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The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNAbinding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved. INTRODUCTION FOXP2 is a transcription factor of interest in the development and evolution of language in humans (1). Broad interest in FOXP2 began with the discovery of its linkage to autosomal dominant transmission of developmental verbal dyspraxia, a deficit of speech articulation, in the large KE family pedigree (2). The trait was linked to a locus on chromosome 7 and eventually to a single nucleotide (residue 553) residing in the DNA-binding domain of FOXP2, a member of the forkhead box family of sequence-specific DNA-binding proteins (2–6). Several unrelated cases having similar phenotypes were also identified and typically involved truncation events of the 30-end of the FOXP2 open reading frame (ORF) (2,7). Affected individuals have normal intelligence and hearing but have jerky, dysfluent and disordered speech (8). FOXP2, therefore, offers an entry point into understanding the molecular underpinnings of the development of patterned syntactic speech. Shortly after the KE phenotype was mapped to FOXP2, analysis of the gene’s sequence conservation revealed an interesting evolutionary history, adding another dimension to its importance in human speech. The mammalian sequence is well conserved except for two mutations in the human lineage (T303N and N325S), both N-terminal to the Zn-finger domain (Figure 1). Conservation analysis revealed an enhanced non-synonymous substitution rate in the hominid lineage, consistent with recent selection (9). In support of this idea, researchers found that FOXP2 locus sequences from a diverse panel of human individuals contain an excess of high-frequency derived alleles and rare intronic alleles indicative of a selective sweep in human ancestors (10,11). Animal models expressing *To whom correspondence should be addressed. Tel: +1 224 213 0560; Email: [email protected] Correspondence may also be addressed to Hao Li. Tel: +1 415 502 8187; Fax: +1 415 514 2617; Email: [email protected] Correspondence may also be addressed to Joseph L. DeRisi. Tel: +1 415 418 3059; Fax: +1 415 514 2617; Email: [email protected] Nucleic Acids Research, 2013, 1–14 doi:10.1093/nar/gkt259 The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research Advance Access published April 26, 2013 at U N IV E R IT Y O F C A L FO R N IA B E R K E L E Y on M ay 1, 2013 http://narrdjournals.org/ D ow nladed from either mutant FOXP2 or lower levels of wild-type protein have borne out the involvement of FOXP2 in vocalization in mice and in zebra finches (12–14). These results suggest that in addition to its developmental role in speech, FOXP2 may have had an evolutionary role in speech and language. Although there exist several possible paths for the molecular evolution of FOXP2 function between ancestral primates and humans, here we investigate the simple possibilities that the selected protein mutations in the human lineage could have altered FOXP2’s-binding activity, driving novel targeting and functions; and/or that the genomic binding sites in humans could have changed, causing modulation of targeting strength and gain and loss of FOXP2-binding targets. Evaluation of these possibilities would be aided by a thorough understanding of the FOXP2 affinity profile, yet there is surprisingly poor agreement over the identity of the FOXP2 DNA-binding motif (Table1). This poor agreement may be due to either the use of different experimental techniques or reliance on previous candidate motifs identified through studies of related proteins (e.g. FOXP1 and FOXP3) (15–17). The lack of a consistent binding site model makes it difficult to predict targets by sequence analysis, which in turn complicates the task of defining evolutionarily novel target repertoires. Here, we clarify FOXP2’s target motif using recently developed microfluidic methods that measure binding affinity of proteins to a library of different DNA sequences (18,19). The resulting detailed binding site model reveals essentially identical affinity profiles for the chimp and human FOXP2 orthologs, suggesting that evolutionary differences between lineages did not involve distinct binding preferences. The derived FOXP2 motif is corroborated by an unbiased search for overrepresented motifs within FOXP2-bound ChIP-seq peaks. We find that most motif sites are conserved, and they tend to be near other transcription factor genes. However, we also find instances of evolutionarily novel FOXP2 target binding sites, including genes involved in synaptic plasticity and neural development, suggesting that changes in cis regulation may underlie novel functions of FOXP2 in human language. MATERIALS AND METHODS Cloning, mutagenesis and expression Full-length FOXP2 coding sequence was initially amplified from HeLa cDNA (primers designed to isoform 1 Ensembl record CCDS5760, included in Supplementary Information) and placed into a PCR2.1topo vector. Point mutations in the derived clone were corrected by site-directed mutagenesis (20). The sequence was confirmed by Sanger sequencing and assembly with phred/phrap. Chimp and human mutant R553H FOXP2 coding versions were constructed by site-directed mutagenesis on this wild-type human plasmid. We removed the first 213 codons by PCR and added flanking promoter, polyA, and His tag sequences necessary for in vitro transcription/translation and MITOMI (see Supplementary Information). The truncation removed the long polyglutamine stretch at the beginning of the protein for improved expression and solubility (Figure 1). A similar truncation was previously used for electromobility shift assay (EMSA) studies (21), as polyglutamine stretches of >40 residues are associated with misfolding and aggregation (22,23). The PCR products were purified by Promega Wizard gel purification and concentrated via vacuum centrifugation to 140 ng/ml. TnT T7 coupled reticulocyte lysate kit from Promega with the addition of 10 mM ZnCl2 was used to produce the protein of interest. We included 3 ml of Fluorotect Green BODIPY charged lysine tRNA in each 75 ml translation reaction for detection of the protein by fluorescence. MITOMI mold and device fabrication MITOMI devices were made as described previously (18,19). Briefly, molds for devices were fabricated on 1 152 214 409 505 722 AA