Kalkan T , Iwasaki Y , Park CY , Thomsen GH .

R01 GM-076599 NIGMS NIH HHS , R01 GM076599 NIGMS NIH HHS

	Figure 1. Comparison of Xenopus and human TRAF4 proteins. Predicted protein sequences of X. laevis (X.l.), X. tropicalis (X.t.), and Homo sapiens (H.s.) TRAF4 proteins are aligned by using Genedoc software (National Resource for Biomedical Supercomputing, Pittsburgh, PA). Identical and similar amino acids conserved among all proteins are shown in black and dark gray boxes, respectively. Lighter shades of gray or no shading represent low levels of amino acid conservation and the lack of conservation, respectively. Domains of TRAF4 are underlined with solid lines: RING finger (red), zinc fingers (blue), coiled-coil or TRAF-N (dark orange), and TRAF-C (light orange). Specific sequence motifs are underlined with dashed lines: first putative nuclear localization signal (NLS; amino acids [a.a.] 11–15) (green), second putative bipartite NLS (a.a. 123–124 and a.a. 136–140) (pink), and PPXY or PY motif (a.a. 305–308) (purple). The predicted X. tropicalis TRAF4 protein shares 95% identity and 98% similarity with the proteins encoded by each X. laevis TRAF4 paralogue. Compared with human TRAF4, the predicted X. laevis TRAF4a and TRAF4b proteins are 77 and 76% identical, respectively, and both are 89% similar to human TRAF4 when conservative amino acid substitutions are considered.
	Figure 2. TRAF4 is ubiquitylated and degraded by Smurf1 in a 26S proteosome-dependent manner. (A) Western blot showing HA-TRAF4 interacts with FLAG-Smurf1-CA. HEK293T cells were transfected with plasmids containing HA-TRAF4, FLAG-Smurf1 wt, and FLAG-Smurf1-CA, as indicated in the figure panel. Lysates were subjected to immunoprecipitation using anti-FLAG antibody and bound HA-TRAF4 was detected by anti-HA antibody. (B) Western blot showing steady-state levels of HA-TRAF4 is regulated by FLAG-Smurf1 in HEK293T cells. HEK293T cells were transfected with plasmids containing HA-TRAF4 and FLAG-Smurf1 wt as indicated in the figure panel. HA-TRAF4 is undetectable in the presence of FLAG-Smurf1 but is restored when proteosome inhibitor MG132 is added. GAPDH is used as loading control (C) Western blot showing ubiquitylation of Myc-TRAF4 by FLAG-Smurf1 (wt) in HEK293T cells. HEK293T cells were transfected with plasmids containing Myc-TRAF4, FLAG-Smurf1 wt, and HA-ubiquitin as indicated in the figure panel. Cells were treated with MG132 before lysis. Lysates were subjected to immunoprecipitation using anti-Myc antibody and HA-ubiquitin was detected with anti-HA antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as loading control.
	Figure 3. Smurf1 regulates endogenous TRAF4. (A) Western blot of Smurf1 and TRAF4 proteins from HeLa cells transfected with control or Smurf1-specific siRNA. Partial reduction of Smurf1 protein resulted in elevated TRAF4. (B) Quantitation of Smurf1 and TRAF4 proteins detected in the Western blot. Smurf1-specific siRNA reduced Smurf1 proteins levels by as much as 55% compared with control siRNA, resulting in a corresponding 55–75% increase in TRAF4. Expression levels are normalized to the average of control triplicates.
	Figure 4. Smurf1 colocalizes with TRAF4. HeLa cells were transfected with expression constructs for TRAF4, catalytically inactive Smurf1C699A (Sf1CA), or both genes, and then examined by immunofluorescent staining. TRAF4 alone localized to clusters of unknown identity (a), whereas Smurf1 alone localized to predominantly the cell surface, including filipodia (b). Coexpression of the two proteins caused Smurf1 and TRAF4 to colocalize, in what seem to be patches at the cell surface (c–f). 4,6-Diamidino-2-phenylindole (DAPI) staining in panels a, b and f.
	Figure 5. Regional and tissue-specific expression of TRAF4 genes during Xenopus embryonic development. (A) Temporal expression of TRAF4a and TRAF4b analyzed by RT-PCR. Ornithine decarboxylase (ODC) was scored as a loading and RNA processing control. (B) In situ RNA hybridization showing spatial expression of TRAF4 (a–q). Side views of four-cell (a), stage 8 (b), and stage 10.5 (c) embryos (animal pole up). Dorsal-posterior view of a stage 13 embryo (d) reveals TRAF4 expression in the neural plate. Sense controls are shown in a′–d′. Sagittal sections of a stage 11 (e) and a stage 12.5 embryo (f) showing TRAF4 expression in the general mesoderm indicated with asterisk (e) and in the presumptive neural plate and the involuting mesoderm pointed with arrow (f). Dorsal-anterior view of a stage 14 embryo (g) and its sense control (g′). TRAF4 is expressed in the developing neural crest (red arrowheads), preplacodal and cement gland region (green arrows) and the posterior neural plate (asterisk). Anterior-lateral (h) and dorsal (k) views of a stage 17 embryo and sense control (h′). Strong expression of TRAF4 is detected in the cranial neural crest (red arrowheads), cement gland (red arrow) and the anterior neural fold hingepoints (green arrowhead) (h). Note the lack of expression in the nonneural ectoderm (asterisk) and weaker expression in the neural plate (h). TRAF4 is expressed in the lateral (arrowhead) and medial trunk neural crest (arrow) (k). Expression of TRAF4 (i) and Slug (j) in the anterior region of stage 17 embryos for comparison. Note that in the neural plate and the transverse neural fold TRAF4 is expressed, whereas Slug is not (asterisks). TRAF4 transcripts are enriched in the anterior neural hingepoints (green arrowheads) (i). Dorsal (anterior to the left) (m) and head (l) views of a stage 21 embryo. TRAF4 is expressed in the somites (red arrow) and the trunk neural crest (green arrow) (m) and in the mandibular (m), hyoid (h) and branchial (b) branches of the migrating cranial neural crest (arrowheads) (l). Lateral views of stage 25 (n) and stage 32 (o) embryos (anterior to the left). Red arrowheads point to TRAF4 expression in the pronephros (pn) and the pronephric duct (pd). TRAF4 also is expressed in the spinal cord (sc) (green arrow) (o). Sense control of a stage 32 embryo is shown in k′. In the head region, TRAF4 expression is detected in neural crest and sensory placode derivatives (p): pharyngeal arches (arrowheads), otic vesicle (ov), olfactory placodes (op), cranial ganglia (cg), vagal ganglia (vg). TRAF4 is expressed in the melanoblasts (arrowheads) (r), which are also neural crest derivatives.
	Figure 6. TRAF4 is a positive regulator of BMP signaling. (A) TRAF4 enhances mesoderm induction by BMP4 in stage 10.5 animal caps. TRAF4 suppresses neural and cement gland induction (B) by a truncated, dominant-negative BMP receptor (tBR) and (C) by Noggin in stage 17 animal caps. (D) On the top is the alignment of 5′UTR regions of TRAF4a and TRAF4b immediately upstream of ATG start codon (red letters). Antisense morpholino oligo (MO) target sequences for TRAF4a (MOa) and TRAF4b (MOb) are indicated by a blue or pink underline, respectively. A western blot on the bottom shows translation of injected TRAF4a mRNA is blocked by MOa but not by MOb. Unspecific bands serve as loading control. (E) Treatment of animal caps with TRAF4 MOa promotes neural induction by a subthreshold dose of Chordin in stage 17 animal caps. In each graph, the y-axis shows the relative expression level of each gene in animal caps expressed as percentage of expression of the same gene in a stage-matched whole embryo. These expression levels are also normalized to an internal control “housekeeping” gene expressed in all cells, ornithine decarboxylase. Results are representative of two to six independent experiments.
	Figure 7. TRAF4 is a positive regulator of Nodal signaling. (A) TRAF4 enhances mesoderm induction by Xenopus nodal 2 (Xnr2) in Xenopus animal caps. Xnr2 mRNA (10 pg) was injected at the two-cell stage into the animal pole of Xenopus embryos. Animal caps were cut at stage 8, and mesoderm marker expression was scored in animal caps harvested at early gastrula, stage 10.5. (B) Treatment of animal caps with TRAF4 MOa, but not a control MO (con mo), reduces mesoderm induction by Xnr2 (5 pg). Coinjection of TRAF4 mRNA lacking the MO target sequence (d5UTR), rescues the response of animal caps to Xnr2. The y-axis shows relative expression levels and normalization was done as explained in Figure 5 legend.
	Figure 8. General effects of TRAF4 loss of function and rescue by TRAF4 mRNA. (A) Unilateral (left-sided) injection of TRAF4a MO (MOa) or TRAFb MO (MOb), but not the control MO (Con MO), causes loss of the neural fold, particularly the neural fold hingepoints on the injected side, which is indicated by arrows (B) Classification of phenotypes caused by injection of TRAF4a MO into dorsal marginal zone, from most severe to minor (1–4) at stage 34: 1, almost complete loss of eyes and pigment, very short axis; 2, rudimentary eyes, severe loss of pigments, short axis; 3, moderate loss of eyes and pigments, short axis; and 4, minor defects in eyes, axis, and pigmentation. Refer to the graph in C for quantification of phenotype frequencies. Note reduced pigments (derivatives of the neural crest) in phenotypes 1 and 2. (C) Developmental defects caused by TRAF4a MO (MOa) were partially rescued by MO-resistant TRAF4a (ΔUTR-TRAF4) mRNA. The graphs show percentage of embryos with phenotypes from 1 to 4, represented by colors next to embryo pictures in B. Results from three independent rescue experiments are shown. Note the shift in percentage of severe phenotypes toward minor phenotypes, when dUTR-TRAF4 mRNA is coinjected with MOa. Injection of GFP mRNA served as a negative control.
	Figure 9. TRAF4 depletion and overexpression affects neural crest markers. (A) Unilateral (left-sided) injection of TRAF4a MO or (B) TRAF4b MO decreases expression of neural crest markers (Foxd3, Slug, and Sox10) at stage 17 on the injected side, which is designated by LacZ staining, red (A) and blue (B) stains. (C) Unilateral injection of TRAF4a mRNA produces ectopic (arrows) and expanded neural crest (brackets), demonstrated by in situ hybridization for the neural crest marker Slug. GFP mRNA was injected as control, red stain corresponds to lineage tracer LacZ which indicates the injected cells.
	Figure 10. Expression patterns of TRAF4 and Smurf1 overlap during midneurula stage. Antero-lateral (a–d) and dorsal (a′–d′) views of stage 17 embryos processed by WISH. Single-probe WISH shows expression of Smurf1 in the neural plate and the surrounding region including the neural crest and the cement gland (b and b′). Sense controls for Smurf1 are shown in a and a′. Single-probe WISH using BCIP as substrate shows TRAF4 expression (c and c′). Double-probe WISH shows expression of Smurf1 (purple) and TRAF4 (blue) overlap in the neural plate, neural crest and cement gland (d and d′). Enlarged anterior and dorsal views of the overlapping expression regions are shown in e and e′, respectively. Arrows point to expression of TRAF4 in the neural crest (blue), which lies within the regions of Smurf1 expression (purple) (e and e′).
	traf4 (TNF receptor-associated factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up
	traf4 (TNF receptor-associated factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior lateral view, anterior left, dorsal up.
	traf4 (TNF receptor-associated factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.

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