Protein-Protein Interaction Among the FoxP Family Members and their Regulation of Two Target Genes, VLDLR and CNTNAP2 in the Zebra Finch Song System.

Ezequiel Mendoza,Constance Scharff

doi:10.3389/fnmol.2017.00112

Abstract

The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP21 contributes to song learning and production in birds. In human cell lines, transcriptional activity of FOXP2 requires homo-dimerization or dimerization with paralogs FOXP1 or FOXP4. Whether FoxP dimerization occurs in the brain is unknown. We recently showed that FoxP1, FoxP2 and FoxP4 (FoxP1/2/4) proteins are co-expressed in neurons of Area X, a song control region in zebra finches. We now report on dimer- and oligomerization of zebra finch FoxPs and how this affects transcription. In cell lines and in the brain we identify homo- and hetero-dimers, and an oligomer composed of FoxP1/2/4. We further show that FoxP1/2 but not FoxP4 bind to the regulatory region of the target gene Contactin-associated protein-like 2 (CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.

Highlights

Forkhead box (Fox) transcription factors comprise 19 highly evolutionary conserved, structurally related families, FoxA to FoxS
We found that zebra finch FoxP proteins when expressed individually in HeLa cells significantly repressed the transcriptional activity under the control of the SV40 promoter (Figure 6; One way analysis of variance (ANOVA); F = 28.79; DF = 7; n = 8; p < 0.0001; Tukey’s Multiple comparison Test; compared to empty vector: FoxP1 p < 0.0001, FoxP2 p = 0.013, and FoxP4 p < 0.0001), in the same range as reported for mice and human FOXP1/2/4
Taken together we show that FoxP1 and FoxP2 can bind to and regulate the Contactin-associated protein-like 2 (CNTNAP2) promoter, with opposing activities

Summary

Introduction

Forkhead box (Fox) transcription factors comprise 19 highly evolutionary conserved, structurally related families, FoxA to FoxS. Many Fox genes perform tissue specific functions during development and mutations can cause cancer and other diseases (Hannenhalli and Kaestner, 2009). The FoxP family consists of only one member in invertebrates (Santos et al, 2011). Gene duplication gave rise to four FoxP subfamily members in vertebrates, FoxP1 to FoxP4 (Song et al, 2016). Expression of these four proteins is specific to particular organs and cell types, with partly overlapping patterns (Lu et al, 2002; Ferland et al, 2003; Mendoza et al, 2015; Spaeth et al, 2015). FoxP1/2/4 are all expressed in the brain (Lu et al, 2002; Teufel et al, 2003), whereas FoxP3 is

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