Cheatle Jarvela AM , Brubaker L , Vedenko A , Gupta A , Armitage BA , Bulyk ML , Hinman VF .

R01 HG003985 NHGRI NIH HHS

	Fig. 1. Sequence alignment for Pm and SpTbr Tbox-DNA-binding domains. (A) Tree topology was determined using a MrBayes model (TOPALI v2.5) and is based on a character alignment that includes the T-box sequences depicted in supplementary figure S1, Supplementary Material online. Lengths of branches are drawn to the scale indicated (0.2 expected substitutions per site), and the numbers indicate support by posterior probability. Bf, Branchiostoma floridae; Dr, Danio rerio; Hp, Hemicentrotus pulcherrimus; Lv, Lytechinus variegatus; Mm, Mus musculus; Pf, Ptychodera flava; Pj, Peronella japonica; Pl, Paracentrotus lividus; Pm, Patiria miniata; Pp, Patiria pectinifera; Sk, Saccoglossus kowalevskii; Sm, Scaphechinus mirabilis; Sp, Strongylocentrotus purpuratus; Xl, Xenopus laevis; Xt, Xenopus tropicalis. (B) Conceptual translation of PmTbr, SpTbr, and MmEomes T-box domains. Highlighted amino acids indicate residues involved in interaction with DNA according to alignment with XlBra crystal (Protein Data Bank ID 1XBR) (Müller and Herrmann 1997). Yellow amino acids indicate identical amino acids, whereas blue denotes nonconserved interactions within the echinoderms. Sequence aligments to XlBra are provided in supplementary figure S1, Supplementary Material online.
	Fig. 2. Position weight matrices depicting binding specificities of Tbr orthologs. Position weight matrices represent the top motifs obtained from PBM data using the Seed-and-Wobble algorithm (Berger et al. 2006; Berger and Bulyk 2009) representing SpTbr and PmTbr data set 1 (supplementary table S1, Supplementary Material online). Secondary motifs represent high-scoring oligomers whose specificity is not captured by the primary motif. Representative 8-mers and their E scores are provided underneath each motif. (A) PmTbr primary binding motif. (B) PmTbr secondary binding motif. (C) SpTbr primary motif. (D) Scatterplot of E scores for each 8-mer in the PmTbr versus the SpTbr data sets. The top 14 8-mer matches to the shared primary position weight matrix are indicated in red, whereas the top 14 matches to the PmTbr secondary motif are blue. All 8-mers and their reverse compliments (supplementary table S1, Supplementary Material online) were assigned sum probability scores based on how well they matched any 8 bp stretch of PmTbr primary position weight matrix (from positions 6–17 shown in A) and PmTbr secondary position weight matrix (from positions 7–18 shown in B). The 14 matches to each site are the top 0.02% of 8-mer matches ranked by sum probability score. E score values indicate the statistical confidence in the seed 8-mer used in position weight matrix construction, where E > 0.45 is considered to be a high-confidence binding event (Berger et al. 2006).
	Fig. 3. Steady-state affinity evaluations for Tbr DNA-binding domains. (A) DNA sequences of oligonucleotide hairpins used in SPR experiments. Nucleotides depicted in red are the predicted protein-binding site. (B) Sensorgrams depicting real-time binding of 100 nM PmTbr and SpTbr DBD to each biotinylated oligonucleotide. Nonspecific binding was determined using a blank flow cell, which had streptavidin but no DNA bound, and was subtracted from all curves. Equilibrium response (Req) was taken from these and curves corresponding to all other protein concentrations at 95 s. Response curves are also buffer subtracted and represent the average of duplicate samples with corresponding error. Results are representative of typical findings from replicate experiments. (C) Req versus concentration plus 1:1 binding fits for Pm and SpTbr’s steady-state affinity for primary and PmTbr secondary binding motifs. Data points indicate the average of duplicate samples plus error from two different concentration series experiments. Errors shown represent standard deviation of data points. (D) Req versus concentration plus 1:1 binding fits to determine Pm and SpTbr’s steady-state affinity for MmEomes secondary binding motif. Primary site binding is also shown because this analysis was performed on a different sensor chip than in C. (E) Dissociation constants of each Tbr for each oligonucleotide plus standard error of the mean. (F) Relative affinity for each ortholog for each DNA Hairpin plus standard error of the mean. All values are relative to the ortholog’s affinity for the primary site. KDs indicate average for two experimental runs, both of which were performed with duplicate scrambled concentration series, with the exception of primary binding site values, which come from data depicted in (C) and (D), and therefore include more experiments.
	Fig. 4. PmTbr can use the primary and secondary sites in vivo to drive reporter gene expression interchangeably except when Tbr levels are reduced. (A) Schematics depicting OtxG mCherry, OtxG GFP, 2 ° Tbr GFP, Tbr Deletion GFP, and Basal promoter GFP reporter gene constructs including the endogenous and mutated Tbr-binding motifs of interest. (B) ChIP PCR using primers pairs surrounding OtxG (OtxG CRM Amplicon) or primers pairs 1 kb up or downstream of OtxG. EtBr-stained gel shows amplicons obtained from total chromatin, preimmune sera mock ChIP, and Anti-PmTbr ChIP. (C–E) qPCR analysis of GFP expression levels driven by constructs indicated. All GFP expression levels have been normalized to mCherry levels that were driven by the coinjected OtxG mCherry construct. (C) Normalized GFP expression levels of OtxG GFP, Basal Promoter GFP, and Tbr Deletion GFP at 28 h. (D) At developmental time points 21 h, 25 h, and 28 h, Tbr is equally able to drive expression from OtxG reporters containing an endogenous primary site and introduced secondary site. The normalized expression level of GFP in OtxG GFP (blue bars) compared with 2 ° Tbr GFP (red bars) is not significantly different. (E) Normalized GFP expression levels resulting from 2 ° Tbr GFP or OtxG GFP coinjected with control MASO (blue bars) or Tbr (red bars) MASOs. In panels, n indicates the number of replicate samples, each consisting of 50 sibling embryos. All error bars indicate standard error of the mean. P values indicate the results of a two-tailed t-test. Details of these tests are provided in the main text. NS indicates not significant by two-tailed t-test.
	Fig. 5. Secondary Tbr reporter has reduced expression compared with OtxG in the ectoderm when Tbr levels are declining. (A–A″) In all panels, blue indicates DAPI nuclear stain and red indicates Tbr localization. (A) Thirty-one hours blastula stage Patiria miniata embryo; (A′) 52-h gastrula stage embryo; and (A″) 65-h late gastrula stage embryo. Arrow heads indicate localization, which is present in only the ciliary band ectoderm by 65 h. (B–E″) In all panels, blue indicates DAPI nuclear stain, red indicates mCherry transcripts labeled by CyIII, and green indicates GFP transcripts labeled by fluorescein. (B), (C), (D), and (E) depict the entire embryo with merged expression, whereas (B′–B″), (C′–C″), (D′–D″), and (E′–E″) are insets of the region of interest for each probe. (B–C″) OtxG GFP and 2 ° Tbr GFP both coexpress spatially with OtxG mCherry at 28 h (D–D″). OtxG GFP reporter coinjected with OtxG mCherry at 56 h. The reporters are still spatially coexpressed at this stage. (E–E″) 2 ° Tbr GFP reporter coinjected with OtxG mCherry at 56 h. GFP expression is reduced compared with OtxG GFP, whereas mCherry levels remain more consistent. (F) Quantification of fluorescent intensities of fluorescein (GFP) relative to CyIII (mCherry) at 28 h and 56 h. N indicates the number of embryos imaged. Error bars indicate standard error of the mean. P values indicate the result of two-tailed t-tests, which are described in the Results.
	Fig. 6. Modular binding of Tbr may allow for diverse transcriptional responses during development and allow for greater evolvability. (A) When PmTbr levels are high, transcription of target genes can be activated via primary and secondary sites. Activated targets are denoted by arrow inputs. However, when PmTbr levels are low (B), only genes regulated via primary sites are activated, whereas those that use secondary sites will have no or reduced transcription, which are shown with no arrows. Because SpTbr has reduced affinity for the secondary site, it will encounter the later scenario, shown in (B), more frequently and may never have an opportunity to activate target genes that are dependent on secondary sites.

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