Intracellular distribution of a speech/language disorder associated FOXP2 mutant

doi:10.1016/j.bbrc.2006.12.130

Biochemical and Biophysical Research Communications

Volume 353, Issue 4, 23 February 2007, Pages 869-874

https://doi.org/10.1016/j.bbrc.2006.12.130 Get rights and content

Abstract

Although a mutation (R553H) in the forkhead box (FOX)P2 gene is associated with speech/language disorder, little is known about the function of FOXP2 or its relevance to this disorder. In the present study, we identify the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. A truncated version of FOXP2 lacking the leu-zip, Zn²⁺ finger, and forkhead domains that was observed in another patient with speech abnormalities demonstrated an aggregated cytoplasmic localization. Furthermore, FOXP2 (R553H) mainly exhibited a cytoplasmic localization despite retaining interactions with nuclear transport proteins (importin α and β). Interestingly, wild type FOXP2 promoted the transport of FOXP2 (R553H) into the nucleus. Mutant and wild type FOXP2 heterodimers in the nucleus or FOXP2 R553H in the cytoplasm may underlie the pathogenesis of the autosomal dominant speech/language disorder.

Section snippets

Materials and methods

DNA construction and site-directed mutagenesis. The full-length of FOXP2 cDNA was isolated from human cDNA library (Stratagene). The enzyme-digested PCR DNA fragments corresponding to full-length FOXP2, and various truncated FOXP2 were subcloned into pEGFP-C1 vector (Clontech), FLAG-tagged vector pcdef3 [26], or pGEX-4T-1 (Amersham).

To generate FOXP2 R553H mutant, site-directed mutagenesis were performed by using QuikChange II Site-Directed Mutagenesis kit (Stratagene) with following primers:

Subcellular distribution of the truncated FOXP2 variants

Full-length FOXP2 was detected in nuclei (Fig. 1). Removal of the glutamine rich N-terminal region containing the polyglutamine tract (polyQ) (resulting construct contains aa 257–715), did not affect FOXP2’s nuclear localization. In contrast, loss of the C-terminal region (1–495), a novel splicing product [15] and (1–327), a novel speech/language disorder variant [16], abolished the nuclear localization of FOXP2. Furthermore, the fragment lacking the C-terminal region caused the protein to

Nuclear translocation signals in FOXP2

The nuclear translocation of FOX proteins, including FOXA2, FOXC1, FOXE1/TTR2, and FOXF2, has been well characterized. In every case, the forkhead domain is necessary for the nuclear localization of these proteins [20], [21], [22], [23]. Sequence alignment reveals that there are 4–5 basic amino acids clustered at the C-terminal end of the forkhead domain. Apparently, these sequences act as NLSs in each case. In FOXP2, the truncation or mutation of the corresponding NLS2 (⁵⁸²KRRSQK⁵⁸⁷) region

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FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it
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Citation Excerpt :
Thus, FOXP2-forkhead associated with POT1, a component of the telomerase ribonucleoprotein [9]. In contrast with FOXP2-forkhead located in the nucleus, the forkhead with (R553H), mutation related to speech-language disorder, partially localizes in both the cytoplasm and the nucleus [2]. To examine the interaction between POT1 and FOXP2(484–598) or FOXP2(484–598) with R553H mutation {FOXP2-R553H-(484–598)} quantitatively, β-galactosidase activity was measured in the yeast Y187 strain co-transformed with pGAD-POT1 and pGBK-FOXP2-forkhead or mutated one (Fig. 1B); The relative β-galactosidase activity of the co-transformed cells with pGAD-POT1 and pGBK-FOXP2-R553H(484–598) was about 2-fold lower than that of the co-transformed cells with pGAD-POT1 and pGBK-FOXP2(484–598).
FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation.
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Intracellular distribution of a speech/language disorder associated FOXP2 mutant

Abstract

Section snippets

Materials and methods

Subcellular distribution of the truncated FOXP2 variants

Nuclear translocation signals in FOXP2

Trends Genet.

Am. J. Hum. Genet.

J. Biol. Chem.

Trends Cogn. Sci.

Am. J. Hum. Genet.

Structure

Cell

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Exp. Cell Res.

The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25

Nat. Genet.

Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia

Nat. Genet.