The C-clamp is required for patterning of the <i>Drosophila</i> embryonic epidermis.

<p>(A) Crossing scheme used to generate embryos containing a P[<i>Da</i>-Gal4] driver and P[UAS-<i>TCF/Pan</i>] transgene in a <i>TCF²/TCF³</i> transheterozygous mutant background. UAS-<i>TCF/Pan</i> (UAS-<i>TCF</i>) encodes for either wild type or a mutant <i>TCF/Pan</i>. (B–C) Darkfield micrographs of the ventral cuticle control (B) or P[<i>Da</i>-Gal4]/+; <i>TCF²/TCF³</i> embryos showing the normal and <i>TCF/Pan</i> mutant phenotypes, respectively. (D–G) Cuticles of <i>TCF²/TCF³</i> mutants expressing wild-type (D), mutant 5 (E), mutant 4 (F) or mutant 8 (G) <i>TCF/Pan</i> cDNAs. (H) Western blots showing comparable levels of TCF/Pan (upper blot) expression for WT and various C-clamp mutants in P[<i>Da</i>-Gal4]; P[UAS-<i>TCF/Pan</i>] embryos with Tubulin used as the loading control (lower blot).</p

Characterization of the DNA-binding and inhibitory functions of the C-clamp.

<p>(A) Sequence of the HMG-Helper site probe and the HMG site oligonucleotide probes used to characterize the ability of C-clamp mutants to bind DNA. (B) Protein fragments containing the HMG domain and wild type (WT) or mutant C-clamps were tested for their ability to bind the HMG-Helper site and the HMG site probes using the Licor EMSA assay described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone-0086180-g001" target="_blank">Figure 1</a>. Ctrl indicates probe only lane. For both probes, WT bound signal was normalized to 100. All mutants were tested at least twice and the averages are reported. Mutants 4, 5 and 8 are in bold, denoting their use in followup experiments. (C, D) EMSA experiments characterizing the defects in Mutants 4, 5 and 8 in binding to the HMG-Helper site (C) and HMG site (D) probes. For each binding reaction, 20 fmol of DNA probe and 9 pmol of protein was used. At these conditions, wild-type protein bound 7–12 times as much HMG-Helper site probe as HMG site probe (data not shown). Data represents means of triplicates±SD. These experiments were repeated three times with similar results. (E) Commassie stained gel of purified WT and C-clamp mutant proteins, demonstrating that each preparation used contained similar amounts of TCF/Pan.</p

Recombinant HMG-C-clamp fragment contains near stoichiometric amounts of Zinc.

<p>Recombinant HMG-C-clamp and HMG domain proteins were purified from <i>E. coli</i> and subjected to ICP-MS. See Materials and Methods for details of ICP-MS analysis.</p

Direct binding of the C-clamp domain to the HMG domain.

<p>Western blots using anti-His tag and anti-GST tag antibodies. (A) Pulldown using GST-HMG or the GST control incubated with wild-type (WT) or Mutant 5 His-C-clamp (see Materials and Methods for details of the binding reaction). The upper blot shows an interaction between GST-HMG and the wild-type C-clamp. The middle and lower blots are 5% input of the total reaction mixture. (B) Pulldowns using GST-HMG (lanes 1–4) or GST control (lanes 5–8) and WT His-C-clamp. The proteins were pretreated with micrococcal nuclease (lanes 2 & 6) or OPA (lanes 4 & 8). The negative controls (lanes 1 & 5 for micrococcal nuclease, and lanes 3 & 7 for OPA treatments) were subjected to the same treatment conditions with nuclease free water being used instead of micrococcal nuclease or OPA. The upper blots show the amount of His-C-clamp pulled down and the middle and lower blots are 5% input of the total reaction. All experiments were repeated three times with similar results.</p

The C-clamp domain requires zinc for binding to the Helper site.

<p>(A) Pretreatment of a recombinant fragment of TCF/Pan containing the HMG and C-clamp domains with the metal chelator 1,10-orthophenanthroline (OPA) inhibits its ability to bind to an oligonucleotide containing a HMG and Helper site. Binding was restored by incubation of the OPA-treated protein with zinc but not other divalent metals. Ctrl indicates a probe only lane and UT refers to protein that was untreated by OPA. For each binding reaction 9 pmoles of protein and 20 fmoles of oligonucleotide were used. (B) Licor quantification of the EMSA data. (C, D) EMSA gel and Licor quantification demonstrating that a protein fragment containing only the HMG domain was insensitive to OPA treatment. 9 (HMG-Cclamp) and 12 (HMG only) pmoles of protein and 20 fmoles of oligonucleotide were used. (E, F) EMSA and Licor quantification demonstrating that recombinant C-clamp protein is sensitive to OPA treatment. 50 pmoles of C-clamp protein and 20 fmoles of oligonucleotide were used. All experiments were carried out at least three times and the bar graph results are the means of at least triplicates±SD.</p

Model depicting a dual role for the C-clamp in enhancing the DNA-binding specificity of TCF/Pan.

<p>The presence of the HMG and C-clamp domains allows TCF/Pan to bind to HMG-Helper site pairs (middle). In addition, the C-clamp may inhibit TCF/Pan from binding to unpaired HMG sites (right). Conversely, the HMG domain may inhibit the C-clamp from binding unpaired Helper sites (left).</p

The cysteine and basic residues of the C-clamp are required for activation of a W-CRM reporter in cell culture.

<p>(A) Amino acid sequences of the C-clamp in wild-type (WT) TCF/Pan and the eight mutants. The amino acids that have been mutated are in red. (B–E) TCF/Pan RNAi rescue assays carried out in Kc cells. Endogenous TCF/Pan was depleted using dsRNA that targets the TCF 3′ UTR, followed by transient transfection of either WT or C-clamp mutant expression constructs containing a heterologous 3′UTR that cannot be targeted by the dsRNA. The Wnt/ß-cat pathway was induced (Wnt on) using Arm* (see text for further description). Ctrl and “Wnt off” refer to controls where there was transfection of an empty expression vector. The <i>nkd-IntE</i> W-CRM reporter was used as an example of a Helper site-dependent W-CRM (B, C), while the synthetic 6xHMG reporter was used as a Helper site-independent Wnt readout (D, E). For some experiments, the mutant TCF/Pan constructs were transfected at eight times the level of wild-type, to ensure that sufficient levels of mutant protein were produced (C, E). Bars are the mean of triplicate transfection SD. Experiments were repeated at least three times with similar results. See Materials and Methods for additional details of the cell culture conditions.</p

The C-clamp domain is sufficient for binding to the Helper site.

<p>(A) EMSA showing that a His-tagged C-clamp domain can bind to a DNA probe containing a Helper site, while a C-clamp protein containing two mutations in the domain (K2A, R4E; same as mutant 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone-0086180-g003" target="_blank">Figure 3A</a>) has greatly reduced binding. Neither protein bound a probe lacking a Helper site. For each binding reaction 50 and 100 pmoles of protein and 20 fmoles of oligonucleotide were used. (B,C) Quantification of the EMSA data using the Licor system. The bar graph results are the means of at least three separate binding reactions±SD. See Materials and Methods for details.</p

The spacing of cysteine residues in the C-clamp is distinct from other Zn-finger motifs.

<p>In a typical C2H2 Zn-finger, the two cysteine and two histidine pairs that coordinate the Zn ion are separated by a stretch of 12 amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone.0086180-Krishna1" target="_blank">[38]</a>. In the treble-clef Zn-fingers found in the estrogen and glucocorticoid receptors, two pairs of cysteines are separated by 9 or 13 residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone.0086180-Krishna1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone.0086180-Grishin1" target="_blank">[51]</a>. A similar organization is found in the Zn2/Cys6-like finger of the yeast copper-regulated transcription factor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086180#pone.0086180-Turner1" target="_blank">[50]</a>. In contrast, the second and third cysteines of C-clamps are closely paired, with the longest (12 residue) spacing found between the first two cysteines.</p

The HMG and C-clamp domains adopt different conformations when bound to distinct binding sites.

<p>(<b>A, B</b>) Recombinant GST-HMG-C-clamp protein was incubated with 20 fold molar excess of control oligonucleotide (SS), a classic HMG and Helper site pair (TH) and a WGAWAW and r-Helper site pair (WH) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004509#pgen.1004509.s015" target="_blank">Table S2</a> for sequences of oligonucleotides). After 20 min to allow binding, the preps were subjected to partial proteolytic digestion with increasing amounts of Glu-C (A) or chymotrypsin (B) and analyzed by SDS-PAGE followed by silver staining. Proteolytic fragments enriched with TH and not WH are indicated with asterisks. (<b>C</b>) Silver stained native gel of GST-HMG-C-clamp and different oligonucleotides at the same concentrations used in the proteolytic digestions, demonstrating that a similar amount of protein is bound to TH and WH, while SS has no detectable binding. Each experiment was performed at least three independent times with similar results.</p