Cic regulates ISC proliferation as a downstream effector of EGFR signaling.

<p>(A–C) Results of the <i>λtop</i> and <i>cic</i> epistasis tests, carried out using the <i>esg</i>^<i>ts</i><i>F/O</i> system, to co-express the indicated transgenes with GFP for 2 days at 29°C. (A) Control of adult midgut (B) <i>λtop</i> overexpresssing midgut (C) <i>λtop</i> and <i>cic</i>^<i>ΔC2</i> co-overexpresssing midgut. GFP+ clones (green) expressing <i>λtop</i> were much smaller when <i>cic</i>^<i>ΔC2</i> was co-overexpressed. Samples stained with anti-PH3 (red) and DAPI (blue). (D-F) Results of the epistasis test between <i>cic</i> and <i>egfr</i>, carried out using the <i>esg</i>^<i>ts</i> system to express the indicated transgenes for 4 days at 29°C. (D) Control adult midgut, (E) <i>Egfr-RNAi</i> expressing midgut, (F) <i>Egfr-RNAi</i> and <i>cic-RNAi</i> co-expressing midgut. The number of GFP+ cells (green) still promoted by depleting <i>cic</i> in EGFR/Ras inactivated background. Samples were stained with anti-PH3 (red) and DAPI to visualize nuclei. (G-H) Results of epistasis tests between <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>, carried out using the <i>esg</i>^<i>ts</i><i>F/O</i> system. The transgenes were induced for 2 days at 29°C (G) <i>Ras</i>^{<i>V12S35</i>} over-expressing midgut (H) <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>^<i>ΔC2</i> co-over expressed midgut. Size of GFP+ clones (green) in <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>^<i>ΔC2</i> co-overexpressing midgut was significantly reduced. Samples were stained with anti-PH3 (red) and DAPI to visualize nuclei. (I-K) ISC mitoses as quantified by scoring PH3+ cells. (I) Quantification of ISCs mitoses for the <i>λtop</i> and <i>cic</i> epistasis test. The increase in mitoses induced by <i>λtop</i> was completely suppressed by <i>cic or cic</i>^<i>ΔC2</i> over expression. (J) Quantification of ISC mitoses from <i>Ras</i>^{<i>V12S35</i>}/<i>cic</i> epistasis tests. The increase in mitosis induced by <i>Ras</i>^{<i>V12S35</i>} was partially suppressed by <i>cic or cic</i>^<i>ΔC2</i> over expression. (K) Quantification of ISCs mitosis in <i>cic</i> and either <i>Egfr or Ras</i> double knock down midguts. The increase in ISC mitoses induced by <i>cic-RNAi</i> is still observed when either <i>Egfr</i> or <i>Ras RNAi</i> is also expressed. Error bars represent standard deviations. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Scale bars represent 50μm (A-H).</p

A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer

<div><p>HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout <i>Drosophila</i> development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.</p></div

<i>cic</i> inactivation promotes ISC proliferation and hyper-activation inhibits ISC proliferation.

<p>(A,B) Knock down of Cic in ISCs using the <i>esg</i>^<i>ts</i><i>; Su(H)-Gal80</i> system. ISCs were marked by YFP (green). Samples were stained with anti-PH3 (red) for mitosis and DAPI (blue) for DNA. (A) Control adult midgut (B) Cic knock down midgut after 4 days induction 29°C. Increases in the number of YFP+ cells are observed in <i>cic</i> depleted midguts as was a large increase in mitotic cells. (C) Midguts were scored for PH3+ cells after 4 days of induction of <i>cic-RNAi</i>. A strong increase in numbers of ISC mitosis was observed in <i>cic</i> knockdown midguts. (D) Clone areas of cic mutant and control WT clones 10, 20, and 30 days after clone induction. Mutant ISCs divided faster and generated bigger clones. (E) Increased number of cells per clone was detected in <i>cic</i> mutant clones. Data was quantified 10 days after <i>cic</i> mutant clones were generated with the MARCM system. (F) Quantification of pH3-positive cells per adult midgut of the indicated genotype. <i>cic</i> transheterozygotes contained significantly more mitotic cells than controls. (G) Quantification of ISC proliferation after 12 hours <i>P</i>.<i>e</i>. infection. A decreased number of PH3+ cells, representing dividing ISCs, was observed in midguts overexpressing either <i>cic</i> or <i>cic</i>^<i>ΔC2</i> after <i>P</i>.<i>e</i>. infection. (H-J) Clones generated by the <i>esg</i>^<i>ts</i><i>F/O</i> system are marked with GFP (green), Cic over-expression was confirmed by anti-Cic (red) staining, and nuclei were visualized by DAPI (blue) staining. (E) Control adult midgut 12 days after clone induction (F) midgut overexpressing Cic (G) midgut overexpressing Cic^ΔC2 12 days after clone induction. The size of clones marked by GFP was reduced after Cic or Cic^ΔC2 overexpression. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars represent standard deviations. Scale bars represent 20 μm in A-B and 50 μm in E-G.</p

Cic regulates Pnt expression through binding to its genomic locus.

<p>(A) Heatmap of mRNA levels indicating RPKM values from RNA-Seq data from control and <i>cic-RNAi</i> expressing, FACS-sorted progenitor cells. (B-D) Relative expression of <i>pnt</i>, <i>pntP1</i>, <i>pntP2</i> and <i>Ets21c</i> as analyzed by qRT-PCR and normalized to <i>β-Tub</i> and <i>Rp49</i>. (B) Fold change of expression from the <i>cic</i> depleted FACS-sorted progenitor cells and ISCs. (C) <i>pnt</i>, <i>pntP1</i>, <i>pntP2</i> and Ets21C were upregulated in <i>cic</i> knock down midguts and downregulated in <i>cic</i> overexpressing midguts. (D) Expression change in <i>cic</i> overexpressed midgut after <i>P</i>.<i>e</i>. infection. Error bars represent standard deviation. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (E) Cic binding sites in the <i>pnt</i> locus, as determined by Cic DamID-Seq of esg+ progenitor cells from control and <i>P</i>.<i>e</i>. infected midguts. Vertical bars represent the log2 ratio of the Dam-fusion signal to the Dam-only signal. Peaks also found in Cic ChIP-Seq from embryos are marked by asterisks. Positions of EMSA probes from the <i>pnt</i> locus are indicated by blue bars. (F) Diagram of probes containing TGAATGAA sites. These sites were replaced with other sequences in probes 1 and 2 to generate probes 1mut and 2mut. (G) DNA binding of Cic and HMG-box mutated Cic to probe 1 or 1mut (left panel). DNA binding of Cic and HMG-box mutated Cic to probe 2 or probe 2mut (right panel). FP indicates “free probes.”</p

Cic targets genes in ISCs found by DamID-Seq.

<p>(A) Graph showing the location of Cic binding relative to annotated transcript TSSs. The distance is from the summit of the Cic peaks to the nearest TSS. Dashed red line showed the summit of the graph is 500bp away from TSS. (B) Box plot showing fold change of peaks in CicDamID and <i>P</i>.<i>e</i>. infected CicDam. (C) Heatmap showing fold enrichment of Cic peaks from Cic DamID-Seq without or with <i>P</i>.<i>e</i>. infection. Y axis represents genes associated with the Cic binding peaks. (D) Expression heatmap of cell cycle regulators and DNA replication related genes from RNA-Seq data from <i>cic-RNAi</i> expressing FACS sorted progenitor cells. The names of the genes that had Cic binding sites by DamID are written in green. (E) Venn diagram showing the overlap between genes upregulated > 1.5 fold upon <i>cic-RNAi</i> (left) and genes associated with Cic binding peaks (right) in ISC/EBs. (F) Graph showing correlation between genes upregulated in Cic-depleted progenitor cells, and the Cic-DamID peaks that changed significantly upon <i>P</i>.<i>e</i>. infection (upper panel). Lower panel show genes ranked by absolute expression change, and then plotted for expression fold change (bottom). (G, H) Cic binding sites in the <i>CycE</i> and <i>stg</i> loci, as determined by Cic DamID-Seq in ISC/EBs from control (above) and <i>P</i>.<i>e</i>. infected (below) midguts. Vertical bars represent the log2 ratio of the Dam-fusion signal to the Dam-only signal. Red arrows indicate TGAATG(G/A)A motifs. (I-K) mRNA level fold changes of <i>stg</i> and <i>CycE</i> analyzed by qRT-PCR and normalized to <i>β-Tub</i> and <i>Rp49</i>. (I) <i>stg</i> and <i>CycE</i> fold enrichment from whole midguts after knocking down or over expressing <i>cic</i> in all cells using the <i>tub</i>^<i>ts</i><i>(tubGal4; tubGal80ts)</i> driver. Transcription of both <i>stg</i> and <i>CycE</i> was induced in <i>cic</i> knock-down midguts and inhibitied in <i>cic</i> over-expressing midgut. (J) <i>stg</i> and <i>CycE</i> expression is upregulated in <i>cic</i>-depleted, FACS-sorted progenitor cells (ISC &EB) and ISCs. (K) <i>stg</i> and <i>CycE</i> expression fold change in <i>cic</i> over expressing midguts after <i>P</i>.<i>e</i>. infection. The induction of <i>stg</i> and <i>CycE</i> by <i>P</i>.<i>e</i>. infection was suppressed by <i>cic</i>^<i>ΔC2</i> overexpression. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars in each graph represent standard deviations.</p

The C1 domain mediates CIC repression and promoter binding in human cells.

<p>(A) Diagram of GFP-tagged human CIC protein constructs tested in reporter and ChIP assays. Mutations in the HMG-box and C1 domains are indicated by vertical lines in both domains. (B) Western blot analysis of wild-type and mutant GFP-CIC fusion proteins stably expressed in Flp-In T-REx 293 cells using antibodies directed against GFP. GAPDH expression served as a loading control. (C) Relative luciferase expression levels driven by a promoter-less vector (<i>Basic</i>) or a synthetic promoter carrying CIC binding sites derived from the <i>ERM/ETV5</i> promoter (<i>ETV5p</i>), in the absence or presence of wild-type (WT) or the indicated mutant GFP-Cic constructs transfected into 293T cells. Luciferase values are expressed relative to the activity of the reporter co-transfected with empty <i>pcDNA5/FRT/TO</i> vector (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#sec010" target="_blank">Materials and methods</a>). (D) ChIP assay using GFP antibodies in Flp-In T-REx 293 cells stably expressing wild-type (WT) or the indicated mutant GFP-CIC fusion proteins. Flp-In T-REx 293 cells stably transfected with an empty vector were used as a control (Empty). Association with the CIC binding elements in the <i>ETV1</i>, <i>ETV4</i> and <i>ETV5</i> promoters was analyzed by quantitative real-time PCR and normalized to the amount of input DNA. Statistical analysis was performed with one-way ANOVA followed by Tukey’s <i>post hoc</i> test; (*P<0.05 and **<i>P</i><0.01); n.s., non significant.</p

EGFR signaling controls Cic subcellular localization in ISCs.

<p>(A, B) Cic and Cic^ΔC2 localized in the nuclei. Transgene expression was induced using the <i>esg</i>^<i>ts</i><i>F/O</i> system at 29°C for 2 days. HA-tagged Cic or Cic^ΔC2 protein was detected by anti-HA antibody (red). Nuclear DNA is marked by DAPI staining (blue) (A) <i>cic-HA</i> overexpressing midgut. (B) <i>cic</i>^<i>ΔC2</i><i>-HA</i> overexpressing midgut. (C, D) Cic but not Cic^ΔC2 protein accumulated in the cytoplasm after <i>P</i>.<i>e</i>. infection. (C) <i>cic-HA</i> overexpressing midgut, exposed to <i>P</i>.<i>e</i>. bacteria for 16 hours. (D) <i>cic</i>^<i>ΔC2</i><i>-HA</i> overexpressing midgut after 16 hours <i>P</i>.<i>e</i>. infection. (E, F) Cic protein accumulated in the cytoplasm when EGFR signaling was activated by <i>Ras</i>^{<i>V12S35</i>}. (E) <i>Ras</i>^{<i>V12S35</i>} and <i>cic-HA</i> overexpressing midgut. (F) <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>^<i>ΔC2</i><i>-HA</i> overexpressing midgut. Cic ^ΔC2 proteins stayed in the nucleus even after overexpressing <i>Ras</i>^{<i>V12S35</i>} to activate MAPK signaling. Scale bars represent 5μm.</p

Cic controls ISC proliferation by regulating <i>pnt</i> transcription.

<p>(A–E) Effects caused by <i>pntP1</i> overexpression and RNAi’s. Transgenes were induced using the <i>esg</i>^<i>ts</i> system at 29°C for 4 days, and samples were stained for GFP (green), DNA (blue) and mitoses (PH3, red). White arrows pointing out PH3 signals. (A) Control adult midgut. (B) <i>pntP1</i> overexpressing midgut after 4 days induction at 29°C. (C) Control midgut after 12 hours <i>P</i>.<i>e</i>. infection. (D) <i>pnt</i> knockdown midgut after 12 hours <i>P</i>.<i>e</i>. infection. Fewer GFP+ and PH3+ cells are observed. (E) <i>Ets21C</i> overexpressing midgut, showing more PH3+ ISCs (arrows) and GFP+ ISCs and EBs (green). (F-H) Ectopic expression or loss of <i>pnt</i> bypasses ISC phenotypes caused by <i>cic</i> overexpression or depletion. (F) <i>pnt</i> and <i>cic</i>^<i>ΔC2</i> co-over-expressing midgut after 4 days induction at 29°C. GFP+ progenitor cells were still able to proliferate. (G) <i>cic</i> knockdown adult midgut and (H) <i>pnt</i>, <i>cic</i> double knockdown midgut. The increased number of progenitor cells marked by GFP upon <i>cic</i> knockdown was decreased by also knocking down <i>pnt</i>. (I-L) Quantification of PH3+ cells in adult midguts of the indicated genotypes. (I) <i>pntP1</i>, <i>pntP2</i> or <i>Ets21C</i> overexpression driven by <i>esg</i>^<i>ts</i> or <i>Dl</i>^<i>ts</i>. All the <i>pntP1</i>, <i>pntP2</i> and <i>Ets21C</i> overexpressing midguts contained more dividing ISCs. (J) <i>pnt</i> or <i>Ets21C</i> knockdown midguts after <i>P</i>.<i>e</i>. infection. ISC mitoses caused by <i>P</i>.<i>e</i>. infection were reduced in <i>pnt</i> or <i>Ets21C</i> knockdown midguts. (K) <i>pnt</i> and <i>cic</i> knock down using <i>Dl</i>^<i>ts</i> system. Fewer mitotic ISCs were observed in the <i>pnt</i> and <i>cic</i> double knockdown midgut than the <i>cic</i> knockdown midgut. (L) <i>pnt</i> and <i>cic</i>^<i>ΔC2</i> co-overexpressing midguts. <i>cic</i>^<i>ΔC2</i> overexpression could not inhibit ISC mitoses caused by <i>pnt</i> overexpression. (M) Quantification of PH3+ cells from adult midguts following <i>P</i>.<i>e</i>. infection. <i>MEK-RNAi</i> completely blocked infection-driven ISC mitoses, but could not inhibit ISC proliferation driven by overexpressed Ets21c. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars in each graph represent standard deviation. Scale bars represent 50μm.</p

Model for Cic control of Drosophila ISC proliferation.

<p>Upon damage, activated EGFR signaling mediates activation of ERK, which phosphorylates Cic, and relocates it to the cytoplasm. As a result, <i>stg</i>, <i>CycE</i>, <i>Ets21C</i> and <i>pnt</i> transcription are relieved from Cic repression, and induce ISC proliferation.</p

The HMG-box and C1 domains are both essential for binding of CIC to DNA.

<p>(A) Diagram of CIC protein constructs tested in EMSA experiments. Constructs 1–3 and 6–17 were transcribed and translated in vitro; constructs 4 and 5 were expressed and purified from bacteria. Construct 2 contains the HMG-box and C1 domains in close proximity, without the intervening sequences that normally separate both domains (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.s004" target="_blank">S4 Fig</a> showing that this arrangement is functional in vivo). Construct 10 represents a minimal (min) version of construct 6 where the HMG-box and C1 domains have been placed immediately next to each other. Dashes in the partial sequences of constructs 15 and 16 indicate deleted residues. (B) EMSA analyses of CIC constructs binding to different wild-type and mutant DNA probes. Numbers indicate the constructs used in the binding reactions; unlabeled lanes contain unprogrammed reticulocyte lysate as a negative control. The probes used are indicated below the gels; <i>1xCBS</i> and <i>2xCBS</i> indicate the presence of 1 or 2 endogenous CIC octameric sites, respectively. <i>hkb 2xCBS mut</i> carries mutated CIC sites. The arrowhead marks the position of free, unbound probe in all the gels. Asterisks indicate the differential mobility of protein:DNA complexes. The sequences of wild-type and mutant probes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.s005" target="_blank">S1 Table</a>.</p