Supplementary Material online

Figure S1. Comparative in silico detection of FOX, FOXP and FOXP2 consensus binding sites in the SRPX2 (A) and uPAR (B) promoter sequences from human, chimpanzee and mouse.
The promoter regions of the human ( Homo sapiens, Hs ) SRPX2 and uPAR genes were taken from the UCSC genome browser web site (http://genome.ucsc.edu/) and were screened in silico for the presence of FOX, FOXP and FOXP2 sites within
1.5 kb of DNA sequence 5’ to the canonical transcription start site (TSS) for each promoter, using the DNA Pattern Find program of the Sequence Manipulation Suite (http://bioinfo.unice.fr/softwares/sms2/dna_pattern.html). Any consensus site found in a given species was compared with the corresponding sequences in the other species using clustalW2 software
(http://www.ebi.ac.uk/Tools/clustalw2/index.html). The corresponding promoter regions from the chimpanzee ( Pan troglodytes ,
Pt ) and mouse ( Mus musculus , Mm ) Srpx2 and Upar genes were taken from the most recent chimpanzee and mouse genome assemblies at http://genome.ucsc.edu/. Consensus FOX-only sites (TRTTKRY, where R = A or G, K = T or G, and Y = T or C) are in orange; consensus FOXP-FOX sites (TATTTRT) are in purple; consensus FOXP2-FOXP-FOX sites (ATTTGT) are in green; consensus FOXP2-only site (AATTTG) is in blue. +1 indicates each corresponding TSS. Blue boxes represent each corresponding first exon. SRP1 to SRP7: consensus binding sites in the SRPX2 promoter region. UP1 to UP6: consensus binding sites in the uPAR promoter region. The SRP1, SRP2, UP2 and UP6 sites that are actual binding sites for FOXP2 are underlined.
Red stars indicate the location of consensus binding sites absent in Homo sapiens , but detected in Pan troglodytes or Mus musculus .

Figure S2. EMSA DNA binding assays. FOXP2 binds to specific sites within the
SRPX2 and uPAR promoters.
All putative FOXP2 binding sites identified within approximately 1.5 kb of DNA sequence 5’ to each corresponding and canonical transcription start site (TSS) ( SRPX2 :
Genbank NM_014467; uPAR : Genbank NM_002659), were tested for ability to be bound by the FOXP2 protein. Radiolabelled probes (see Table 1) were incubated with nuclear extracts isolated from HEK293T cells transfected with FOXP2 (+) or an empty vector control (-)(as detailed in materials and methods). A probe known to be bound by FOXP2 was used as a positive control (CONSENSUS). The SRwt1, SRwt2, UPwt2 and UPwt6 probes were specifically bound by FOXP2 (arrow shows the position of the gel shift indicative of protein-DNA binding) and thus were analyzed further (see Figure 3 of main text).
1

2

SRP5
SRP6
SRP7 uPAR
UP1
UP2
UP3
UP4
UP5
UP6
Table S1. List of probes used for EMSA DNA binding assays.
The wild-type (wt) and mutated binding site in each probe is highlighted (wt consensus FOX-only sites in orange; wt consensus FOXP-FOX sites in purple; wt consensus FOXP2-FOXP-FOX sites in green; wt consensus FOXP2-only site in blue; all corresponding mutated sites in red).
Wild-type probe (5' to 3') Mutated probe (5' to 3')
SRPX2
SRP1 AGCTttgg tgtttgt gag tgtttat gaaat
SRP2 AGCTgtttttttt gtaaaca atataatc
SRP3
SRP4
AGCTggttacc aatttg cttgcat
AGCTcaataca t atttgt cacaggt
AGCTttgg tgggagt
AGCTgtttttttt gag gttccca tgggaat atataatc gaaat
AGCTgtttcacca
AGCTcaacaag
AGCTcatgct
AGCTacattca tatttac cgtcaaa
AGCTcaaaa acaaacaaacaaaca
AGCTtgcagac
AGCTttataaa
AGCTcaag
AGCTtca t tgttggc atttgt tgtttgat atcaata ataaata tatttatttatttat caggc tgaatta gtctgt acattca cagacat tatt ataaataaataaataaata aaaac ataa
AGCTcaaaa
AGCTtca actccctccctccca aaaac atccctccctccctcccta ataa
3