FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it

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Abstract

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.

Highlights

► We isolated protection of telomeres 1 (POT1) as a FOXP2-associated protein by a yeast two-hybrid. ► FOXP2 associated and co-localized with POT1 in the nuclei. ► FOXP2(R553H) also co-localized with POT1 in both the cytoplasm and nuclei. ► FOXP2(R553H) partially prevented the nuclear translocation of POT1. ► FOXP2(R553H) mutation may be associated with the pathogenesis of speech-language disorder.

Introduction

FOXP2 is a forkhead box, approximately 110 amino acids in length with a winged-helix DNA binding domain, -containing transcriptional factor [1]. Nuclear localization signals (NLSs) are found at the C-terminal and N-terminal ends of the forkhead [2]. FOXP2 also has leucine zipper and zinc finger domains [1]. FOXP2 expresses in the various tissues including lung and brain [1]. This protein is believed to have diverse roles during development, such as morphogenesis, differentiation, and proliferation. In the brain, the expression of the human and mouse homologues is very similar during development and in adulthood [3], [4].

The missense mutation (R553H) in the forkhead-containing domain in the FOXP2 gene has been found in patients of speech-language disorder, which exhibits autosomal dominant trait and severe abnormalities in speech and language [5]. The relationship between this FOXP2 mutation and the pathogenesis of speech-language disorder has been extensively studied. FOXP2(R553H) demonstrates reduced DNA binding and a defect in nuclear localization [2], [6]; some of the FOXP2(R553H) is translocated into the nucleus in the dimeric form, while the rest of the FOXP2(R553H) remains in the cytoplasm [2]. Therefore, in addition to a defect in FOXP2(R553H) DNA binding activity, the binding partners of FOXP2 may fail to function in the nucleus if they are associated with FOXP2(R553H) in the cytoplasm [6].

Little is known about the molecular mechanism of FOXP2-mediated regulation, although FOXP2 negatively regulates gene expression by associating with the co-repressor C-terminal binding protein (CtBP) via binding motif located at N-terminal region [7]. 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 bait. We have identified protection of telomeres 1 (POT1) as the associated protein, and demonstrate that FOXP2(R553H) prevents the nuclear translocation of POT1.

Section snippets

DNA construction

POT1 cDNA was isolated from a human fetal cDNA library by yeast two-hybrid screening. The enzyme-digested PCR DNA fragments corresponding to full-length FOXP2, FOXP2(484–715), FOXP2(484–598) containing forkhead domain (FOXP2-forkhead), FOXP2(484–598) with R553H mutation (FOXP2-R553H-forkhead), FOXP2(576–715), FOXP2-mutated NLS (mNLS) [2], and full-length POT1 were subcloned into the following vectors: pGAD, pGBK, pEGFP-C1 (Clontech), and FLAG vector pcDEF3 [8].

Yeast two-hybrid assay

Yeast two-hybrid assays were

Isolation of POT1 via yeast two-hybrid screening

We examined FOXP2-associated protein by yeast two-hybrid assay. We used the C-terminal region of FOXP2 including the forkhead domain, FOXP2(484–715), as bait (Fig. 1A) and isolated POT1 cDNA (from the eighth amino acid to the 3′-UTR). Yeast AH109 co-transformed with pGAD-POT1 and pGBK-FOXP2(484–715) or pGBK-FOXP2(484–598) containing forkhead domain grew on SD-Leu/-Trp/-His/-Ade plates, while cells co-transformed with pGAD-POT1 and pGBK-FOXP2(576–715) lacking forkhead domain did not (Fig. 1A).

Discussion

POT1 is a component of the telomerase ribonucleoprotein complex that is essential for the replication of chromosome termini [9]. A six-protein complex (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1), known as shelterin, protects the telomeres of human chromosomes [10]. POT1 directly binds the single-stranded 3′extension at the chromosome end [11], [12], which is bridged with TIN2 through protein–protein interactions via TPP1 [10], [13]. POT1-TPP1 switches from inhibiting to allowing telomerase access

Acknowledgments

Authors thank Dr. Akifumi Mizutani for introduction of yeast-two-hybrid assay in the initial time of this study. This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) of the Ministry of Education, Culture, Sports, Science and Technology, Japan (21200011, 21700377); Grants-in-Aid for Health Labour Scientific Research of the Ministry of the Health, Labour and Welfare, Japan; and a Grant-in-Aid for Intramural Research (20B-13) for Neurological and Psychiatric Disorders of

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