Figure 1 (facing page). Identification of CNTNAP2 as a Direct Neural Target Bound by Human FOXP2.

In Panel A, a 300-bp clone was identified through shotgun cloning of gene fragments identified by FOXP2-chromatin immunoprecipitation and localized to intron 1 of the human CNTNAP2 gene in 7q35. Semiquantitative PCR analysis indicated consistent enrichment of this region in multiple independent experiments in a neuronlike cell line immunoprecipitated with an N-terminal FOXP2 antibody (lane 2), as compared with a control sample without the antibody (lane 3) and input DNA samples (lane 1). Lane 4 shows the water control sample. Two FOXP2 consensus binding sites were identified (highlighted in red). In Panel B, electrophoretic mobility shift assays (EMSAs) using nuclear extracts from transfected HEK293T cells assessed the ability of FOXP2 protein to bind to the 5′ consensus binding site (highlighted in red). Efficient binding to the CNTNAP probe was observed when FOXP2 was present but not when either un-transfected cells or cells expressing a mutant form of FOXP2 (R553H) were used.¹⁷ Binding to the labeled probe was efficiently reduced by competition with an un-labeled probe (CNTNAP) but not by a mutant form of the probe (CNTNAP-M) or an irrelevant binding site (NFK). The arrow shows the position of the shift caused by FOXP2 binding to the CNTNAP probe.