primers are indicated as dark-green or light-green triangles, respectively. Three primers 4750F, Crk i1F1, and 6034R are used for routine genotyping by genomic PCR (see Table S3). To illustrate the location of this conditional mutation, the Myo1c gene is shown in a green box. Numbers above the lines and boxes are the position of the nucleotide, whereas unique restriction sites are also indicated. (B) Initial embryonic stem (ES) colony screening for homologous recombination on the 3′ side. Genomic PCR was performed between Crk iR7 primer (outside of the targeting construct; see above) and another primer in the FneoF cassette (the FneoF cassette has been deleted; see above). Four colonies were positive among 96 ES colonies picked (two positives are shown; arrow). M, 1-kb ladder lane. (C) Homologous recombination was also confirmed at the 5′ homology side. XbaI digestion generated a 382-bp fragment (closed arrow) from the PCR product amplified from the heterozygote between 555F and Crk e1R primers (open arrow, ∼5 kb; see panel (A) for the locations of the primers) because an XbaI site was introduced in the mutated allele within exogenous sequence 1 shown in panel (A) immediately downstream of the NotI site (at position 5,000 base, A). This XbaI site does not exist the fragment amplified from the wild-type gene. Therefore, the presence of the short XbaI fragment is diagnostic for homologous recombination within the long arm. The results illustrated in panels (B, C) indicate that double reciprocal homologous recombination took place at both 5′ and 3′ sides of Crk exon 1 in ES cells (#20 was chosen as a representative in this panel). A germ line derived from ES clone #20 was used for the analysis presented in this article.

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    Figure 1. Embryonic phenotypes from deficiencies of Crk and Crkl in mice.

    (A, B, C, D) Histologic sections from an E16.5 embryo lacking Crk (Crkd/d) showed defects, including a cleft palate (arrow in panel A), cervical/extra-thoracic thymic lobes (red arrows in panel B, ts in panel C), d-transposition of aorta and pulmonary trunk associated with double-outlet right ventricle (C) and ventricular septal defect (arrow in panel D). We also noted a condition known as an interrupted aortic arch type B (IAA-B) in panel (D) and other sections (not shown). Asterisk in panel (A) indicates a dilated blood vessel. Accompanied panels (A′, B′, C′, and D′) show sections from a wild-type littermate corresponding to sections (A, B, C, and D), respectively. Abbreviations used in the panels are as follows: ns, nasal septum; ps, palatal shelf; to, tongue; rcc, right common carotid artery; lcc, left common carotid artery; ric, right internal carotid artery; lic, left internal carotid artery; rec, right external carotid artery; lec, left external carotid artery; cv, cervical vertebra; tv1, thoracic vertebra 1; tv2, thoracic vertebra 2; es, esophagus; t, trachea; ts, thymus; st, top of the sternum (manubrium); co, ribs (costae); ao, aorta; pt, pulmonary trunk; rv, right ventricle; and lv, left ventricle. (E, F, G, H, I) Compound heterozygosity for Crk and Crkl deficiency resulted in an embryonic phenotype at E16.5. Timed mating was set up between Meox2cre/+ and Crkf/f;Crklf2/f2 parents to drive cre-dependent recombination in the epiblast. Compound heterozygotes (Crkf/+;Crklf2/+;Meox2cre/+) showed severe edema and subcutaneous hemorrhage at E16.5 (left, E), associated with a cleft palate (F) and abnormal great arteries and heart (G, H). Ink injection into the right ventricle revealed an abnormal pattern of the great arteries such as enlarged aorta without forming a left-sided arch of aorta (ao, G) as well as a ventricular septal defect (G), as ink flowed into the left ventricles from the right ventricle (dotted ellipses, G). When viewed from the left side (panel H), pulmonary trunk abnormally branched into the left common carotid artery via the ductus arteriosus connected to the descending aorta. A similar case of interrupted arch of aorta type B was found in another compound heterozygote in the same litter (I). Asterisk indicates an abnormal outflow tract externally suspected to be a persistent truncus arteriosus. The compound heterozygote also exhibited a small cervical thymic lobe, which was removed before examination of the great arteries. cp, cleft palate; pl, palate (closed); rcc, right common carotid artery; lcc, left common carotid artery; ao, aorta; rv, right ventricle; lv, left ventricle; vsd, ventricular septal defect; da, descending aorta. (J, K, L, M) Early developmental defects were observed in E8.5 and E9.5 mouse embryos when combined Crk and Crkl deficiency was induced in the mesoderm driven by Mesp1cre. The genotypes of the individual embryos shown (numbered from 1 through 11) are indicated below the panels (K, L). Panels (J, K) show lateral views of embryos isolated at E9.5. Panel (L) shows dorsal views of two E8.5 embryos. Note that Crkf/f;Crklf2/+;Mesp1cre/+ and Crkf/+;Crklf2/f2;Mesp1cre/+ embryos were phenotypically similar (embryo 1 compared with embryos 2, 3, 7, and 8). Asterisks indicate enlarged hearts without proper looping and chamber development. Arrowheads indicate the position of the posterior most somite visually identifiable, thereby indicating a delay in somitogenesis in Crkf/f;Crklf2/+;Mesp1cre/+ and Crkf/+;Crklf2/f2;Mesp1cre/+ embryos compared with cre-negative control embryos. ht, heart; al, allantois. Panel (M) shows embryos 6, 7, and 8 in yolk sac. Note a delay in vascular remodeling in embryos 7 and 8, compared with the cre-negative control embryo (embryo 6).

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    Figure S2. Crk and Crkl in situ RNA hybridization at E10.5 mouse embryos.

    (A, B) Two paralogous genes Crk and Crkl (Crk-like) were expressed broadly, but not uniformly, in mid-gestation mouse embryos at E10.5 (A and B, respectively). In situ hybridization showed similar expression patterns for both genes with particularly high levels in head and pharyngeal structures, including mesoderm and neural crest cells. In the trunk, Crk and Crkl were detected in limb buds as well as in somitic structures. The ventral parts of the brain and neural tube expressed both Crk and Crkl, whereas Crk expression extended more dorsally than Crkl in the brain. In contrast, Crk and Crkl expression was notably lower in the developing heart at this developmental stage. These results confirm that Crk and Crkl are co-expressed highly in head and pharyngeal mesenchyme cell types, although our analysis did not exhaustively determine their precise localization. A1, pharyngeal arch 1; a2, pharyngeal arch 2; fl, forelimb bud; hl, hind limb bud; ht, heart; n, nasal process.

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    Figure S3. Crkl exon 2 deletion results in edema, thymic, and great artery defects at E16.5.

    (A) Crkl exon 2 deletion was triggered in the epiblast in embryos isolated from genetic cross between Crkl f2/f2 and Crkl f2/+;Meox2cre/+ parents. Three embryos shown have the genotypes indicated. Crkld2 denotes an exon 2 deletion upon cre-dependent recombination. Embryos #1 and 2 show severe and mild edema, respectively. (A, B) Crkl exon 2 homozygous deletion resulted in missing thymus in embryo #1 and hypoplastic thymic lobes in embryo #2, whereas embryo #4 (not shown in A) had thymic lobes only mildly affected, compared with control Crkl f2/f2 embryo (#3 in A). Thymic lobes are outlined by dotted lines. In addition to missing thymus, embryo #1 showed an interrupted arch of aorta type B, and abnormal origin of the right subclavian artery, whereas embryos #2 and 4 had normal pattern of the great arteries. Rt, right thymic lobe; lt, left thymic lobe; rcc, right common carotid artery; lcc, left common carotid artery; rsa, right subclavian artery; ao, aorta; aa, arch of aorta; da, ductus arteriosus; ra, right atrium; la, left atrium; vt, ventricle.

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    Figure 2. Crk;Crkl deficiency induces multiple defects in MEFs.

    (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;R26creERT2/+ 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.

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    Figure S4. Crk-deficient primary MEFs are motile, but impaired in media acidification and population morphology.

    (A) Primary MEFs (Crk f/f;R26creERT2/+) were treated with either vehicle or 0.25 μM 4-hydroxytamoxifen (4OHT) for 24 h, and then washed, replated, and cultured for additional 48 h without 4OHT before plating at a low density on glass coverslips coated with type I collagen. The position of the cell nucleus was tracked every 5 min for 8 h 1 d after plating on the coverslip. Each colored line represents movements of a single cell (a total of 10 cells in each group). (A, B) The bar graph is a quantitative representation of the average motility speed from the data shown in panel (A). Bars indicate a range of SD. (C) The media pH indicator (phenol red) showed that Crk deficiency caused a higher pH of the culture media. MEFs were replated into four-well plate at high-density 48 h after deficiency induction (24 h of 4OHT treatment followed by 48 h of culture without the agent). (D) The morphology of confluent culture was different between 4OHT-treated and CTRL groups. Note that whereas many cells in the control group show an elongated or polarized shape, cells in the 4OHT-treated group have more polygonal shapes, giving a flat cobblestone-like appearance as a population.

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    Figure S5. Decreased proliferation of MEFs deficient for Crk and Crkl.

    Crk;Crkl double deficiency resulted in an decrease in the number of MEFs in the S phase, whereas cells in the G1 phase increased. Cell cycle profiles were determined by propidium iodide binding in 4OHT-treated groups compared with control groups without 4OHT treatment as described in Fig 2A. It is also noteworthy that cell cycle progression was inhibited without passage compared with those split on Day 2. Cells were fed with fresh media every day with or without passage. The bracket under the G1 peak indicates the gate used for cell size estimates shown in Fig 2F.

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    Figure 3. Crk and Crkl deficiencies affect numerous pathways.

    (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).

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    Figure S6. General analysis of differential gene (DE) expression.

    (A) Principal component analysis was conducted using DESeq2. Red arrows indicate shifts after 4-hydroxytamoxifen treatment (4OHT) upon induced deficiency of either or both Crk and Crkl compared with a vehicle control as paired samples. See Table S2 footnote about the pairwise structure. Note that 4OHT-induced gene deficiency generated a parallel shift in each genotype subgroup (groups 1–4 in each genotype). (B) MA plots demonstrate relationship between the expression level (counts) and relative change (log fold change) for each dot representing a single gene. Red dots indicate the genes with DE with FDR < 0.05. The degree of DE is presented in log2(fold change). Note that general degrees of DE is greatest in the Crk;Crkl deficiency group, followed by Crk and Crkl groups, consistent with the degree of shift/separation in the PCA plots above. The Crk gene showed the greatest degree of change after 4OHT treatment as designed with a value of log2(fold change) smaller than −3.0 (arrows); actual log2 (fold change) values were −6.41 and −6.83 for Crk in the Crk and Crk;Crkl deficiency groups, respectively. The Crkl gene was transcribed without exon 2 after deficiency induction, thus picking up an early termination codon immediately after the SH2 domain as reported previously (Haller et al, 2017). Although such SH2-only Crkl proteins could be possible, shorter proteins were not detected from the allele after deficiency induction in Western blot analysis (Fig 2A).

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    Figure 4. Glucose metabolism is a common target of Crk and Crkl deficiency.

    (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.

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    Figure 5. Chromatin immunoprecipitation (ChIP)-Seq analysis.

    (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.

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    Figure S7. H3K27Ac and Pol2 ChIP-Seq signals within transcription start site ± 2 kb regions in the subset red genes.

    (A) Boxplots show H3K27Ac and Pol2 ChIP signals as read counts normalized to genomic content without transformation. The whiskers show the range between the maximum and minimum data points within 1.5× IQR above the upper quartile or below the lower quartile. The dots outside of the whiskers were outliers. Note that the data distributions do not fit Gaussian distributions. Statistical analysis was performed by Mann–Whitney U tests. (B) Boxplots show the same ChIP-Seq signals upon square root transformation (sqrtRPGC). Statistical analysis was then performed by post hoc Tukey tests upon two-way ANOVA.

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    Figure 6. Deficiency for Crk and Crkl results in aberrant glucose metabolism and Igf1 signaling.

    (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.

  • Figure S8.
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    Figure S8. Effects of glucose concentrations on MEFs.

    (A) Effects of low glucose was evaluated between 0 and 5 mM in the presence of 10% dialyzed FBS with or without 1 mM pyruvate supplement. Although both control and Crk;Crkl-deficient MEFs died without glucose within 24 h, more control MEFs survived compared with Crk;Crkl-deficient MEFs. The presence of pyruvate did not rescue MEFs deprived of glucose in control or Crk;Crkl-deficient MEFs (CTRL and 4OHT, respectively). (B) Effects of low glucose was evaluated between 0 and 1 mM. Note that although both control and Crk;Crkl-deficient MEFs tolerated low glucose down to 0.1 mM for a period of 24 h, the 0.1 mM concentration appeared to restrict cell proliferation particularly in control MEFs. From these results, we decided to use 0.1 mM as a glucose starvation concentration.

  • Figure S9.
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    Figure S9. Crk/Crkl deficiency and 2-deoxy-D-glucose induce cell blebbing.

    (A) Immunofluorescent images of vinculin show blebbing morphology induced by Crk/Crkl deficiency and 2-deoxy-D-glucose (2DG). MEFs were stained with anti-vinculin, DAPI, and CellMask 24 h after incubation with or without 2DG in glucose-free DMEM supplemented with 5 mM D-glucose and 10% dialyzed FBS. Vinculin signal intensity ratio (nuc/cyto), average edge curvature, and area ratio (cell/nuc) were obtained per single cell (shown in red, light blue, and magenta, respectively). Note that only cells successfully segmented are labeled. (B) Yellow arrows indicate cells identified as “blebbing” using a combination of three thresholds determined in panel (B). (B) To standardize automated identification, a total of 100 cells were visually identified as either “blebbing” or “nonblebbing” (green or orange dots, respectively) in five randomly selected images from each group. All cells were then combined and plotted on the XY plane between the area ratio and cell edge curvature or between vinculin signal intensity ratio and curvature. According to the XY-plots, thresholds were determined (dotted lines): single cell curvature greater than 0.029 (pixel−1), area ratio (cell/nuc) smaller than 4.5, and vinculin intensity ratio (nuc/cyto) smaller than 1.15. Blue arrows indicate outliers which could be removed by additional filters described in the Materials and Methods section.

  • Figure S10.
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    Figure S10. Overexpression of Crk or Crkl elevates phopho-S6 levels in 293 cells.

    Human embryonic kidney 293 cells were transfected with plasmids by which expression of mouse Crkl or Crk fused to an EGFP was overexpressed. Overexpression of either Crk or Crkl elevated phospho-S240/244 ribosomal S6 levels that coincided with levels of S6 proteins. Cell lysates were harvested 48 h after transfection. Note that albeit at lesser degrees (detection sensitivities approximately fourfold to fivefold lower), the anti-CRK monoclonal antibody cross-reacted with EGFP-Crkl fusion protein besides detecting EGFP-Crk and endogenous CRK. Numbers below the names of the plasmids are shown in μg.

  • Figure 7.
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    Figure 7. An activated Rapgef1 partially rescues aspects of Crk/Crkl deficiency in MEFs.

    (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 p130Cas (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.

  • Figure S11.
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    Figure S11. Automated image quantification for focal adhesions.

    (A, B) Segmentation results show a field of view of control and Crk/Crkl-deficient MEFs (A and B, respectively) after automated segmentations for focal adhesions (Fak immunostaining), nucleus (DAPI), and cell body (CellMask). Despite empirical optimization performed for selected images, segmentations were not 100% accurate. White arrowheads indicate focal adhesions identifiable by visual inspections that were failed to be included in automated segmentation. Asterisks indicate out-of-focus cells. However, such segmentation errors were rare and did not affect statistic evaluations. (C) An X-Y plot show a weak correlation between cell area and numbers of focal adhesions per cell. To exclude extreme values likely from failed segmentations, we set a 99 percentile upper cutoff for focal adhesions, whereas a lower cutoff was set to remove the cells with four or smaller number of focal adhesions as they appeared to correlate with out-of-focus cells (asterisks in panel B), which may be in part attributable to uneven thickness of plastic in 96-well plate. (D) The number of focal adhesions per cell was plotted in a box plot after a log10 transformation because the raw numbers did not follow a Gaussian distribution (Fig 7G). After transformation, the results were analyzed by two-way ANOVA and Tukey’s post hoc tests.

  • Figure S12.
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    Figure S12. H3K27Ac and Pol2 ChIP-Seq signal profiles in Myc and Vegfa.

    (A) Association of H3K27Ac and Pol2 was reduced in broad regions along the Myc gene in the Crk/Crkl deficiency–induced groups (K27ac_4OHT and Pol2_4OHT groups compared to corresponding CTRL groups). The regions that show large differences generally match the regulatory sequences identified in ENSEMBL Regulatory Build (tss_8830 and proximal_30145) as well as in the transcription factor binding profile reported in the UCSC Genome Browser (bar with gray-black shades; the darker the shade, the higher the peak level is). The arrow beneath the map indicates the direction of transcription. (B) H3K27Ac and Pol2 association was reduced in the Vegfa gene in the Crk/Crkl deficiency–induced groups (K27ac_4OHT and Pol2_4OHT groups compared with corresponding CTRL groups; see the regions in the blue and green rectangles, respectively). Notable differences were observed in the area highlighted in the red rectangle for both H3K27Ac and Pol2, which matches the transcription factor binding peak profile. The blue bidirectional arrow indicates the position of the Hypoxia Response Element previously identified (Forsythe et al, 1996). A targeted deletion of the Hypoxia Response Element (Vegfatm2Pec/tm2Pec) generated viable mice. The mutation affected Vegfa expression in response to hypoxia in neural tissues but not in fibroblasts (Oosthuyse et al, 2001). The arrow beneath the map indicates the direction of transcription.

Supplementary Materials

  • Figures
  • Table S1 Genetic interaction between Tbx1 and Crk in mice.

  • Table S2 RNA-Seq read depths and mapping efficiency.

  • Supplemental Data 1.

    The xlsx file includes a table of “canonical pathways” identified by Ingenuity Pathway Analysis based on the RNA-Seq data obtained from the primary MEF samples. The values are shown in −log10(P-value).[LSA-2019-00635_Supplemental_Data_1.xlsx]

  • Supplemental Data 2.

    The xlsx file includes a table of the differential expression (DE) genes identified by DESeq2 at a p.adj (FDR) smaller than 0.05 in the subsets a-d (subsets “red,” “orange,” “yellow,” and “green” shown in Fig 3B). Each DE gene is listed with KEGG terms. The DE genes either down-regulated or up-regulated are highlighted by light blue or orange shade, respectively.[LSA-2019-00635_Supplemental_Data_2.xlsx]

  • Supplemental Data 3.

    The xlsx file includes a table of metabolites identified by CE-TOF/MS metabolome analysis using the primary MEF samples.[LSA-2019-00635_Supplemental_Data_3.xlsx]

  • Table S3 PCR primers used in this study.

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The CRK family controls glucose metabolism and cell size
Akira Imamoto, Sewon Ki, Leiming Li, Kazunari Iwamoto, Venkat Maruthamuthu, John Devany, Ocean Lu, Tomomi Kanazawa, Suxiang Zhang, Takuji Yamada, Akiyoshi Hirayama, Shinji Fukuda, Yutaka Suzuki, Mariko Okada
Life Science Alliance Feb 2020, 3 (2) e201900635; DOI: 10.26508/lsa.201900635

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The CRK family controls glucose metabolism and cell size
Akira Imamoto, Sewon Ki, Leiming Li, Kazunari Iwamoto, Venkat Maruthamuthu, John Devany, Ocean Lu, Tomomi Kanazawa, Suxiang Zhang, Takuji Yamada, Akiyoshi Hirayama, Shinji Fukuda, Yutaka Suzuki, Mariko Okada
Life Science Alliance Feb 2020, 3 (2) e201900635; DOI: 10.26508/lsa.201900635
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Volume 3, No. 2
February 2020
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