CHIRP-Seq: FoxP2 transcriptional targets in zebra finch brain include numerous speech and language-related genes - PubMed (original) (raw)

Gregory L Gedman et al. Res Sq. 2024.

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Abstract

Background: 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.

Results: 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 validated 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.

Conclusions: 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.

Keywords: ChIP-Seq; Chromatin-immunoprecipitation; FoxP2; language; songbird; speech; vocal learning; zebra finch.

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Conflict of interest statement

Competing Interests The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1. Updated zebra finch reference genome assembly improves sequence alignment and gene assignments.

A) Average sequence alignment statistics across all samples using the original (blue) and updated (orange) genomic assemblies. All comparisons were significant at p < 5e-8 or greater. B) Total number of called FOXP2 peaks by condition using the original and updated genome assemblies. C) Total number of high-confidence peaks and genes associated with peaks for the adult male condition, comparing the original and updated assemblies. D) Total percentage of genes associated with peaks that were found using one or both assemblies.

Figure 2

Figure 2. FOXP2 promoter binding and gene regulation varies across conditions.

A) Feature plot of called peaks for all conditions. Total peaks for each condition are displayed as a proportion of each annotated feature. B) Total number of unique peaks (blue) and associated unique genes (green) for each condition in the experiment. Abbreviations: NS = Non-singer, S = Singer

Figure 3

Figure 3. FoxP2 and CASK protein signals co-localize within striatal and cerebellar neurons.

A) Nissl stain of sagittal section of the male zebra finch brain at the level of the cerebellum and Area X (dotted white circle). B) Nissl stain of the zebra finch telencephalon at the level of striatal Area X (dotted white circle) which is visible bilaterally. C-F) Photomicrographs show immunostain signals for DNA (DAPI-blue), FoxP2 (green) and CASK (red) as well as a merged image (far right panel in each row). C) As expected, cerebellar Purkinje neurons do not show strong DAPI signals (left panel; white arrowheads) but do co-stain for FoxP2 and CASK D). Striatal neurons from an adult male zebra finch housed with a female show co-localization of FoxP2 and CASK signals (white arrowheads). In striking contrast, those from a male who sang alone (E) show undetectable FoxP2 signals and strong CASK signals whereas those from a non-singer (F) show robust FoxP2 signals and undetectable CASK. Abbreviations: A=Anterior, D=Dorsal, L=lateral

Figure 4

Figure 4. FOXP2 regulates genes involved in human speech and language in adult male zebra finches.

Dot plot highlighting speech/language related GO terms from gprofileR. Color of each dot denotes significance after multiple test corrections (Enrichment P-value; FDR < 0.05) and the size of each dot denotes the number of genes found in the given term and condition. Adult and juvenile female birds are not displayed as they had no enrichment for the plotted terms. A full list of significant GO terms for each condition can be found in Table S2.

Figure 5

Figure 5. Adult male zebra finch regulatory network highlights molecular targets of FOXP2, many of which are involved in human speech/language dysfunction.

Protein-protein interaction network for all genes associated with human speech/language dysfunction in the adult male zebra finch (Fig. 4). Network constructed using StringDB (v10.0). Lines between nodes (genes) denote confidence of interaction with all connections >40% confidence. All unplaced nodes are clustered on the left of the network. Orange boxes highlight important genes in the ubiquitination pathway.

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