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Fig. S1. GRHL1 is highly conserved between vertebrate and invertebrate species. (A) Sequence alignment of Xenopus, human and murine GRHL1 orthologues. The DNA-binding (unbroken line) and dimerization (broken) domains conserved with other Grainyhead-like factors are indicated. (B) Analysis of overall identity and relative homology of the XGRHL1 polypeptide, when compared with the human (hGRHL1), murine (mGRHL1), Drosophila (dGRH) and C. elegans (ceGRH) orthologues. The high degree of identity/homology of the DNA binding and dimerization domains are also illustrated.
Fig. S2. The dominant-negative mutant of XGrhl1 (D227XGrhl1) has similar functional characteristics to the wild-type factor. (A) An N-terminal truncation of XGRHL1 (D227XGRHL1) does not affect protein:protein interactions with the full-length molecule. Schematic of full-length and truncated [putative dominant negative (DN)] XGRHL polypeptides used in this study is shown at top. The D227XGRHL1 removes the entire activation domain (amino acids 2-227). Radiolabeled full-length XGRHL1 (35SXGRHL1) was incubated with glutathione-S-transferase (GST; lane 3) or GST fused in frame with XGRHL1 (lane 2) or the D227 mutant (lane 5). A GST-human GRHL1 chimera was also tested (lane 4). As a reference, 5% of the 35SXGRHL1 polypeptide was loaded (lane 1). (B) XGRHL1 and D227XGRHL1 have similar affinities for a Grhl DNA-binding consensus motif. Aliquots (10-40 ng) of recombinant XGRHL1 or D227XGRHL1 were incubated with a radiolabeled probe encoding a previously defined consensus sequence for Drosophila grh binding. Excess free probe migrates near the bottom of the gel. (C) Expression of the Drosophila dominant-negative mutant D447grh in dissociated animal cap explant cells blocks XGrhl1-induced gene expression specifically. Dispersed animal cap explant cells harvested at stage 9 from (1) wild-type embryos, or embryos injected (2) with XGrhl1 encoding transcripts or (3) D447grh and XGrhl1 transcripts were allowed to reaggregate and mature until stage 18. Aggregates were harvested and RNA was prepared. Ectopic expression of D447grh results in specific downregulation of XGrhl1-responsive epidermal genes, including XK81A1, Dlx3, Dlx5 and XAP-2. ODC was used as a control for RNA recovery. (D) Expression of the Xenopus dominant-negative mutant D227XGrhl1 in dissociated animal cap explant cells blocks XGrhl1-induced gene expression specifically. Dispersed animal cap explant cells harvested at stage 9 from (1) wild-type embryos, or embryos injected (2) with XGrhl1 encoding transcripts or (3) D227XGrhl1 and XGrhl1 transcripts were allowed to reaggregate and mature until stage 18. Aggregates were harvested and RNA was prepared. Ectopic expression of D227XGrhl1 results in specific downregulation of XGrhl1-responsive epidermal genes, including XK81A1, Dlx3, Dlx5 and XAP-2.
Fig. S3. Inhibition of in vitro XGrhl1 translation by an XGrhl1-specific morpholino activity. (A) Antisense-sequence encoding XGrhl1-specific morpholinos (MO) interact with the translation start site block translation of XGrhl1 mRNA in vitro. XGrhl1-encoding RNA transcripts were incubated with increasing amounts of XGrhl1 translation initiation site-targeted antisense oligomers (M01, M02) or a control MO (CMO) for 30 minutes. The reactions were incubated subsequently with a translation mix in the presence of 35S-labeled methionine, the products separated on SDS-PAGE gels and analyzed by autoradiography. (B) XGrhl1-targeted morpholinos-induced translational silencing is rescued partially by a mutated XGrhl1 transcript in vitro. XGrhl1 transcripts were incubated with XGrhl1 translation initiation site targeted MO antisense oligomer M01 (10 mM) in the presence or absence of excess XGrhl1-encoding transcripts (WtXGr; lane 3), or a mutant full-length XGrhl1 (M-XGrhl1)-encoding transcript in which codons at the third or wobble position encoding amino acids 3-8 were altered to reduce the efficiency of MO-binding (lane 2). A control MO (CMO) was also tested (lanes 1 and 3). In vitro 35S-labeled translated products of these reactions were analyzed by SDS-PAGE electrophoresis.
Fig. S4. XGrhl1 binds specifically to a distinct motif in the proximal XK81A1 keratin promoter. (A) Electrophoretic mobility shift analysis (EMSA) of XGRHL1 binding to the XK81A1 promoter. A region ~200 bp upstream of the XK81A1 transcriptional start site with significant homology with a Drosophila GRH consensus sequence is shown at the top. Recombinant XGRHL1 was incubated with a probe encoding the –202 to –135 bp region of the proximal XK81A1 promoter. The retarded band (lane 1) is super-shifted (arrow) specifically by polyclonal antisera directed against full-length XGRHL1. (B) The –202 to-173 bp region of the XK81A1 promoter binds XGRHL1. A series of radiolabeled overlapping double stranded oligonucleotide probes (lanes 2-4, 8) encompassing the –202 to –135 bp region were evaluated by EMSA for their ability to bind XGRHL1. Sequences of the probes are shown above. The red type indicates potential XGRHL1 binding motifs. Green type indicates the previously defined XAP-2 binding sequence. Further mutational analysis of the core –202/–173 bp XGRHL1 binding sequence delineated a 4 bp mutant (blue type, underlined) that blocks factor binding. (C) An excess of the M2 XK81A1 promoter mutant blocks XGRHL1 binding specifically. A radiolabeled –202/–173 bp XK81A1 encoding probe was incubated with XGRHL1 in the absence (lane 1), or presence of increasing concentrations (fold excess) of unlabeled wild-type probe (lanes 2-5) or similar concentrations of the M2 mutant.
References
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Sasai,Y., Lu, B., Piccolo, S.and De Robertis, E. M. (1996). Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps. EMBO J. 15, 4547-4555.
Wilson, P.A., Lagna, G., Suzuki, A. and Hemmati-Brivanlou, A. (1997). Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1. Development 124, 3177-3184.
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