Lar analysis of the remaining sequences that do not shift MidT-box could not generate a binding motif using MEME (data not shown). However, manual inspection of the sequences showed that most had sequences that resemble consensus T-box sites, suggesting that they may have been included in the selected oligonucleotides because of weak affinity for MidTbx. 25033180 The most frequent nucleotide at each position results in CAAGGTGTCAAGGCG as a consensus MidTbx binding motif. This motif is comprised of two regions where MidTbx displays a strong preference for particular nucleotides. Region 1 (Figure 3A ?black underline) spans positions 3? and consists of an AGGTGT sequence identical to the T-half-site. Region 2 (Figure 3A ?blue underline) consists of a CG at positionsand 15. The two regions are separated by 5 bases where there is a less strict AN-3199 supplier requirement for particular nucleotides. The nucleotides in region 1 (3?) resemble the T-half-site that MidTbx is able to bind specifically (Figure 1C). Furthermore, region 1 is similar to sites selected by other T-box family members [5,6,7,8,9]. In region 1, MidTbx has a strong requirement for a GTG at positions 5? with the T at position 6 occasionally being substituted for a C. This substitution is not correlated to the presence of another nucleotide at other positions within the motif. Positions 3 and 4 appear to be more variable, most commonly consisting of purines while position 8 is often a T or A. This demonstrates that MidTbx binds to a motif 76932-56-4 site recognized by other members of the T-box family. Region 2, consisting of CG at positions 14 and 15 has not yet been found in the binding motif of other T-box genes. We considered whether the presence of the CG was an artifact since it often appears in the primer sequence included in our analysis (Figure 3B). Two lines of evidence demonstrate that this is likely not the case. First, analysis of the same 27 clones without the primer sequences using MEME still produces a motif with a CG at positions 14 and 15 (not shown). This demonstrates that MidTbx selects a CG dinucleotide at positions 14 and 15 within the random 26 nucleotide core or primer sequence. Second, the number of nucleotides between region 2 and region 1 is invariant between the clones. If region 2 was not specifically selected by MidTbx, we would expect region 1 to vary in its location with respect to the primer sequence. However, since the spacing between region 1 and 2 is always exactly 5 nucleotides, we argue that there is a real preference for a CG dinucleotide at positions 14 and 15. However, there is not an absolute requirement for a CG in region 2 since MidTbx can bind to clones 43, 50 and 72 which lack it (Figure 3). In addition, MidTbx can bind the T-site and the Tbx20 motif, which both lack a CG (Figure 1C). The mouse Tbx20 site does contain a CG dinucleotide (GGAGGTGTGAGGCGA), but it is not as frequently represented as the CG in the Mid consensus and it is shifted over by one position such that it corresponds to nucleotides 13 and 14 numbered with respect to the Mid motif. Furthermore, the CG in the mouse Tbx20 motif is likely an artifact since it falls in the primer region. In order to test whether region 2 is necessary for binding, we generated a 15 base oligonucleotide corresponding to our consensus (CAAGGTGTCAAGGCG). The bases in region 1 or region 2 (underlined) were 
mutated in order to assess their effect on binding (Figure 3C). We found that mutating region 1 disrupted binding of MidTbx.Lar analysis of the remaining sequences that do not shift MidT-box could not generate a binding motif using MEME (data not shown). However, manual inspection of the sequences showed that most had sequences that resemble consensus T-box sites, suggesting that they may have been included in the selected oligonucleotides because of weak affinity for MidTbx. 25033180 The most frequent nucleotide at each position results in CAAGGTGTCAAGGCG as a consensus MidTbx binding motif. This motif is comprised of two regions where MidTbx displays a strong preference for particular nucleotides. Region 1 (Figure 3A ?black underline) spans positions 3? and consists of an AGGTGT sequence identical to the T-half-site. Region 2 (Figure 3A ?blue underline) consists of a CG at positionsand 15. The two regions are separated by 5 bases where there is a less strict requirement for particular nucleotides. The nucleotides in region 1 (3?) resemble the T-half-site that MidTbx is able to bind specifically (Figure 1C). Furthermore, region 1 is similar to sites selected by other T-box family members [5,6,7,8,9]. In region 1, MidTbx has a strong requirement for a GTG at positions 5? with the T at position 6 occasionally being substituted for a C. This substitution is not correlated to the presence of another nucleotide at other positions within the motif. Positions 3 and 4 appear to be more variable, most commonly consisting of purines while position 8 is often a T or A. This demonstrates that MidTbx binds to a motif recognized by other members of the T-box family. Region 2, consisting of CG at positions 14 and 15 has not yet been found in the binding motif of other T-box genes. We considered whether the presence of the CG was an artifact since it often appears in the primer sequence included in our analysis (Figure 3B). Two lines of evidence demonstrate that this is likely not the case. First, analysis of the same 27 clones without the primer sequences using MEME still produces a motif with a CG at positions 14 and 15 (not shown). This demonstrates that MidTbx selects a CG dinucleotide at positions 14 and 15 within the random 26 nucleotide core or primer sequence. Second, the number of nucleotides between region 2 and region 1 is invariant between the clones. If region 2 was not specifically selected by MidTbx, we would expect region 1 to vary in its location with respect to the primer sequence. However, since the spacing between region 1 and 2 is always exactly 5 nucleotides, we argue that there is a real preference for a CG dinucleotide at positions 14 and 15. However, there is not an absolute requirement for a CG in region 2 since MidTbx can bind to clones 43, 50 and 72 which lack it (Figure 3). In addition, MidTbx can bind the T-site and the Tbx20 motif, which both lack a CG (Figure 1C). The mouse Tbx20 site does contain a CG dinucleotide (GGAGGTGTGAGGCGA), but it is not as frequently represented as the CG in the Mid consensus and it is shifted over by one position such that it corresponds to nucleotides 13 and 14 numbered with respect to the Mid motif. Furthermore, 
the CG in the mouse Tbx20 motif is likely an artifact since it falls in the primer region. In order to test whether region 2 is necessary for binding, we generated a 15 base oligonucleotide corresponding to our consensus (CAAGGTGTCAAGGCG). The bases in region 1 or region 2 (underlined) were mutated in order to assess their effect on binding (Figure 3C). We found that mutating region 1 disrupted binding of MidTbx.
