The positional distribution of the C-terminal eh1-like motifs in Fox proteins of the B, E, H and Q subclasses (A) and the A D, C and I subclasses (B)

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators"</p><p>http://www.biomedcentral.com/1471-2164/8/201</p><p>BMC Genomics 2007;8():201-201.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1939712.</p><p></p> Size of polylinker represents the distance between the first residue of the eh1 motif and the conserved C-terminal residue of the winged helix DNA-binding domain

The diagrams summarize the amino acid compositions of the eh1-like motifs identified in Fox proteins

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators"</p><p>http://www.biomedcentral.com/1471-2164/8/201</p><p>BMC Genomics 2007;8():201-201.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1939712.</p><p></p> The amino acid usage frequency of eh1-like motifs identified in invertebrate (A) and vertebrate (B) Fox proteins. The diagrams were generated with the WebLogo program [44]

A phylogenetic tree for proteins of the FoxE subclass and the FoxC and FoxD outgroups

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators"</p><p>http://www.biomedcentral.com/1471-2164/8/201</p><p>BMC Genomics 2007;8():201-201.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1939712.</p><p></p> A neighbor-joining method was used to construct the tree topology and bootstrapping values are shown at each branch point (percentage of 1000 bootstrap samples) using the MEGA 3.1 software. Gaps were deleted in pairwise comparisons. The distance scale below the tree represents the number of substitutions per site. The C and D families are collapsed for better illustration. Protein sequences that lack a recognizable eh1-like motif are represented by blue triangles. Proteins and subclasses that contain an eh1-like motif are represented by red circles

Positional fluctuations of eh1-like motifs in the ortholog and paralog groups of vertebrate Fox proteins

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators"</p><p>http://www.biomedcentral.com/1471-2164/8/201</p><p>BMC Genomics 2007;8():201-201.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1939712.</p><p></p> (A) Positional fluctuations of the eh1-like motifs of the ortholog and paralog groups of the A, C and D subclasses. (B) Positional fluctuations of the eh1-like motifs of the ortholog and paralog groups of the B, E, H and Q subclasses. Polylinker represents the distance between the first residue of the eh1-like motif and the conserved C-terminal residue of the winged helix DNA-binding domain. The paralog groups within a Fox subclass are indicated on the -axis

Conserved amino acids in the extreme C-terminus of FoxD4/FoxD4L1 proteins.

<p>(A) CLUSTALW alignment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061845#pone.0061845-Thompson1" target="_blank">[64]</a>, viewed in ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061845#pone.0061845-Gouet1" target="_blank">[65]</a>, of the extreme C-terminal region of human FoxD4 (UniProtKB/Swiss Prot accession number Q12950), human FoxD4L1 (Q9NU39), mouse FoxD4 (Q60688), <i>Danio</i> FoxD4L1 (O73784) and <i>Xenopus laevis</i> FoxD4L1 (Q9PRJ8). The black boxes highlight identical amino acids, the light boxes highlight conserved amino acids and the bold letters indicate identical amino acids within a conserved region. The blue line denotes the amino acids in the <i>Xenopus</i> sequence predicted to form an α-helix, and the red line denotes Motif 6 (Fig. 1A). Arrows denote amino acid substitutions in the C-terminal mutants used in this study (L>A; Q>R; GARQ>GARG; GARQ>GARP). (B) Amino acid changes made in the C-terminal mutants used in this study.</p

Conserved amino acids in the Acidic Blob region of FoxD4/FoxD4L1 proteins that were mutated for this study.

<p>(A) CLUSTALW alignment of the N-terminal region including the Acid Blob (AB, denoted by red line), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061845#pone-0061845-g003" target="_blank">Figure 3</a>. The highly conserved IDIL sequence is predicted to form a short β-strand (green line). Six amino acids, denoted by the blue line, were deleted in the AB1 construct. The amino acid substitutions made in the AB2 and AB4 constructs are noted. (B) Predicted protein folding within the Acidic Blob of the wild-type (Wt) and AB mutated <i>Xenopus</i> FoxD4L1 proteins. Red lines denote the short β-strand, and the blue ribbon denotes a 1.7 turn α-helix predicted to form by the 6 alanine residues. Dashes over the aspartic (D) and glutamic (E) acid residues indicate negative charges.</p

Predicted structures in FoxD proteins.

<p>Legend: The N-terminus of each FoxD protein contains the conserved IDVV/IDIL/IDVL sequence at the amino acid (aa) location indicated, closely followed by a glycine (G) residue. Psipred predicts these regions to be random coil, whereas Porter predicts most of them to form a β-strand at the amino acids indicated. The C-terminus of each FoxD protein contains a conserved Eh-1 motif at the amino acid (aa) location indicated. At locations downstream of this motif, the proteins are predicted to either be random coil or to form an α-helical structure at the indicated locations.</p

The ability to up-regulate <i>gem</i> and <i>zic2</i> is lost in the AB4 mutant.

<p>(A) The FoxD4L1-AB mutant expressing clones, marked by nuclear βGal (pink dots), are located in the neural ectoderm. For AB1 and AB2, the βGal labeled cells are more intensely stained (darker blue) than their neighboring cells (e) expressing endogenous level of <i>gem</i> or <i>zic2</i>. For AB4, the βGal labeled cells are stained at the same intensity as the neighboring cells (e). Insets are higher magnifications of the clone, the position of which is indicated on the whole embryo by a bracket. For <i>gem</i>, images are dorsal views with vegetal pole to the top; for <i>zic2</i>, images are vegetal views with dorsal to the top. (B) The percentage of embryos in which the FoxD4L1-AB mutants caused up-regulation of <i>gem</i> or <i>zic2</i> in the dorsal neural ectoderm. The data for the ΔAB mutant (14aa deletion in Fig. 8A) is shown for comparison. Numbers above each bar indicates sample size; * indicates significant difference from wild type (WT) at the p<0.001 level. Data for WT and ΔAB are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061845#pone.0061845-Neilson1" target="_blank">[39]</a>.</p

Grg4 binds to the FoxD4L1 C-term mutants.

<p>(A–D) Myc-tagged versions of wild-type (WT), as well as mutants harboring amino acid substitutions downstream of the Eh-1 domain (QR, GARG, LA, GARP) in FoxD4L1 were expressed in <i>Xenopus</i> oocytes along with HA-tagged wild-type <i>Xenopus</i> Grg4. Co-immunoprecipitation (IP) and Western blot (WB) analyses of oocyte lysates expressing HA- and Myc-tagged constructs are indicated. (A) All four constructs bind with Grg4. The control panels (B–D) show that the IPs contain similar levels of FoxD4L1 wild-type and mutant proteins (B), as do the direct lysates (C). Grg4 expressing lysates also show similar levels of this protein (D). Note: Although the co-expression of Grg4 along with the wild-type and mutant Fox constructs shows similar protein levels and binding in the IPs, it is worth noting that there is a marked reduction in expression of all Fox proteins in the presence of Grg4. This may be due to degradation, rather than competition for ribosomes that affects translation, since Grg4 levels are not affected.</p

The C-terminus of FoxD4/FoxD4L1 from frog and mammals contains a novel specific conserved motif, which we term the Fox homology motif 2 (FH2).

<p>(A) The sequence logo of the 10 amino acid FH2 motif. (B) The FH2 motif is outlined in red on the FoxD4/FoxD4L1 sequence alignment.</p