Human-specific transcriptional regulation of CNS development genes by FOXP2

Abstract

The signalling pathways controlling both the evolution and development of language in the human brain remain unknown. So far, the transcription factor FOXP2 (forkhead box P2) is the only gene implicated in Mendelian forms of human speech and language dysfunction. It has been proposed that the amino acid composition in the human variant of FOXP2 has undergone accelerated evolution, and this two-amino-acid change occurred around the time of language emergence in humans. However, this remains controversial, and whether the acquisition of these amino acids in human FOXP2 has any functional consequence in human neurons remains untested. Here we demonstrate that these two human-specific amino acids alter FOXP2 function by conferring differential transcriptional regulation in vitro. We extend these observations in vivo to human and chimpanzee brain, and use network analysis to identify novel relationships among the differentially expressed genes. These data provide experimental support for the functional relevance of changes in FOXP2 that occur on the human lineage, highlighting specific pathways with direct consequences for human brain development and disease in the central nervous system (CNS). Because FOXP2 has an important role in speech and language in humans, the identified targets may have a critical function in the development and evolution of language circuitry in humans.

Figure 1

FOXP2 and FOXP2^chimp differentially regulate genes in SH-SY5Y cells. a) Schematic of human FOXP2 showing its major functional protein domains and the two amino acid changes in the mutant FOXP2^chimp. b) Representative immunoblot for FLAG-tagged FOXP2 and FOXP2^chimp stable overexpression in SH-SY5Y cells. c–e) Immunofluorescent staining of antibodies against FLAG epitope (green) and FOXP1 (red), and DAPI (blue) for nuclei. c) Vector cells demonstrate no FLAG expression, while both FOXP2 (d) and FOXP2^chimp (e) expressing cells have FLAG-tagged FOXP2 in the cell nucleus. Scale bars are 5 microns. f) Subcellular fractionation followed by immunoblotting. g–h) Quantitative RT-PCR of genes that were differentially expressed in cells expressing FOXP2 compared to FOXP2^chimp. Asterisks indicate P≤0.05 and error bars are ± s.e.m. (two-tailed Student’s t-test, n=3 or 4).

Figure 2

FOXP2 and FOXP2^chimp differentially transactivate target promoters independent of FOXP1 or FOXP4 interaction. a) Immunoblotting for FLAG or FOXP1 following immunoprecipitation with either FLAG or FOXP1 Abs. b) Mass spectrometry results from SH-SY5Y or 293T cells overexpressing FOXP2 or FOXP2^chimp. The first number indicates the number of spectra and the second is the number of unique peptides. c) Cell growth analysis does not show a significant difference in proliferation between cells expressing FOXP2 or FOXP2^chimp over time (P≤0.05). Error bars are ± s.e.m. (two-tailed Student’s t-test, n=3). d–e) Dual luciferase assays in 293T cells transiently transfected with promoter fragments driving luciferase and either FOXP2 or FOXP2^chimp. Asterisks indicate P≤0.05 and error bars are ± s.e.m. (two-tailed Student’s t-test, n=3–6).

Figure 3

Visualization of one of the modules containing FOXP2 and FOXP2^chimp differentially expressed genes. Five hundred pairs of genes with the greatest topological overlap are shown. Positive correlations are depicted in red and negative correlations are depicted in blue. The gene symbols for hub genes are accentuated in large bold text.