13 research outputs found
Fusion of CIC to a heterologous DNA binding domain bypasses the requirement for C1.
<p>(A) Structure of <i>Drosophila</i> CIC and CIC derivatives in which the HMG-box has been replaced by the bHLH domain of Hairy. The CIC(bHLH) and CIC(bHLH)<sup>ÎC1</sup> proteins are tagged with the HA epitope and are thus discernable from endogenous CIC. (B) Expression of CIC(bHLH)-HA in embryos stained with anti-HA antibody. The inset shows a higher magnification view of nuclear CIC(bHLH)-HA accumulation. (C-E) <i>Sxl</i> mRNA expression in female wild-type (C) and transgenic embryos expressing CIC(bHLH) (D) and CIC(bHLH)<sup>ÎC1</sup> (E). <i>Sxl</i> appears clearly repressed in both transgenic embryos.</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
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 Tukeyâs <i>post hoc</i> test; (*P<0.05 and **<i>P</i><0.01); n.s., non significant.</p
Patterns of <i>CIC</i> mutations in human OD and <i>CIC-DUX4</i> sarcomas.
<p>(A) Diagram of the CIC protein showing a set of curated mutations from the COSMIC database (<a href="http://cancer.sanger.ac.uk/cosmic" target="_blank">http://cancer.sanger.ac.uk/cosmic</a>). Only mutations corresponding to gliomas are shown. The tumor suppressor role of CIC in OD is thought to involve the repression of CIC targets such as the <i>ETV/PEA3</i> family genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref029" target="_blank">29</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref030" target="_blank">30</a>]. Note that missense mutations tend to cluster in the HMG-box and C1 domains. In contrast, nonsense and frameshift mutations (indicated as âOther mutationsâ) are distributed along the entire length of the protein, which is also consistent with a requirement for an intact C-terminal region. (B) Structure and function of oncogenic CIC-DUX4 fusions, which usually include most of the CIC protein (including the C1 domain) coupled to the C-terminal trans-activation domain of DUX4 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref031" target="_blank">31</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006622#pgen.1006622.ref066" target="_blank">66</a>]. The double homeodomain region of DUX4 is indicated by boxes.</p
Distinct modes of target recognition by sequence-specific HMG-box proteins.
<p>The diagram summarizes the main DNA-binding mechanisms used by each HMG-box sub-family. Sox proteins usually bind their Sox sites in combination with partner factors that recognize adjacent DNA sequences, but can also form homo- and heterodimers via specific dimerization motifs such as those present in SoxD and SoxE family members. Some TCF factors also exhibit bi-partite DNA recognition via the HMG-box and the C-clamp domain that binds GC-rich sequences known as Helper sites. In contrast, CIC proteins appear to bind individual octameric sites through their HMG-box and C1 domains, acting independently of other specific DNA sites and partner proteins.</p
CIC recognizes individual octameric sites and does not depend on helper sites for selecting its targets.
<p>(A) Alignment of sequences flanking functional CBSs from selected <i>D</i>. <i>melanogaster</i> (<i>Dm</i>), <i>D</i>. <i>virilis</i> (<i>Dv</i>) and mouse (<i>Mm</i>) CIC target genes. The CBSs are highlighted in yellow. Conserved flanking motifs are shaded in different colors. (B) Sequences of probes containing intact or mutated CBSs. (C) Diagram of recombinant <i>Drosophila</i> (Dm) and human (Hs) CIC constructs used in the EMSA experiments; both constructs were produced in bacteria. (D) EMSA analyses using the DNA probes and proteins shown in panels B and C, respectively. Numbers indicate the constructs used in the binding reactions; unlabeled lanes are negative controls without added protein. Free probes are indicated by an arrowhead. (E) Diagram of a control <i>bnk-lacZ</i> reporter and a modified version carrying two CBSs (<i>bnk 2CBS-lacZ</i>). The positions of the inserted CBSs are indicated below the reporters, with conserved motifs among <i>Drosophila</i> species shaded in grey. (F, G) Patterns of expression of <i>bnk-lacZ</i> and <i>bnk 2CBS-lacZ</i> reporters.</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 1â3 and 6â17 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
Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).
<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p
Immunostaining of histological sections of Tyr::<i>Cre</i><sup>ERT2</sup>; <i>Braf<sup>CA/+</sup></i> and CMV-<i>Cre</i><sup>T/+</sup>; <i>Kras</i><sup>+/LSLG12Vgeo</sup> adenomas (<i>Kras</i><sup>+/LSLG12Vgeo</sup> 6 months) and Tyr::<i>Cre</i><sup>ERT2</sup>; <i>Braf<sup>CA/+</sup></i>; <i>Lkb1</i><sup>flox/+</sup> and CMV-<i>Cre</i><sup>T/+</sup>; <i>Kras</i><sup>+/LSLG12Vgeo</sup> adenocarcinomas (<i>Kras</i><sup>+/LSLG12Vgeo</sup> 11 months) with CC10, SP-C, E-Cadherin, Ki67, p53 and LKB1 antibodies.
<p>(*) indicates airways. Bars 500 ”m and 80 ”m for magnifications.</p
Neonatal activation of BRAF<sup>V600E</sup> through the expression of Tyr::<i>Cre</i><sup>ERT2</sup> upon 4OHTx treatment drives aberrant proliferation of lung cells.
<p>(<b>A</b>) Schematic representation of mouse treatment and expressed proteins. (<b>B</b>) Representative images of Cre-recombinase staining in 4-days-old wild type (WT, nâ=â3) and Tyr::<i>Cre</i><sup>ERT2</sup>; <i>Braf<sup>CA/+</sup></i> (nâ=â3) mice lungs treated with 4OHTx. Bars 80 ”m. (<b>C</b>) Anti-p-ERK1/2 staining of untreated (â4OHTx) and treated (+4OHTx) 4-days-old Tyr::<i>Cre</i><sup>ERT2</sup>; <i>Braf<sup>CA/+</sup></i> mice lungs. (<b>D</b>) Schematic representation of the genetic strategy to identify tyrosinase-promoter driven Cre-recombinase lung expressing cells. Representative images of EYFP, SP-C and CC10 expressing cells in Tyr::<i>Cre</i><sup>ERT2</sup>;ROSA-lsl-<i>EYFP</i> mice 3 days after 4OHTx treatment (nâ=â3 mice) are shown. Bars 80 ”m. (<b>E</b>) Ki67 staining of histologically normal lungs in 8-days-old mice showed increased proliferation index in 4OHTx treated Tyr::<i>Cre</i><sup>ERT2</sup>; <i>Braf<sup>CA/+</sup></i> mice compared to untreated. Bars 500 ”m. Quantification of samples is shown below. 20X fields (nâ=â8 and nâ=â11 from 3 different untreated and 4OHTx treated mice respectively) were quantified. <i>p</i>-value was calculated performing Mann-Whitneyâs test.</p