CHIRP-Seq: FOXP2 transcriptional targets in zebra finch brain include numerous speech and language-related genes

Vocal learning is a rare, convergent trait that is fundamental to both human speech and birdsong. The Forkhead Box P2 (FOXP2) transcription factor appears necessary for both types of learned signals, as human mutations in FOXP2 result in speech deficits, and disrupting its expression in zebra finches impairs male-specific song learning. In juvenile and adult male finches, striatal FOXP2 mRNA and protein decline acutely within song-dedicated neurons during singing, indicating that its transcriptional targets are also behaviorally regulated. The identities of these targets in songbirds, and whether they differ across sex, development and/or behavioral conditions, are largely unknown.

Here we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to identify genomic sites bound by FOXP2 in male and female, juvenile and adult, and singing and non-singing birds. Our results suggest robust FOXP2 binding concentrated in putative promoter regions of genes. The number of genes likely to be bound by FOXP2 varied across conditions, suggesting specialized roles of the candidate targets related to sex, age, and behavioral state. We interrogated these binding targets both bioinformatically, with comparisons to previous studies, and biochemically, with immunohistochemistry using an antibody for a putative target gene. Gene ontology analyses revealed enrichment for human speech- and language-related functions in males only, consistent with the sexual dimorphism of song learning in this species. Fewer such targets were found in juveniles relative to adults, suggesting an expansion of this regulatory network with maturation. The fewest speech-related targets were found in the singing condition, consistent with the well-documented singing-driven down-regulation of FOXP2 in the songbird striatum.

Overall, these data provide an initial catalog of the regulatory landscape of FOXP2 in an avian vocal learner, offering dozens of target genes for future study and providing insight into the molecular underpinnings of vocal learning.