TAZ/WWTR1 Mediates the Pulmonary Effects of NKX2-1 Mutations in Brain-Lung-Thyroid Syndrome

TAZ/WWTR1 Mediates the Pulmonary Effects of NKX2-1 Mutations in Brain-Lung-Thyroid Syndrome Abstract Context Identification of a frameshift heterozygous mutation in the transcription factor NKX2-1 in a patient with brain-lung-thyroid syndrome (BLTS) and life-threatening lung emphysema. Objective To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis. Methods Mutational study by DNA sequencing, generation of expression vectors, site-directed mutagenesis, protein–DNA-binding assays, luciferase reporter gene assays, confocal microscopy, coimmunoprecipitation, and bioinformatics analysis. Results We identified a mutation [p.(Val75Glyfs*334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs*75)] in the carboxy-terminal domain in other patients with BLTS but without signs of respiratory distress. Both mutations showed similar protein expression profiles, subcellular localization, and deleterious effects on thyroid-, brain-, and lung-specific promoter activity. Coexpression of the coactivator TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1) restored the transactivation properties of p.(Ala276Argfs*75) but not p.(Val75Glyfs*334) NKX2-1 on a lung-specific promoter, although both NKX2-1 mutants could interact equally with TAZ/WWTR1. The retention of residual transcriptional activity in the carboxy-terminal mutant, which was absent in the amino-terminal mutant, allowed the functional rescue by TAZ/WWTR1. Conclusions Our results support a mechanistic model involving TAZ/WWTR1 in the development of human congenital emphysema, suggesting that this protein could be a transcriptional modifier of the lung phenotype in BLTS. Brain-lung-thyroid syndrome (BLTS; Online Mendelian Inheritance in Man no. 610978) is a dominantly inherited disease caused by mutations in the NK2 homeobox 1 (NKX2-1) gene, also known as TTF1 or TITF1. NKX2-1 encodes a homeodomain transcription factor expressed in thyroid, lung, and forebrain during embryogenesis (1). Loss of function of NKX2-1 leads to congenital hypothyroidism associated with neurologic disorders and respiratory distress of variable severity (2, 3). More than 118 different mutations in NKX2-1 have been reported to date, including missense, nonsense, or splicing mutations, small insertions or deletions, and deletions of the whole gene (4). BLTS shows a striking phenotypic heterogeneity, both in the number of organs affected and in the severity of the disease. Most patients present with benign hereditary chorea and congenital hypothyroidism; however, only 50% of patients will develop the full-blown syndrome (5). Although the neurologic and thyroid disorders appear isolated (4, 6), a very few documented cases have expressed only the lung defect (7). The lethality associated with BLTS is always caused by respiratory failure (8, 9). The intrinsic pathogenic mechanisms leading to each of the distinct phenotypes in BLTS are obscure. The thyroid gland will be, in general terms, hypoplastic, likely owing to a reduction of thyroid-stimulating hormone (TSH) receptor in thyrocytes, because TSHR is a direct transcriptional target of NKX2-1 (10). Brain basal ganglia (striatum and putamen) also show volumetric and metabolic abnormalities as revealed by advanced magnetic resonance imaging (11). No lung hypoplasia has been described in BLTS; however, diffuse alterations of the lung architecture with low alveolar counts, alveolar remodeling, type II pneumocytes hyperplasia, and septal thickening by interstitial fibrosis are typical (8, 12). The phenotypic heterogeneity of BLTS, in particular, the severity of lung insufficiency, can be explained by aberrant protein interactions with coactivators or corepressors resulting from the intrinsic characteristics of each particular NKX2-1 mutant (13). NKX2-1 contains three domains: an amino-terminal transactivation domain (N-TAD), a DNA-binding homeodomain, and a carboxy-terminal transactivation domain (C-TAD), with each domain acting as a module. Structural and functional studies have shown that distinct portions of the protein are endowed with diverse developmental functions (14). The N- and C-terminal domains interact with other proteins, forming complexes on promoter or regulatory DNA regions to control the expression of target genes in a gene-specific and tissue-specific manner (15, 16). NKX2-1 has been shown to interact with a broad range of proteins, including ubiquitous (AP-1, CBP/p300, C/EBP, RARs, ACTR/NCOA3, SRC1/NCOA1, STAT3, TAP26/BR22) and tissue-restricted [PAX8, DREAM, FOXA1/HNF3α, FOXP2, GATA6, NFIB, TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1)] transcription factors or cofactors (15, 17–28). The precise topology of interactions between TADs and these target molecules remains to be characterized in detail (23). The interaction between NKX2-1 and PAX8, an important transcription factor in the thyroid, has been extensively studied and illustrates the complex relationship between proteins for gene promoter regulation (15, 29). Accordingly, the activity of some transcriptionally inactive NKX2-1 mutants can be rescued by their interaction with PAX8 on the thyroglobulin (TG) promoter (5); however, this thyroid phenotypic rescue is not observed with other NKX2-1 mutations (30). In contrast, some PAX8 mutants are rescued by their interaction with NKX2-1 on the TG promoter (31). Overall, these findings suggest that the specific location of the mutation determines the capacity of transcriptional partners to, at least partially, restore transcriptional activities. One of the aforementioned proteins interacting with NKX2-1 is TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain-containing transcription regulator protein 1). TAZ forms part of the Hippo signaling pathway, which, together with YAP, controls the size of organs and tissue homeostasis and regeneration (32–34). TAZ also regulates the activity of several transcription factors involved in development and disease (35, 36). It is widely expressed, with the highest levels in kidney and lung (37). Moreover, the expression patterns of TAZ and NKX2-1 overlap in a spatiotemporal fashion in respiratory epithelial cells of embryonic and adult mouse lung. Additionally, TAZ interacts with the N-TAD of NKX2-1, acting as a transcriptional coactivator of NKX2-1 in the activation of the surfactant protein C gene (25). Similar coactivation activity was reported in the thyroid, where TAZ was coexpressed with NKX2-1 and PAX8 during murine thyroid development and throughout adult life, acting as a transcriptional coactivator of NKX2-1 and PAX8 over the TG promoter (15). Transgenic mice lacking TAZ present with pathological changes in the kidney and lung that resemble human polycystic disease and pulmonary emphysema (38). Although several years have passed since the identification of the first mutation in the NKX2-1 gene, the molecular alterations responsible for such variable phenotypes are still far from being understood. Approaches that consider how such alterations affect the interaction of NKX2-1 with protein partners in a specific tissue are needed to fully understand the causes of this complex disease. In the present study, we identified a de novo frameshift heterozygous mutation in NKX2-1 in one patient with severe BLTS and life-threatening lung emphysema. The mutation is one of the most identified N-terminal mutations to date, leading to a highly aberrant protein. We performed a functional comparative study with a previously identified frameshift mutation located at the most C-terminal domain that was described in two sisters with thyroid and choreic complications but not lung involvement (30). Through the present analysis, we have demonstrated that TAZ is a phenotype modifier protein in BLTS, at least in some NKX2-1 mutations. Patient and Methods Case report The subject of the present study was born from a healthy white nonconsanguineous couple at 40 weeks of gestation after a normal pregnancy. The Apgar score was 9 of 9. He had a birth weight of 2935 g and a birth length of 51 cm. On the second day of life, he developed tachypnea, intercostal inspiratory retractions, flaring nostrils, and poor peripheral perfusion. Septicemia was suspected, and antibiotic treatment was started, without clinical improvement. No bacterial infection could be confirmed. He maintained tachypnea, cyanosis, and bilateral lung crackles on respiratory auscultation and required oxygen support. A chest radiograph showed diffuse ground glass opacity (Fig. 1A), compatible with severe hyaline membrane disease. Figure 1. View largeDownload slide Clinical study of lung function and anthropometry. (A) Neonatal chest radiograph showing diffuse ground glass opacity, air bronchogram, and a reticular-interstitial pattern, compatible with severe hyaline membrane disease. (B) Computed tomography scan at 12 years showing extensive hypoattenuation and decreased vascularization areas, suggestive of lung destruction. Bronchial wall thickening and linear images are suggestive of reduced lung parenchyma. Pathologic findings of lung biopsy showing (C) lung parenchyma with large areas of emphysema and (D) interstitial fibrosis and bronchiolar epithelial alteration. (E) Spirometry at 12 years of age showing severe impairment of pulmonary function by forced expiratory volume at 1 second. The airflow (in L/s) was measured and compared with the standards for age and sex at five time points within 1 second. The area in red shows severe impairment of pulmonary function compared with the standard values. (F) Sustained underweight in relation to undernutrition due to feeding/gastric surgery difficulties. (G) Sustained undergrowth and delayed puberty compatible with constitutional delay of growth and development (retarded bone age). The percentiles are indicated as follows: 50%, blue; 10% to 90%, green; and 3% to 97%, red, according to the Spanish Auxology Study of 2010. Figure 1. View largeDownload slide Clinical study of lung function and anthropometry. (A) Neonatal chest radiograph showing diffuse ground glass opacity, air bronchogram, and a reticular-interstitial pattern, compatible with severe hyaline membrane disease. (B) Computed tomography scan at 12 years showing extensive hypoattenuation and decreased vascularization areas, suggestive of lung destruction. Bronchial wall thickening and linear images are suggestive of reduced lung parenchyma. Pathologic findings of lung biopsy showing (C) lung parenchyma with large areas of emphysema and (D) interstitial fibrosis and bronchiolar epithelial alteration. (E) Spirometry at 12 years of age showing severe impairment of pulmonary function by forced expiratory volume at 1 second. The airflow (in L/s) was measured and compared with the standards for age and sex at five time points within 1 second. The area in red shows severe impairment of pulmonary function compared with the standard values. (F) Sustained underweight in relation to undernutrition due to feeding/gastric surgery difficulties. (G) Sustained undergrowth and delayed puberty compatible with constitutional delay of growth and development (retarded bone age). The percentiles are indicated as follows: 50%, blue; 10% to 90%, green; and 3% to 97%, red, according to the Spanish Auxology Study of 2010. The neonatal screening program revealed primary congenital hypothyroidism with filter paper TSH values of 70 mU/L (normal, <10 mU/L), confirmed in serum samples on the 10th day of life (TSH, 224 mU/L; and free thyroxine, 0.6 ng/dL; Table 1). Ultrasonography and scintigraphy showed a normal in situ thyroid gland at birth. Treatment with levothyroxine (37.5 μg/d) was started at day 10 of life, leading to normalization of the thyroid hormone parameters by the 30th day of life. Table 1. Patient’s Hormonal Status and Bone Age at Different Stages of Life Measurement  Age  NA  10 da  22 d  4 mo  10 y, 11 mo  11 y, 10 mo  12 y, 7 mo  13 y, 2 mo  14 y, 3 mo  TSH, µU/mL  70 (1–10)  224 (1–10)  13.5 (0.5–6.5)  3.7 (0.5–6.5)  —  —  —  3.8 (0.57–5.92)  —  Free T4, ng/dL  —  0.6 (0.9–2.3)  2.8 (0.9–2.3)  1.4 (0.77–1.78)  —  —  —  1.6 (0.72–2)  —  IGF-1, ng/mL)  —  —  —  —  89.8 (80–420)  123 (111–551)  87.8 (143–693)  197 (183–850)  226 (237–996)  IGFBP3, µg/mL)  —  —  —  —  2.27 (2.1–7.7)  2.64 (2.4–8.4)  2.58 (2.7–8.9)  —  —  Bone age  —  —  —  —  9 y  —  10 y  —  —  FSH/LH, mIU/mL; (Tanner 1 [<4/<1]; Tanner >1 [1.5–12.4/1.7–8.6])  —  —  —  —  1.4/0.7  1.5/0.5  —  1.8/1.1  1.74/0.73  Testosterone, ng/dL (Tanner 1 [<25]; adulthood [280–800])  —  —  —  —  17  13  59  263.8    Measurement  Age  NA  10 da  22 d  4 mo  10 y, 11 mo  11 y, 10 mo  12 y, 7 mo  13 y, 2 mo  14 y, 3 mo  TSH, µU/mL  70 (1–10)  224 (1–10)  13.5 (0.5–6.5)  3.7 (0.5–6.5)  —  —  —  3.8 (0.57–5.92)  —  Free T4, ng/dL  —  0.6 (0.9–2.3)  2.8 (0.9–2.3)  1.4 (0.77–1.78)  —  —  —  1.6 (0.72–2)  —  IGF-1, ng/mL)  —  —  —  —  89.8 (80–420)  123 (111–551)  87.8 (143–693)  197 (183–850)  226 (237–996)  IGFBP3, µg/mL)  —  —  —  —  2.27 (2.1–7.7)  2.64 (2.4–8.4)  2.58 (2.7–8.9)  —  —  Bone age  —  —  —  —  9 y  —  10 y  —  —  FSH/LH, mIU/mL; (Tanner 1 [<4/<1]; Tanner >1 [1.5–12.4/1.7–8.6])  —  —  —  —  1.4/0.7  1.5/0.5  —  1.8/1.1  1.74/0.73  Testosterone, ng/dL (Tanner 1 [<25]; adulthood [280–800])  —  —  —  —  17  13  59  263.8    Abbreviations: NA, not applicable; IGF-1, insulinlike growth factor-1; IGFBP3, insulinlike growth factor-binding protein 3; FSH, follicle-stimulating hormone; LH, luteinizing hormone; T4, levothyroxine. a After the treatment began, the values began to move toward the normal range. Treatment was started at day 10 of life with 37.5 µg/d of levothyroxine, which was increased to 50 µg/d at 3 months. The dose was gradually increased to 100 µg/d when the patient was 13 years old. Reference values (in parentheses) are specific to age and sex (39). Determinations were performed using electrochemiluminescence immunoassays. View Large Generalized choreoathetotic movements were observed at 8 months of age associated with hypotonia and psychomotor delay. Mutation analysis Genetic analysis was performed after provision of informed consent by the patient’s parental representatives and was approved by the institutional ethics review board committee. Genomic DNA of the subject was obtained from peripheral blood leukocytes using the Chemagic MSM I automated nucleic acid isolation system (PerkinElmer Chemagen Technologie GmbH, Baesweiler, Germany). The three coding exons and intron flanking regions from all four transcript variants of the NKX2-1 gene were amplified using a total of nine primer pairs and sequenced directly in both directions with BigDye Terminator V3.1 (Applied Biosystems, Life Technologies Corp., Carlsbad, CA) on an AB 3730XL DNA Analyzer (Applied Biosystems). The eight coding exons and intron flanking regions from TAZ/WWTR1 gene were also amplified and sequenced as described (primer sequences available on request). Plasmids and subcloning The plasmids used in the present work were as follows: wild-type (WT) NKX2-1 (3) and 825delC cDNA mutant (C-mut) (30) of human NKX2-1 cloned into pcDNA3; WT mouse TAZ/WWTR1 cDNA cloned into pCMV5 (TAZ) (25); WT mouse TAZ fused to a Flag-tag and cloned into pEF-BOS (TAZ-N-FLAG) (37); WT human PAX8 cloned into pcDNA3 (31) and reporter vectors hTGenh/prom-Luc (3), hSP-Bprom-Luc (B-500) (40), and mLhx6prom-Luc (41) using the pGL3 Basic vector backbone (Promega Corp., Madison, WI). Site-directed mutagenesis The human N-terminal NKX2-1 mutant (223dupG) was generated by site-directed mutagenesis using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA) with primers 5′-TACCACATGACGGCGGCGGGGGGTGCCCCAGCTCTCGCA-3′ and 5′-TGCGAGAGCTGGGGCACCCCCCGCCGCCGTCATGTGGTA-3′ in WT NKX2-1 (the WT sequence has five Gs in the underlined region). The fidelity of the mutated construct was confirmed by sequencing. In vitro synthesis of NKX2-1 and electrophoretic mobility shift assay Proteins were synthesized from 1 µg of each WT or mutant NKX2-1 vector (223dupG and 835delC) by in vitro transcription/translation using the TNT-Coupled Reticulocyte Lysate System (Promega Corp.). Protein–DNA binding was examined by electrophoretic mobility shift assay using 3 µL of in vitro–translated proteins mixed with 50,000 cpm of 32P-labeled oligonucleotide C derived from the TG promoter. Assays were performed as previously described (30, 42). Immunoblot analysis Samples of 30 µg of total extracts from cells transfected with plasmids, quantified according to the Bradford method (Bio-Rad Laboratories, Hercules, CA), were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Ponceau S staining was used to check for equal protein loading. Membranes were blocked and incubated with the following antibodies: NKX2-1 antibody (1:1500 dilution; EP1584Y; Epitomics, Burlingame, CA); TAZ antibody (1:200 dilution; sc-48805; Santa Cruz Biotechnology, Santa Cruz, CA); or FLAG-Tag antibody (1:1500 dilution; NBP1-06712; Novus Biologicals, Littleton, CO). Immunoreactive bands were visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, IL). For TNT-reticulocyte immunoblotting, 7 µL of in vitro–translated proteins was used. In vitro transactivation assay HeLa cells were grown in Dulbecco’s modified Eagle medium (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum. For transactivation assays, 105 cells were plated in MW12 culture dishes 24 hours before transfection. The cells were transfected using FuGENE6 (Promega Corp.) with a total of 1 µg of DNA per well, including 600 ng of the specific reporter plasmid (hTG, hSP-B, or mLhx6) and different amounts of expression vectors (NKX2-1 WT, 223dupG or 825delC mutants, TAZ, and PAX8) and empty vector as indicated to 400 ng. To correct for transfection efficiency, 10 ng of Renilla-encoding pRL-CMV vector was added in all cases. Cells were harvested 24 hours after transfection, lysed, and analyzed for luciferase (LUC) and Renilla activity using the dual-LUC reporter assay system (Promega Corp.). The ratio between the LUC and Renilla activities was expressed relative to the ratio obtained in cells transfected with reporter and empty expression vector (pcDNA3). Transfections were performed in triplicate and repeated at least three times; the LUC activity values are presented as the mean ± standard deviation. Statistical analysis was performed using Student’s t test to obtain the P value associated with the observed fold of activation differences. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05, P < 0.01, and P < 0.001. Immunofluorescence assay MDCK1 epithelial and NIH3T3 fibroblast cells were seeded on coverslips in 60-mm-diameter culture dishes. The cells were transfected 24 hours later with 5 µg of an empty plasmid (as a control for immunostaining specificity) or the same amount of the NKX2-1 WT, the N-terminal (223dupG) or the C-terminal (825delC) mutant expression vectors. Twenty-four hours later, the cells were fixed in 70% methanol at −20°C for 10 minutes, washed three times with phosphate-buffered saline (PBS)-0.05% Tween-20 (PBS-Tween) for 5 minutes, and blocked with PBS-Tween containing 5% goat serum for 1 hour at room temperature (RT). The cells were then incubated with the anti–NKX2-1 antibody for 1 hour at RT, washed three times in PBS-Tween for 5 minutes, incubated for 1 hour at RT with an Alexa Fluor 488-conjugated secondary antibody, washed three times with PBS-Tween for 5 minutes, with the last wash containing 4′,6-diamidino-2-phenylindole, and mounted on ProLong® Gold Antifade Reagent from Molecular Probes (Invitrogen-Life Technologies Ltd., Paisley, UK). The cells were observed under a Leica TCS SP2 confocal microscope using 63× magnification under oil immersion (Leica Corp., Deerfield, IL). Immunoprecipitation Aliquots of 25 µL of Dynabeads® Protein G (Novex®; Life Technologies AS, Oslo, Norway) were washed for 10 minutes with PBS-0.02% Tween-20 and bound with 3 to 5 µg of each antibody for 1 hour with rotation at 4°C. The bead–antibody complexes were cross-linked by bis-sulfosuccinimidyl suberate (BS3; Thermo Fisher Scientific). Next, 1 mg of total extracts from transfected HEK293T cells with TAZ-N-FLAG, together with pcDNA3.1, WT, or mutant NKX2-1 vectors were added in a final volume of 1 mL and incubated overnight with rotation at 4°C. The bead–antibody–antigen complexes were washed three times using 300 μL of PBS-0.02% Tween-20 and eluted in 25 μL of Laemmli 2× Sample Buffer at 95°C for 15 minutes. The eluted samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with the specific antibody. Finally, 30 µL of total extract from the same transfected HEK293T cells was used as an input. Proximity ligation assay HeLa cells were seeded on coverslips and transfected 24 hours later by calcium phosphate coprecipitation with 2 μg of PAX8 and 2 μg of NKX2-1 WT, N-mut, or C-mut expression vectors. After 24 hours, the cells were fixed in 4% paraformaldehyde for 10 minutes at RT, permeabilized, and blocked in 1% bovine serum albumin for 30 minutes. Next, the cells were incubated for 1 hour with the NKX2-1 antibody and a PAX8 antibody (AB53490; Abcam, Cambridge, UK). To study protein interactions, we used the DuoLink-PLA Kit (Sigma-Aldrich Corp.). In brief, the cells were washed and incubated with anti-rabbit and anti-mouse secondary antibodies coupled to complementary oligonucleotides. Subsequently, the oligonucleotides were annealed and amplified by polymerase chain reaction and detected through hybridization of complementary fluorescence-labeled oligonucleotides. Cells were washed three times, with the third wash containing 4′,6-diamidino-2-phenylindole for nuclear staining, and mounted as described. The cells were observed under a 63× objective in a LSM710 Zeiss confocal microscope. Bioinformatic analysis The sequences were analyzed using the Sequencher V4.1.4 (Genes Code Corp., Ann Arbor, MI). The protein subcellular localization prediction was performed with WoLF PSORT (available at: http://wolfpsort.org/) (43), NucPred from Stockholm Bioinformatics Center (available at: http://www.sbc.su.se/~maccallr/nucpred/cgi-bin/single.cgi) (44), and NLStradamus (available at: http://www.moseslab.csb.utoronto.ca/NLStradamus/) (45). Results Clinical evolution of the patient During the first 2 years of life, the patient developed pulmonary impairment with recurrent lung infections that led to the need for mechanical ventilation and tracheotomy. Nissen fundoplication surgery was performed because of a diagnosis of gastroesophageal reflux; however, no clinical improvement was observed. The patient continued to require nocturnal oxygen until age 2.5 years and received chronic inhaled corticosteroid treatment. Serial computed tomography scans were performed during follow-up and showed a destroyed lung, with hypoattenuation and decreased vascularization areas (Fig. 1B; Supplemental Fig. 1A). A lung biopsy performed at 12 months revealed wide emphysematous areas with destroyed alveolar structure (Fig. 1C) and interstitial fibrosis (Fig. 1D). Clinically, at 14 years of age, he could perform moderate physical activity; he did not require any treatment, although his pulmonary function was severely affected (Fig. 1E). At 17 years of age, he was registered for lung transplantation; however, the disease had stabilized, and surgery was cancelled. It was difficult to measure the patient’s tolerance to efforts owing to the limited motility caused by the involuntary choreic movements. Pulmonary functional tests showed moderate-to-severe dysfunction with an obstructive pattern. The last thoracic computed tomography scan from 2014 showed diffuse affection that was more intense in the superior and middle lobes and in the lingular segment. At the last follow-up examination, the patient was receiving corticosteroids and bronchodilators and had clinical stability with no exacerbation during the previous year. Levothyroxine treatment was continued; the dose was progressively increased to 50 µg/d at 3 months and subsequently gradually increased to 100 μg/d at 14 years. At that time, a thyroid ultrasound scan showed a homogeneous and small thyroid gland. The choreoathetotic movements and psychomotor characteristics were as follows: head control was achieved at 18 months, sitting at 2 years, walking with help at 2 years, 9 months, and autonomous walking at 3.5 years. He pronounced his first words at 2 years and composed sentences at 5 years. Choreoathetosis remained stable with slight improvement at adolescence. At 14 years of age, he showed mild cognitive impairment with adequate school performance and generalized chorea of a nonprogressive course. The cerebral magnetic resonance imaging findings were normal. Since the early neonatal period, the patient presented with a low weight gain curve (Fig. 1F), which became more severe through the years, reaching −2.5 at the chronological age of 14.7 years. This was attributed to chronic undernutrition resulting from feeding difficulties related to the performance of the gastric fundoplication. His height curve was also poor (−2.3 standard deviation; Fig. 1G). The insulinlike growth factor-1 and insulinlike growth factor-binding protein 3 levels were within the low-normal range (Table 1). During follow-up, his bone age was delayed by 2 years, and he started puberty at age 13.1 years with adequate follicle-stimulating hormone, luteinizing hormone, and testosterone values (Table 1). Identification and characterization of an NKX2-1 human mutation Using polymerase chain reaction and direct sequencing of the patient’s DNA, we identified a de novo heterozygous mutation in the NKX2-1 gene (Fig. 2A). The parents were healthy and presented with no NKX2-1 mutations. The mutation was a duplication of a guanine at position 223 of the cDNA (according to the ENST00000498187 transcript), which generates a frameshift, leading to an aberrantly long protein of 407 amino acids, retaining only the first 75 amino acids of the WT protein [c.223dupG/p.(Val75Glyfs*334); Fig. 2B and 2C]. The mutant protein lacks the homeodomain and the C-TAD but conserves most of the N-TAD and the first four of seven phosphorylated serine residues (46). This is one of the most identified amino-terminal frame shift mutations thus far in the NKX2-1 gene. Amino acid prediction of the mutant anticipates a severe impact on protein function based on its reduced conservation of amino acid sequence with respect to the WT protein and the absence of critical domains of this transcription factor. Figure 2. View largeDownload slide Identification of a mutation in the NKX2-1 gene. WT and mutant NKX2-1 protein primary structure, expression, stability, and subcellular localization. (A) NKX2-1 sequence chromatograms from the patient and his unaffected parents. The arrow indicates duplication of a guanine at position 223 in exon 2 of the NKX2-1 complementary DNA (according to the ENST00000498187 transcript). The heterozygous de novo mutation generates a double peak sequence beyond the insertion site, which is absent in the chromatogram of parents, indicating a de novo mutation. (B) Amino acid sequence comparison between NKX2-1 WT protein, the N-terminal mutant frameshift described in the present study [p.(Val75Glyfs*334)], shown in bold, and a C-terminal mutant frameshift [p.(Ala276Argfs*75)], previously described (30). The homeobox (gray box), cysteines for dimerization (plus sign), serines involved in phosphorylation (asterisk), the nuclear localization signal (NLS; underlined), the limit between exon 1 and exon 2 (arrow), the putative TAZ interaction motifs LPPY (yellow) and pSP (green) are shown. The nonsense region of the N-mut (red bold letters) and C-mut (red letters), the newly generated NLS (red underline) and putative pSP motifs (light blue) are also indicated. The most important functional amino acids are absent in the N-mut protein, barring the four phosphorylated serines. (C) Diagrams representing WT, N-mut, and C-mut NKX2-1 proteins. The homeodomain (HD) is shown in black, N-TAD and C-TAD in gray, and the nonsense aberrant region of N-mut and C-mut by diagonal lines. The relative position of NKX2-1 antibody epitope is indicated. (D) Immunoblot of WT, N-mut, or C-mut NKX2-1 proteins. Proteins were transcribed and translated from their respective vectors (including pcDNA3.1 empty vector as control) using TNT reticulocytes. The anti–NKX2-1 antibody detects all three proteins because of the N-terminal location of the epitope (C). Note that the N-mut is larger than that of the other two proteins, according to its predicted sequence. (E) Analysis of WT and mutant NKX2-1 protein stability. Immunoblots of 30 μg total protein extracts from HeLa cells not transfected or transfected with WT, N-mut, or C-mut NKX2-1 expression vectors and lysed at five different time points, as indicated in each lane. All three proteins are expressed, with a maximum peak at 24 hours and are still present at 48 hours after transfection. Tubulin was used as a loading control. (F) Subcellular localization of WT, N-mut, or C-mut NKX2-1 by confocal microscopy. MDCK1 cells were seeded on coverslips, transfected with empty pcDNA3.1 (control), WT, N-mut, or C-mut NKX2-1 expression vectors and stained for immunofluorescence with an anti–NKX2-1 antibody. 4′,6-Diamidino-2-phenylindole was used to stain nuclear DNA. Ab, antibody; COOH, carboxyl. Figure 2. View largeDownload slide Identification of a mutation in the NKX2-1 gene. WT and mutant NKX2-1 protein primary structure, expression, stability, and subcellular localization. (A) NKX2-1 sequence chromatograms from the patient and his unaffected parents. The arrow indicates duplication of a guanine at position 223 in exon 2 of the NKX2-1 complementary DNA (according to the ENST00000498187 transcript). The heterozygous de novo mutation generates a double peak sequence beyond the insertion site, which is absent in the chromatogram of parents, indicating a de novo mutation. (B) Amino acid sequence comparison between NKX2-1 WT protein, the N-terminal mutant frameshift described in the present study [p.(Val75Glyfs*334)], shown in bold, and a C-terminal mutant frameshift [p.(Ala276Argfs*75)], previously described (30). The homeobox (gray box), cysteines for dimerization (plus sign), serines involved in phosphorylation (asterisk), the nuclear localization signal (NLS; underlined), the limit between exon 1 and exon 2 (arrow), the putative TAZ interaction motifs LPPY (yellow) and pSP (green) are shown. The nonsense region of the N-mut (red bold letters) and C-mut (red letters), the newly generated NLS (red underline) and putative pSP motifs (light blue) are also indicated. The most important functional amino acids are absent in the N-mut protein, barring the four phosphorylated serines. (C) Diagrams representing WT, N-mut, and C-mut NKX2-1 proteins. The homeodomain (HD) is shown in black, N-TAD and C-TAD in gray, and the nonsense aberrant region of N-mut and C-mut by diagonal lines. The relative position of NKX2-1 antibody epitope is indicated. (D) Immunoblot of WT, N-mut, or C-mut NKX2-1 proteins. Proteins were transcribed and translated from their respective vectors (including pcDNA3.1 empty vector as control) using TNT reticulocytes. The anti–NKX2-1 antibody detects all three proteins because of the N-terminal location of the epitope (C). Note that the N-mut is larger than that of the other two proteins, according to its predicted sequence. (E) Analysis of WT and mutant NKX2-1 protein stability. Immunoblots of 30 μg total protein extracts from HeLa cells not transfected or transfected with WT, N-mut, or C-mut NKX2-1 expression vectors and lysed at five different time points, as indicated in each lane. All three proteins are expressed, with a maximum peak at 24 hours and are still present at 48 hours after transfection. Tubulin was used as a loading control. (F) Subcellular localization of WT, N-mut, or C-mut NKX2-1 by confocal microscopy. MDCK1 cells were seeded on coverslips, transfected with empty pcDNA3.1 (control), WT, N-mut, or C-mut NKX2-1 expression vectors and stained for immunofluorescence with an anti–NKX2-1 antibody. 4′,6-Diamidino-2-phenylindole was used to stain nuclear DNA. Ab, antibody; COOH, carboxyl. Phenotypic comparison of patients harboring p.(Val75Glyfs*334) and p.(Ala276Argfs*75) We previously identified a frameshift mutation in NKX2-1 [c.825delC/p.(Ala276Argfs*75)] in two sisters of a Spanish family. Unlike the present patient, the two sisters had congenital hypothyroidism and benign chorea but no pulmonary distress (30). The mutant protein from the previous study was shorter than the WT protein but retained much of the normal sequence of the WT and is one of the most described C-terminal frameshift mutations (Fig. 2B and 2C). To search for specific pathogenic mechanisms that might explain the strikingly different lung phenotypes, normal and severe respiratory insufficiency, between the patients with BLTS, we performed a functional comparative study of the two NKX2-1 mutants: p.(Val75Glyfs*334) (herein referred to as N-mut) and p.(Ala276Argfs*75) (herein referred to as C-mut). Protein expression, stability, and subcellular localization of NKX2-1 mutants We first used an in vitro transcription and translation system to synthesize WT, N-mut, and C-mut NKX2-1 proteins. All three proteins generated were detected with a specific N-terminal NKX2-1 antibody, and all three were correctly expressed with a molecular weight compatible with the expected length of the sequence (Fig. 2D). We evaluated their stability in cultured cells and found that the expression of all three was maximal at 24 hours and remained detectable at 48 hours after transfection (Fig. 2E), suggesting that no mutant protein was prematurely degraded with respect to WT. We also performed an in silico analysis using WoLF PSORT, NucPred, and NLStradamus bioinformatic programs, which predicted that the WT and the two mutants would contain consensus nuclear localization sequences (Fig. 2B). Therefore, similar to the WT protein, the mutant proteins retain the capacity to reach the nucleus, which was verified by confocal microscopy of MDCK1 cells transfected with each of the three NKX2-1 constructs (Fig. 2F). Nuclear location was confirmed in a different cell line (NIH3T3; Supplemental Fig. 1B). Transcriptional activity of N-mut and C-mut on three tissue-specific promoters Given the correct nuclear localization of the two mutants, we next studied their transcriptional activity. We first used an electrophoretic mobility shift assay to assess the ability of the mutant proteins to bind DNA using the NKX2-1 binding sites within the TG promoter (oligo C) as a probe (42). The N-mut protein failed to bind to the specific probe (Fig. 3A, lanes 6 to 8). In contrast, strong binding was detected with the WT protein (lanes 3 to 5), and a reduced binding ability was observed for the C-mut protein (lanes 9 to 11), as previously described (30). PCCl3 nuclear extracts were used as a positive control (lanes 12 to 14). Figure 3. View largeDownload slide DNA-binding, transcriptional, and interacting properties of WT and mutant NKX2-1 proteins. (A) WT and mutant NKX2-1 proteins translated from TNT reticulocytes and control PCCl3 nuclear extracts were incubated with the 32P-labeled C oligonucleotide derived from the TG promoter (lanes 3, 6, 9, and 12). Empty pcDNA3.1 vector was used as control for nonspecific bands (lane 2). For competition, a 100-fold excess of unlabeled related C (lanes 4, 7, 10, and 13) or unrelated Sp1 (lanes 5, 8, 11, and 14) oligonucleotides were used. TNT reticulocyte lysates (3 μL) or 7 μg of nuclear extracts were added to each lane. The WT (lanes 3 and 5) and C-mut (lanes 9 and 11) bind the specific probe; however, the N-mut fails to bind (lanes 6 and 8). PCCl3 nuclear extracts were used as a positive control (lanes 12 and 14). The C-mut NKX2-1/DNA complex migrates faster than the WT NKX2-1/DNA complex, in agreement with its smaller size. A nonspecific band related to the TNT lysates was observed in all lanes but was not competed for by the competitor oligos. (B) Transcriptional capacity of N-mut and C-mut over brain-, thyroid-, and lung-specific promoters compared with WT NKX2-1. Light gray bars represent isolated expression of each of the NKX2-1 proteins. Black bars represent transactivation through coexpression of each of them with TAZ. HeLa cells were transiently transfected with 200 ng of WT, N-mut, or C-mut NKX2-1 expression vectors with or without 200 ng of TAZ vector (or empty pcDNA3.1 vector as control). Transcriptional activation was assessed by transfecting 600 ng of each reporter vector: the hTG enhancer/promoter (thyroid), the mLhx6 promoter (brain), and the hSP-B promoter (lung). LUC activity was normalized to Renilla activity to adjust for transfection efficiency. Experiments were performed at least three times in triplicate and expressed as mean ± standard deviation. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). The ns and asterisks over the bars indicate comparison of each condition in the presence or absence of TAZ; ns and number signs over individual conditions indicate comparison with the empty vector. (C) The interaction between NKX2-1 variants and TAZ was analyzed by coimmunoprecipitation assays. HEK293T cells were cotransfected with WT, N-mut, or C-mut NKX2-1 and TAZ-N-FLAG (or pcDNA3.1 as control) expression vectors. Dynabeads® Protein G, cell extracts and antibodies (anti-NKX2-1, anti-TAZ, or anti-FLAG) were allowed to interact overnight at 4°C. Bead–antibody–protein complexes were washed and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. WT, N-mut, or C-mut NKX2-1 was detected by immunoblotting with anti-NKX2-1. TAZ was detected with both an anti-TAZ antibody and an anti-FLAG antibody. The immunoblots represent the (Left) inputs (30 μg of total extracts) and (Right) immunoprecipitated proteins. (Top) Schematic representation of epitope location for antibodies in WT, N-mut, and C-mut NKX2-1 and TAZ proteins. (D) Competition of WT NKX2-1 and mutant variants. HeLa cells were transfected with empty vector or NKX2-1 WT (2 μg), together with increasing amounts of (top) N-mutant protein (0 to 3 μg), Renilla, and TG or (bottom) SP-B promoters driving LUC expression. The results are expressed as relative fold activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). Asterisks indicate comparisons with empty vector; ns and number signs over each condition indicate comparison with the WT vector. (E) PAX8 and mutant NKX2-1 interaction. HeLa cells showed interaction of PAX8 cotransfected with (left) NKX2-1 WT, (center) N-mut, and (right) C-mut. The nucleus stained blue with 4′,6-diamidino-2-phenylindole. Red dots indicate protein–protein interactions detected by polylactide (magnification ×63 images shown). (F) PAX8 rescue of NKX2-1 mutants on the TG promoter. HeLa cells were transfected with equal amounts of the NKX2-1 variants in the presence or absence of PAX8 and with TG promoter-driven LUC and Renilla expression vectors. The results are expressed as the relative fold TG promoter activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk); ns and asterisk over the bars indicates comparisons of each condition in the presence or absence of PAX8 and ns and numbered sign indicate comparison vs the empty vector condition. α, antibody; IB, immunoblotting; IP, immunoprecipitation. Figure 3. View largeDownload slide DNA-binding, transcriptional, and interacting properties of WT and mutant NKX2-1 proteins. (A) WT and mutant NKX2-1 proteins translated from TNT reticulocytes and control PCCl3 nuclear extracts were incubated with the 32P-labeled C oligonucleotide derived from the TG promoter (lanes 3, 6, 9, and 12). Empty pcDNA3.1 vector was used as control for nonspecific bands (lane 2). For competition, a 100-fold excess of unlabeled related C (lanes 4, 7, 10, and 13) or unrelated Sp1 (lanes 5, 8, 11, and 14) oligonucleotides were used. TNT reticulocyte lysates (3 μL) or 7 μg of nuclear extracts were added to each lane. The WT (lanes 3 and 5) and C-mut (lanes 9 and 11) bind the specific probe; however, the N-mut fails to bind (lanes 6 and 8). PCCl3 nuclear extracts were used as a positive control (lanes 12 and 14). The C-mut NKX2-1/DNA complex migrates faster than the WT NKX2-1/DNA complex, in agreement with its smaller size. A nonspecific band related to the TNT lysates was observed in all lanes but was not competed for by the competitor oligos. (B) Transcriptional capacity of N-mut and C-mut over brain-, thyroid-, and lung-specific promoters compared with WT NKX2-1. Light gray bars represent isolated expression of each of the NKX2-1 proteins. Black bars represent transactivation through coexpression of each of them with TAZ. HeLa cells were transiently transfected with 200 ng of WT, N-mut, or C-mut NKX2-1 expression vectors with or without 200 ng of TAZ vector (or empty pcDNA3.1 vector as control). Transcriptional activation was assessed by transfecting 600 ng of each reporter vector: the hTG enhancer/promoter (thyroid), the mLhx6 promoter (brain), and the hSP-B promoter (lung). LUC activity was normalized to Renilla activity to adjust for transfection efficiency. Experiments were performed at least three times in triplicate and expressed as mean ± standard deviation. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). The ns and asterisks over the bars indicate comparison of each condition in the presence or absence of TAZ; ns and number signs over individual conditions indicate comparison with the empty vector. (C) The interaction between NKX2-1 variants and TAZ was analyzed by coimmunoprecipitation assays. HEK293T cells were cotransfected with WT, N-mut, or C-mut NKX2-1 and TAZ-N-FLAG (or pcDNA3.1 as control) expression vectors. Dynabeads® Protein G, cell extracts and antibodies (anti-NKX2-1, anti-TAZ, or anti-FLAG) were allowed to interact overnight at 4°C. Bead–antibody–protein complexes were washed and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. WT, N-mut, or C-mut NKX2-1 was detected by immunoblotting with anti-NKX2-1. TAZ was detected with both an anti-TAZ antibody and an anti-FLAG antibody. The immunoblots represent the (Left) inputs (30 μg of total extracts) and (Right) immunoprecipitated proteins. (Top) Schematic representation of epitope location for antibodies in WT, N-mut, and C-mut NKX2-1 and TAZ proteins. (D) Competition of WT NKX2-1 and mutant variants. HeLa cells were transfected with empty vector or NKX2-1 WT (2 μg), together with increasing amounts of (top) N-mutant protein (0 to 3 μg), Renilla, and TG or (bottom) SP-B promoters driving LUC expression. The results are expressed as relative fold activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). Asterisks indicate comparisons with empty vector; ns and number signs over each condition indicate comparison with the WT vector. (E) PAX8 and mutant NKX2-1 interaction. HeLa cells showed interaction of PAX8 cotransfected with (left) NKX2-1 WT, (center) N-mut, and (right) C-mut. The nucleus stained blue with 4′,6-diamidino-2-phenylindole. Red dots indicate protein–protein interactions detected by polylactide (magnification ×63 images shown). (F) PAX8 rescue of NKX2-1 mutants on the TG promoter. HeLa cells were transfected with equal amounts of the NKX2-1 variants in the presence or absence of PAX8 and with TG promoter-driven LUC and Renilla expression vectors. The results are expressed as the relative fold TG promoter activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk); ns and asterisk over the bars indicates comparisons of each condition in the presence or absence of PAX8 and ns and numbered sign indicate comparison vs the empty vector condition. α, antibody; IB, immunoblotting; IP, immunoprecipitation. We then measured the transcriptional activity of the mutant proteins using representative target promoters specific for the tissues affected in BLTS: Lhx6 (LIM homeobox 6) from brain (basal ganglia), surfactant protein B (SP-B) from lung, and TG from thyroid. Compared with that of the WT protein, the activity of the N- and C-mut proteins over the three promoters was null (Fig. 3B). Although the lack of ability to bind and activate its target promoters suggested that loss of function of the N-mut protein was responsible for the BLTS in the present patient, it does not explain the phenotypic differences between this patient with severe lung disease and the two previously reported patients with normal respiratory function. TAZ/WWTR1 specifically rescues C-mut, but not N-mut, on the lung SP-B promoter TAZ is a transcriptional coactivator that interacts through its WW domain with a large number of transcription factors (37). Among them, it cooperates with NKX2-1 to activate surfactant protein C expression in embryonic and adult mouse lung (25). This observation prompted us to evaluate whether TAZ could be a modifier protein over the transcriptional activity of the NKX2-1 mutant proteins. We found that TAZ coexpression partially rescued the ability of the NKX2-1 C-mut protein to activate the TG and Lhx6 promoters, and, importantly, SP-B promoter activity was completely recovered (Fig. 3B). Coexpression of TAZ with the NKX2-1 N-mut had no effect on the activity of any of the promoters. These findings do much to explain why patients with the C-terminal mutation have a milder brain and thyroid phenotype, with no lung involvement (30), than the present patient with severe pulmonary disease. To rule out the possibility that a mutation in TAZ was present in the patient with the N-terminal mutation, the entire coding region of TAZ/WWTR1 was sequenced, and no pathogenic mutations were identified (data not shown). Overall, these data strongly suggest that TAZ could be a genetic modifier of the severity of the pulmonary phenotype and could explain in part the clinical variability caused by the different NKX2-1 mutations. Physical interaction between TAZ/WWTR1 and NKX2-1 mutants The coactivating function of TAZ is believed to be via binding of its WW domain to the (L/P)PXY amino acid motif present in a large number of transcription factors (37). Accordingly, Park et al. (25) demonstrated that TAZ interacts with the N-TAD of NKX2-1 and suggested that the LPPY amino acid sequence at position 97-100 of NKX2-1 (Fig. 2B) is responsible for this interaction (25). Nevertheless, this motif was found to be not essential for the binding between TAZ and NKX2-1 in another study (47). To investigate to which extent NKX2-1 mutants retain their ability to interact with TAZ, we cotransfected the three NKX2-1 expression vectors, together with FLAG-tagged TAZ into HEK293T cells and measured their interactions by immunoprecipitation. Immunoblotting of immunoprecipitated complexes demonstrated that all three NKX2-1 proteins interacted with TAZ (Fig. 3C), confirming that this binding does not require the LPPY motif of NKX2-1, which is absent in the N-mut protein. One possible candidate binding motif would be pS-P, present in the first 74 amino acids of the NKX2-1 (Fig. 2B), as described by Kanai et al. (37), who showed that TAZ binds to the (L/P)PXY motif but also to pS/T-P residues with slightly lower affinity. Dominant negative activity of the N-mut NKX2-1 protein Our previous work showed that the NKX2-1 C-mut exerts a dominant negative effect over the WT protein and decreases its ability to activate the TG but not the SP-B promoter (30). Because it is possible that truncated versions of NKX2-1 retain their ability to interact with partner proteins and impair the function of the WT protein, we analyzed the activity of TG and SP-B promoters in HeLa cells transfected with the WT protein and increasing amounts of the N-mut protein. We found that the N-mut protein clearly interfered with the ability of the WT protein to activate the TG promoter (Fig. 3D). A similar trend was found for the SP-B promoter, although the difference was not statistically significant. Interaction of PAX8 with NKX2-1 mutants TAZ plays a general role in the activation of NKX2-1 in every tissue in which NKX2-1 has a function. In the thyroid, the cooperation between NKX2-1 and PAX8 to activate thyroid-specific promoters is well known (15). Thus, we analyzed whether the different NKX2-1 mutants retain their ability to bind PAX8. Thus, HeLa cells were transfected with PAX8, together with the WT, N-mut, or C-mut NKX2-1, and a proximity ligation assay was performed to detect protein–protein interactions at a single molecule level. Both mutant proteins were found to bind PAX8 to an extent similar to that of the WT protein (Fig. 3E). To study whether this interaction is functional, we measured the ability of the different mutants to activate the TG promoter in the presence of PAX8. We found that activation of the TG promoter was much weaker by PAX8 than by NKX2-1 (Fig. 3F). PAX8 could partially rescue the ability of the C-mut to activate the TG promoter; however, the increase in the activity of the promoter in the presence of the N-mut and PAX8 was minor. This could explain the more aggressive thyroid phenotype in the patient carrying the N-terminal mutation. Discussion In the present work, we have described a heterozygous mutation in the NKX2-1 transcription factor in a child who developed severe pulmonary complications at the second day of life. Congenital hypothyroidism was diagnosed in the patient in the neonatal screening program, who presented with choreic movements and psychomotor delay from the eight month of life. Therefore, the patient developed full-blown and early expressed BLTS. Patients with this syndrome have been described previously with or without pulmonary dysfunction (48), and the mutations they harbor localize in different domains of NKX2-1. The mutation we have reported is one of the most identified N-terminal frameshift mutations to date in NKX2-1. To understand the molecular basis of the disorder in our patient and to shed further light on the disease, we performed a comparative study using a C-terminal NKX2-1 mutation described previously in two patients with BLTS who did not present with pulmonary involvement (30). Our results showed that both mutant proteins have a similar expression profile and are able to reach the nucleus. Nevertheless, although the C-mut protein retains residual DNA binding activity, the N-mut protein with an N-terminal domain mutation, resulting in loss of the DNA binding domain, is incapable of binding DNA. Both N- and C-mut proteins failed to transactivate specific brain, lung, and thyroid promoters; however, cotransfection of the coactivator factor TAZ/WWTR1 fully rescued the ability of the C-mut, but not the N-mut, protein to transactivate a lung-specific promoter. This tissue-specific action explains, at least in part, the phenotype of the two patients with the C-terminal mutation who did not present with lung complications. This result could also provide an explanation for the phenotype of the patient with the N-terminal mutation, who had severe lung complications. We initially predicted that the N-mut protein, which has lost the LPPY motif, would be unable to interact with TAZ. However, coimmunoprecipitation assays showed that this was not the case. We propose that such an interaction could occur through another candidate-binding motif, the pSer-Pro (pS/T-P), which is conserved in the first 74 amino acids of the NKX2-1 N-mut and has been reported to also bind to TAZ, albeit with lower affinity (37). The most plausible explanation for the molecular mechanism underlying the functional rescue of TAZ on NKX2-1 C-mut and not in N-mut is illustrated in the model depicted in Fig. 4A. As a coactivator, TAZ does not bind DNA directly but rather mediates the transcriptional effects of DNA-binding transcription factors through efficient assembly of the whole transcriptional protein complex. Therefore, TAZ interacts with specific transcription factors through its N-terminal WW domain and with the basal transcriptional machinery through its C-terminal extended coiled–coiled region, acting as a TAD, to stimulate the expression of target genes (35). The results obtained in the present study suggest that two requisites are necessary for NKX2-1 mutants to be amenable for functional rescue by TAZ: (1) to retain sufficient capacity to interact with the WW domain of TAZ; and (2) to retain at least a weak DNA binding ability. As shown in the present study, the NKX2-1 N-mut could only fulfill the first of these requirements, restricting the capacity of TAZ for functional rescue. This contrasts with the C-mutant protein, which can both weakly bind DNA and interact with TAZ. A review of all the mutants of NKX2-1 described to date indicated no correlation is obvious between the mutated region of the protein and the associated phenotype, highlighting the variable penetrance of the different mutations. A summary of the NKX2-1 mutants and the phenotypes associated is shown in Fig. 4B, and the most relevant information for all the mutations described in the reported data can be found in Supplemental Table 1. To compile Fig. 4B, we considered only the mutants from studies that addressed the state of the three organs affected by the syndrome. Together with gene deletions, frameshift mutations more frequently present with the full phenotype, and mutations that generate a stop codon or amino acid substitutions show a weaker association with the full-blown phenotype, which is present only in mutations before or in the homeodomain. Frameshift mutations after the homeodomain that generate a partially aberrant protein can also cause the full phenotype, which might indicate interference between the aberrant tail and the native region. As we have demonstrated in the present study, expression of aberrant NKX2-1 interferes with the ability of the WT to activate target promoters. It is particularly challenging to predict the behavior of the mutants in terms of phenotype probabilities. The structure of the protein and the interaction with principal partner proteins such as TAZ and PAX8 should be studied in detail for every mutant to ascertain the pathogenic mechanism underlying the mutation. Thus, it would be interesting to analyze the functional state of the C-terminal mutants that develop the full phenotype. Our results suggest that NKX2-1 mutations that completely prevent the interaction with TAZ could be inactive on lung-specific target genes, causing severe pulmonary disease. The possibility of such an event among the described NKX2-1 mutations is unknown but could be low because even in the absence of the LPPY motif, the N-mut protein retained sufficient interacting capacity with TAZ, likely through a Ser-Pro motif (37). Figure 4. View largeDownload slide (A) Mechanistic model for the interaction between WT and mutant NKX2-1 with TAZ/WWTR1. Proposed model shown to describe the behavior of WT, N-mut, and C-mut NKX2-1 proteins to explain the differences among the patients studied. (B) Diagrams showing the distribution of the different NKX2-1 mutations described in the reported data and the associated phenotypes. (Upper left) Frameshift mutations, (upper right) STOP codon mutations, (bottom left) amino acid substitutions, and (bottom right) gene deletions. B, brain; BL, brain-lung; BLT, brain-lung-thyroid; BT, brain-thyroid; BTM, basal transcriptional machinery; C, carboxy-terminal; HD, homeodomain; N, amino-terminal; WW, tryptophan-rich protein-binding domain. Figure 4. View largeDownload slide (A) Mechanistic model for the interaction between WT and mutant NKX2-1 with TAZ/WWTR1. Proposed model shown to describe the behavior of WT, N-mut, and C-mut NKX2-1 proteins to explain the differences among the patients studied. (B) Diagrams showing the distribution of the different NKX2-1 mutations described in the reported data and the associated phenotypes. (Upper left) Frameshift mutations, (upper right) STOP codon mutations, (bottom left) amino acid substitutions, and (bottom right) gene deletions. B, brain; BL, brain-lung; BLT, brain-lung-thyroid; BT, brain-thyroid; BTM, basal transcriptional machinery; C, carboxy-terminal; HD, homeodomain; N, amino-terminal; WW, tryptophan-rich protein-binding domain. Regarding the lung phenotype of the patient, it has been recently reported that TAZ is involved in the pathogenesis of pulmonary fibrosis through multifaceted effects on lung fibroblasts (49). Moreover, TAZ is an integral component of the Wnt/β-catenin cascade (50), which has been consistently linked to the pathogenesis of fibrotic disorders (51). With this information, we speculate that TAZ might not only mediate the transcriptional activity of NKX2-1 over surfactant protein promoters but also could be involved in the abnormal alveolarization of the lung (52) and the interstitial fibrosis (53) as the other relevant pathological hallmark of pulmonary disease in the present patient. In support of this concept, transgenic mice lacking TAZ have pathological changes in the lung that resemble human pulmonary emphysema and fibrosis signs (38). Furthermore, interaction with PAX8, a transcription factor that cooperates with NKX2-1 to modulate the expression of thyroid-specific genes, rescues the activity of the C-mutant NKX2-1 but not of the N-mutant NKX2-1, which might explain in part the more severe thyroid phenotype of the present patient with the N-terminal mutation. In conclusion, we found that the c.223dupG/p.(Val75Glyfs*334) mutation in the NKX2-1 gene was responsible for BLTS in a patient with severe lung emphysema. It is one of the most described amino-terminal mutations to date and generates a highly aberrant protein. The described mutation does not alter the nuclear localization of the mutant protein but blocks its DNA-binding capacity and its ability to activate specific promoters of brain, lung, and thyroid. The TAZ coactivator completely recovers the transcriptional activity of a C-terminal mutant [c.825delC/p.(Ala276Argfs*75)] in lung but not in the N-terminal mutant identified in the present study. These data explain the severity of the lung phenotype in the present patient and suggest that TAZ could be a modifier protein of the phenotype in BLTS in patients with NKX2-1 mutations. Abbreviations: BLTS brain-lung-thyroid syndrome C-mut p.(Ala276Argfs*75) LUC luciferase N-mut p.(Val75Glyfs*334) NKX2-1 NK2 homeobox 1 PAX8 paired box 8 PBS phosphate-buffered saline RT room temperature SP-B surfactant protein B TAD transactivation domain TAZ transcriptional coactivator with PDZ-binding motif TAZ-N-FLAG WT mouse TAZ fused to a Flag-tag and cloned into pEF-BOS TG thyroglobulin TSH thyroid-stimulating hormone WT wild-type WWTR1 WW domain-containing transcription regulator protein 1. Acknowledgments We thank all family members for their collaborative participation in this study. We are grateful to Dr. M. Zannini for providing pCMV5-mTAZ and to Dr. S.A. Anderson for providing p5′-Lhx6-IRES-GFP. We are also grateful to Dr. Kenneth McCreath for helpful comments on the manuscript and to Javier Perez from the Imaging Science Service (Biomedical Research Institute) for help with the illustrations. Financial Support: This work was supported by funding from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (grants PI1002160 and PI16/00830 to J.C.M.) and Ministerio de Economía y Competitividad of Spain, Fondo Europeo de Desarrollo Regional (grants SAF2013-44709R and SAF2016-75531R to P.S.). M.A.Z. and P.S. were supported by CIBERONC. C.M.M. was supported by a postdoctoral fellowship from the Research Program of the European Society for Pediatric Endocrinology. Disclosure Summary: The authors have nothing to disclose. References 1. Fernández LP, López-Márquez A, Santisteban P. Thyroid transcription factors in development, differentiation and disease. Nat Rev Endocrinol . 2015; 11( 1): 29– 42. Google Scholar CrossRef Search ADS PubMed  2. 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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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Abstract

Abstract Context Identification of a frameshift heterozygous mutation in the transcription factor NKX2-1 in a patient with brain-lung-thyroid syndrome (BLTS) and life-threatening lung emphysema. Objective To study the genetic defect that causes this complex phenotype and dissect the molecular mechanism underlying this syndrome through functional analysis. Methods Mutational study by DNA sequencing, generation of expression vectors, site-directed mutagenesis, protein–DNA-binding assays, luciferase reporter gene assays, confocal microscopy, coimmunoprecipitation, and bioinformatics analysis. Results We identified a mutation [p.(Val75Glyfs*334)] in the amino-terminal domain of the NKX2-1 gene, which was functionally compared with a previously identified mutation [p.(Ala276Argfs*75)] in the carboxy-terminal domain in other patients with BLTS but without signs of respiratory distress. Both mutations showed similar protein expression profiles, subcellular localization, and deleterious effects on thyroid-, brain-, and lung-specific promoter activity. Coexpression of the coactivator TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1) restored the transactivation properties of p.(Ala276Argfs*75) but not p.(Val75Glyfs*334) NKX2-1 on a lung-specific promoter, although both NKX2-1 mutants could interact equally with TAZ/WWTR1. The retention of residual transcriptional activity in the carboxy-terminal mutant, which was absent in the amino-terminal mutant, allowed the functional rescue by TAZ/WWTR1. Conclusions Our results support a mechanistic model involving TAZ/WWTR1 in the development of human congenital emphysema, suggesting that this protein could be a transcriptional modifier of the lung phenotype in BLTS. Brain-lung-thyroid syndrome (BLTS; Online Mendelian Inheritance in Man no. 610978) is a dominantly inherited disease caused by mutations in the NK2 homeobox 1 (NKX2-1) gene, also known as TTF1 or TITF1. NKX2-1 encodes a homeodomain transcription factor expressed in thyroid, lung, and forebrain during embryogenesis (1). Loss of function of NKX2-1 leads to congenital hypothyroidism associated with neurologic disorders and respiratory distress of variable severity (2, 3). More than 118 different mutations in NKX2-1 have been reported to date, including missense, nonsense, or splicing mutations, small insertions or deletions, and deletions of the whole gene (4). BLTS shows a striking phenotypic heterogeneity, both in the number of organs affected and in the severity of the disease. Most patients present with benign hereditary chorea and congenital hypothyroidism; however, only 50% of patients will develop the full-blown syndrome (5). Although the neurologic and thyroid disorders appear isolated (4, 6), a very few documented cases have expressed only the lung defect (7). The lethality associated with BLTS is always caused by respiratory failure (8, 9). The intrinsic pathogenic mechanisms leading to each of the distinct phenotypes in BLTS are obscure. The thyroid gland will be, in general terms, hypoplastic, likely owing to a reduction of thyroid-stimulating hormone (TSH) receptor in thyrocytes, because TSHR is a direct transcriptional target of NKX2-1 (10). Brain basal ganglia (striatum and putamen) also show volumetric and metabolic abnormalities as revealed by advanced magnetic resonance imaging (11). No lung hypoplasia has been described in BLTS; however, diffuse alterations of the lung architecture with low alveolar counts, alveolar remodeling, type II pneumocytes hyperplasia, and septal thickening by interstitial fibrosis are typical (8, 12). The phenotypic heterogeneity of BLTS, in particular, the severity of lung insufficiency, can be explained by aberrant protein interactions with coactivators or corepressors resulting from the intrinsic characteristics of each particular NKX2-1 mutant (13). NKX2-1 contains three domains: an amino-terminal transactivation domain (N-TAD), a DNA-binding homeodomain, and a carboxy-terminal transactivation domain (C-TAD), with each domain acting as a module. Structural and functional studies have shown that distinct portions of the protein are endowed with diverse developmental functions (14). The N- and C-terminal domains interact with other proteins, forming complexes on promoter or regulatory DNA regions to control the expression of target genes in a gene-specific and tissue-specific manner (15, 16). NKX2-1 has been shown to interact with a broad range of proteins, including ubiquitous (AP-1, CBP/p300, C/EBP, RARs, ACTR/NCOA3, SRC1/NCOA1, STAT3, TAP26/BR22) and tissue-restricted [PAX8, DREAM, FOXA1/HNF3α, FOXP2, GATA6, NFIB, TAZ/WWTR1 (transcriptional coactivator with PDZ-binding motif/WW domain-containing transcription regulator protein 1)] transcription factors or cofactors (15, 17–28). The precise topology of interactions between TADs and these target molecules remains to be characterized in detail (23). The interaction between NKX2-1 and PAX8, an important transcription factor in the thyroid, has been extensively studied and illustrates the complex relationship between proteins for gene promoter regulation (15, 29). Accordingly, the activity of some transcriptionally inactive NKX2-1 mutants can be rescued by their interaction with PAX8 on the thyroglobulin (TG) promoter (5); however, this thyroid phenotypic rescue is not observed with other NKX2-1 mutations (30). In contrast, some PAX8 mutants are rescued by their interaction with NKX2-1 on the TG promoter (31). Overall, these findings suggest that the specific location of the mutation determines the capacity of transcriptional partners to, at least partially, restore transcriptional activities. One of the aforementioned proteins interacting with NKX2-1 is TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain-containing transcription regulator protein 1). TAZ forms part of the Hippo signaling pathway, which, together with YAP, controls the size of organs and tissue homeostasis and regeneration (32–34). TAZ also regulates the activity of several transcription factors involved in development and disease (35, 36). It is widely expressed, with the highest levels in kidney and lung (37). Moreover, the expression patterns of TAZ and NKX2-1 overlap in a spatiotemporal fashion in respiratory epithelial cells of embryonic and adult mouse lung. Additionally, TAZ interacts with the N-TAD of NKX2-1, acting as a transcriptional coactivator of NKX2-1 in the activation of the surfactant protein C gene (25). Similar coactivation activity was reported in the thyroid, where TAZ was coexpressed with NKX2-1 and PAX8 during murine thyroid development and throughout adult life, acting as a transcriptional coactivator of NKX2-1 and PAX8 over the TG promoter (15). Transgenic mice lacking TAZ present with pathological changes in the kidney and lung that resemble human polycystic disease and pulmonary emphysema (38). Although several years have passed since the identification of the first mutation in the NKX2-1 gene, the molecular alterations responsible for such variable phenotypes are still far from being understood. Approaches that consider how such alterations affect the interaction of NKX2-1 with protein partners in a specific tissue are needed to fully understand the causes of this complex disease. In the present study, we identified a de novo frameshift heterozygous mutation in NKX2-1 in one patient with severe BLTS and life-threatening lung emphysema. The mutation is one of the most identified N-terminal mutations to date, leading to a highly aberrant protein. We performed a functional comparative study with a previously identified frameshift mutation located at the most C-terminal domain that was described in two sisters with thyroid and choreic complications but not lung involvement (30). Through the present analysis, we have demonstrated that TAZ is a phenotype modifier protein in BLTS, at least in some NKX2-1 mutations. Patient and Methods Case report The subject of the present study was born from a healthy white nonconsanguineous couple at 40 weeks of gestation after a normal pregnancy. The Apgar score was 9 of 9. He had a birth weight of 2935 g and a birth length of 51 cm. On the second day of life, he developed tachypnea, intercostal inspiratory retractions, flaring nostrils, and poor peripheral perfusion. Septicemia was suspected, and antibiotic treatment was started, without clinical improvement. No bacterial infection could be confirmed. He maintained tachypnea, cyanosis, and bilateral lung crackles on respiratory auscultation and required oxygen support. A chest radiograph showed diffuse ground glass opacity (Fig. 1A), compatible with severe hyaline membrane disease. Figure 1. View largeDownload slide Clinical study of lung function and anthropometry. (A) Neonatal chest radiograph showing diffuse ground glass opacity, air bronchogram, and a reticular-interstitial pattern, compatible with severe hyaline membrane disease. (B) Computed tomography scan at 12 years showing extensive hypoattenuation and decreased vascularization areas, suggestive of lung destruction. Bronchial wall thickening and linear images are suggestive of reduced lung parenchyma. Pathologic findings of lung biopsy showing (C) lung parenchyma with large areas of emphysema and (D) interstitial fibrosis and bronchiolar epithelial alteration. (E) Spirometry at 12 years of age showing severe impairment of pulmonary function by forced expiratory volume at 1 second. The airflow (in L/s) was measured and compared with the standards for age and sex at five time points within 1 second. The area in red shows severe impairment of pulmonary function compared with the standard values. (F) Sustained underweight in relation to undernutrition due to feeding/gastric surgery difficulties. (G) Sustained undergrowth and delayed puberty compatible with constitutional delay of growth and development (retarded bone age). The percentiles are indicated as follows: 50%, blue; 10% to 90%, green; and 3% to 97%, red, according to the Spanish Auxology Study of 2010. Figure 1. View largeDownload slide Clinical study of lung function and anthropometry. (A) Neonatal chest radiograph showing diffuse ground glass opacity, air bronchogram, and a reticular-interstitial pattern, compatible with severe hyaline membrane disease. (B) Computed tomography scan at 12 years showing extensive hypoattenuation and decreased vascularization areas, suggestive of lung destruction. Bronchial wall thickening and linear images are suggestive of reduced lung parenchyma. Pathologic findings of lung biopsy showing (C) lung parenchyma with large areas of emphysema and (D) interstitial fibrosis and bronchiolar epithelial alteration. (E) Spirometry at 12 years of age showing severe impairment of pulmonary function by forced expiratory volume at 1 second. The airflow (in L/s) was measured and compared with the standards for age and sex at five time points within 1 second. The area in red shows severe impairment of pulmonary function compared with the standard values. (F) Sustained underweight in relation to undernutrition due to feeding/gastric surgery difficulties. (G) Sustained undergrowth and delayed puberty compatible with constitutional delay of growth and development (retarded bone age). The percentiles are indicated as follows: 50%, blue; 10% to 90%, green; and 3% to 97%, red, according to the Spanish Auxology Study of 2010. The neonatal screening program revealed primary congenital hypothyroidism with filter paper TSH values of 70 mU/L (normal, <10 mU/L), confirmed in serum samples on the 10th day of life (TSH, 224 mU/L; and free thyroxine, 0.6 ng/dL; Table 1). Ultrasonography and scintigraphy showed a normal in situ thyroid gland at birth. Treatment with levothyroxine (37.5 μg/d) was started at day 10 of life, leading to normalization of the thyroid hormone parameters by the 30th day of life. Table 1. Patient’s Hormonal Status and Bone Age at Different Stages of Life Measurement  Age  NA  10 da  22 d  4 mo  10 y, 11 mo  11 y, 10 mo  12 y, 7 mo  13 y, 2 mo  14 y, 3 mo  TSH, µU/mL  70 (1–10)  224 (1–10)  13.5 (0.5–6.5)  3.7 (0.5–6.5)  —  —  —  3.8 (0.57–5.92)  —  Free T4, ng/dL  —  0.6 (0.9–2.3)  2.8 (0.9–2.3)  1.4 (0.77–1.78)  —  —  —  1.6 (0.72–2)  —  IGF-1, ng/mL)  —  —  —  —  89.8 (80–420)  123 (111–551)  87.8 (143–693)  197 (183–850)  226 (237–996)  IGFBP3, µg/mL)  —  —  —  —  2.27 (2.1–7.7)  2.64 (2.4–8.4)  2.58 (2.7–8.9)  —  —  Bone age  —  —  —  —  9 y  —  10 y  —  —  FSH/LH, mIU/mL; (Tanner 1 [<4/<1]; Tanner >1 [1.5–12.4/1.7–8.6])  —  —  —  —  1.4/0.7  1.5/0.5  —  1.8/1.1  1.74/0.73  Testosterone, ng/dL (Tanner 1 [<25]; adulthood [280–800])  —  —  —  —  17  13  59  263.8    Measurement  Age  NA  10 da  22 d  4 mo  10 y, 11 mo  11 y, 10 mo  12 y, 7 mo  13 y, 2 mo  14 y, 3 mo  TSH, µU/mL  70 (1–10)  224 (1–10)  13.5 (0.5–6.5)  3.7 (0.5–6.5)  —  —  —  3.8 (0.57–5.92)  —  Free T4, ng/dL  —  0.6 (0.9–2.3)  2.8 (0.9–2.3)  1.4 (0.77–1.78)  —  —  —  1.6 (0.72–2)  —  IGF-1, ng/mL)  —  —  —  —  89.8 (80–420)  123 (111–551)  87.8 (143–693)  197 (183–850)  226 (237–996)  IGFBP3, µg/mL)  —  —  —  —  2.27 (2.1–7.7)  2.64 (2.4–8.4)  2.58 (2.7–8.9)  —  —  Bone age  —  —  —  —  9 y  —  10 y  —  —  FSH/LH, mIU/mL; (Tanner 1 [<4/<1]; Tanner >1 [1.5–12.4/1.7–8.6])  —  —  —  —  1.4/0.7  1.5/0.5  —  1.8/1.1  1.74/0.73  Testosterone, ng/dL (Tanner 1 [<25]; adulthood [280–800])  —  —  —  —  17  13  59  263.8    Abbreviations: NA, not applicable; IGF-1, insulinlike growth factor-1; IGFBP3, insulinlike growth factor-binding protein 3; FSH, follicle-stimulating hormone; LH, luteinizing hormone; T4, levothyroxine. a After the treatment began, the values began to move toward the normal range. Treatment was started at day 10 of life with 37.5 µg/d of levothyroxine, which was increased to 50 µg/d at 3 months. The dose was gradually increased to 100 µg/d when the patient was 13 years old. Reference values (in parentheses) are specific to age and sex (39). Determinations were performed using electrochemiluminescence immunoassays. View Large Generalized choreoathetotic movements were observed at 8 months of age associated with hypotonia and psychomotor delay. Mutation analysis Genetic analysis was performed after provision of informed consent by the patient’s parental representatives and was approved by the institutional ethics review board committee. Genomic DNA of the subject was obtained from peripheral blood leukocytes using the Chemagic MSM I automated nucleic acid isolation system (PerkinElmer Chemagen Technologie GmbH, Baesweiler, Germany). The three coding exons and intron flanking regions from all four transcript variants of the NKX2-1 gene were amplified using a total of nine primer pairs and sequenced directly in both directions with BigDye Terminator V3.1 (Applied Biosystems, Life Technologies Corp., Carlsbad, CA) on an AB 3730XL DNA Analyzer (Applied Biosystems). The eight coding exons and intron flanking regions from TAZ/WWTR1 gene were also amplified and sequenced as described (primer sequences available on request). Plasmids and subcloning The plasmids used in the present work were as follows: wild-type (WT) NKX2-1 (3) and 825delC cDNA mutant (C-mut) (30) of human NKX2-1 cloned into pcDNA3; WT mouse TAZ/WWTR1 cDNA cloned into pCMV5 (TAZ) (25); WT mouse TAZ fused to a Flag-tag and cloned into pEF-BOS (TAZ-N-FLAG) (37); WT human PAX8 cloned into pcDNA3 (31) and reporter vectors hTGenh/prom-Luc (3), hSP-Bprom-Luc (B-500) (40), and mLhx6prom-Luc (41) using the pGL3 Basic vector backbone (Promega Corp., Madison, WI). Site-directed mutagenesis The human N-terminal NKX2-1 mutant (223dupG) was generated by site-directed mutagenesis using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA) with primers 5′-TACCACATGACGGCGGCGGGGGGTGCCCCAGCTCTCGCA-3′ and 5′-TGCGAGAGCTGGGGCACCCCCCGCCGCCGTCATGTGGTA-3′ in WT NKX2-1 (the WT sequence has five Gs in the underlined region). The fidelity of the mutated construct was confirmed by sequencing. In vitro synthesis of NKX2-1 and electrophoretic mobility shift assay Proteins were synthesized from 1 µg of each WT or mutant NKX2-1 vector (223dupG and 835delC) by in vitro transcription/translation using the TNT-Coupled Reticulocyte Lysate System (Promega Corp.). Protein–DNA binding was examined by electrophoretic mobility shift assay using 3 µL of in vitro–translated proteins mixed with 50,000 cpm of 32P-labeled oligonucleotide C derived from the TG promoter. Assays were performed as previously described (30, 42). Immunoblot analysis Samples of 30 µg of total extracts from cells transfected with plasmids, quantified according to the Bradford method (Bio-Rad Laboratories, Hercules, CA), were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Ponceau S staining was used to check for equal protein loading. Membranes were blocked and incubated with the following antibodies: NKX2-1 antibody (1:1500 dilution; EP1584Y; Epitomics, Burlingame, CA); TAZ antibody (1:200 dilution; sc-48805; Santa Cruz Biotechnology, Santa Cruz, CA); or FLAG-Tag antibody (1:1500 dilution; NBP1-06712; Novus Biologicals, Littleton, CO). Immunoreactive bands were visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, IL). For TNT-reticulocyte immunoblotting, 7 µL of in vitro–translated proteins was used. In vitro transactivation assay HeLa cells were grown in Dulbecco’s modified Eagle medium (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum. For transactivation assays, 105 cells were plated in MW12 culture dishes 24 hours before transfection. The cells were transfected using FuGENE6 (Promega Corp.) with a total of 1 µg of DNA per well, including 600 ng of the specific reporter plasmid (hTG, hSP-B, or mLhx6) and different amounts of expression vectors (NKX2-1 WT, 223dupG or 825delC mutants, TAZ, and PAX8) and empty vector as indicated to 400 ng. To correct for transfection efficiency, 10 ng of Renilla-encoding pRL-CMV vector was added in all cases. Cells were harvested 24 hours after transfection, lysed, and analyzed for luciferase (LUC) and Renilla activity using the dual-LUC reporter assay system (Promega Corp.). The ratio between the LUC and Renilla activities was expressed relative to the ratio obtained in cells transfected with reporter and empty expression vector (pcDNA3). Transfections were performed in triplicate and repeated at least three times; the LUC activity values are presented as the mean ± standard deviation. Statistical analysis was performed using Student’s t test to obtain the P value associated with the observed fold of activation differences. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05, P < 0.01, and P < 0.001. Immunofluorescence assay MDCK1 epithelial and NIH3T3 fibroblast cells were seeded on coverslips in 60-mm-diameter culture dishes. The cells were transfected 24 hours later with 5 µg of an empty plasmid (as a control for immunostaining specificity) or the same amount of the NKX2-1 WT, the N-terminal (223dupG) or the C-terminal (825delC) mutant expression vectors. Twenty-four hours later, the cells were fixed in 70% methanol at −20°C for 10 minutes, washed three times with phosphate-buffered saline (PBS)-0.05% Tween-20 (PBS-Tween) for 5 minutes, and blocked with PBS-Tween containing 5% goat serum for 1 hour at room temperature (RT). The cells were then incubated with the anti–NKX2-1 antibody for 1 hour at RT, washed three times in PBS-Tween for 5 minutes, incubated for 1 hour at RT with an Alexa Fluor 488-conjugated secondary antibody, washed three times with PBS-Tween for 5 minutes, with the last wash containing 4′,6-diamidino-2-phenylindole, and mounted on ProLong® Gold Antifade Reagent from Molecular Probes (Invitrogen-Life Technologies Ltd., Paisley, UK). The cells were observed under a Leica TCS SP2 confocal microscope using 63× magnification under oil immersion (Leica Corp., Deerfield, IL). Immunoprecipitation Aliquots of 25 µL of Dynabeads® Protein G (Novex®; Life Technologies AS, Oslo, Norway) were washed for 10 minutes with PBS-0.02% Tween-20 and bound with 3 to 5 µg of each antibody for 1 hour with rotation at 4°C. The bead–antibody complexes were cross-linked by bis-sulfosuccinimidyl suberate (BS3; Thermo Fisher Scientific). Next, 1 mg of total extracts from transfected HEK293T cells with TAZ-N-FLAG, together with pcDNA3.1, WT, or mutant NKX2-1 vectors were added in a final volume of 1 mL and incubated overnight with rotation at 4°C. The bead–antibody–antigen complexes were washed three times using 300 μL of PBS-0.02% Tween-20 and eluted in 25 μL of Laemmli 2× Sample Buffer at 95°C for 15 minutes. The eluted samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with the specific antibody. Finally, 30 µL of total extract from the same transfected HEK293T cells was used as an input. Proximity ligation assay HeLa cells were seeded on coverslips and transfected 24 hours later by calcium phosphate coprecipitation with 2 μg of PAX8 and 2 μg of NKX2-1 WT, N-mut, or C-mut expression vectors. After 24 hours, the cells were fixed in 4% paraformaldehyde for 10 minutes at RT, permeabilized, and blocked in 1% bovine serum albumin for 30 minutes. Next, the cells were incubated for 1 hour with the NKX2-1 antibody and a PAX8 antibody (AB53490; Abcam, Cambridge, UK). To study protein interactions, we used the DuoLink-PLA Kit (Sigma-Aldrich Corp.). In brief, the cells were washed and incubated with anti-rabbit and anti-mouse secondary antibodies coupled to complementary oligonucleotides. Subsequently, the oligonucleotides were annealed and amplified by polymerase chain reaction and detected through hybridization of complementary fluorescence-labeled oligonucleotides. Cells were washed three times, with the third wash containing 4′,6-diamidino-2-phenylindole for nuclear staining, and mounted as described. The cells were observed under a 63× objective in a LSM710 Zeiss confocal microscope. Bioinformatic analysis The sequences were analyzed using the Sequencher V4.1.4 (Genes Code Corp., Ann Arbor, MI). The protein subcellular localization prediction was performed with WoLF PSORT (available at: http://wolfpsort.org/) (43), NucPred from Stockholm Bioinformatics Center (available at: http://www.sbc.su.se/~maccallr/nucpred/cgi-bin/single.cgi) (44), and NLStradamus (available at: http://www.moseslab.csb.utoronto.ca/NLStradamus/) (45). Results Clinical evolution of the patient During the first 2 years of life, the patient developed pulmonary impairment with recurrent lung infections that led to the need for mechanical ventilation and tracheotomy. Nissen fundoplication surgery was performed because of a diagnosis of gastroesophageal reflux; however, no clinical improvement was observed. The patient continued to require nocturnal oxygen until age 2.5 years and received chronic inhaled corticosteroid treatment. Serial computed tomography scans were performed during follow-up and showed a destroyed lung, with hypoattenuation and decreased vascularization areas (Fig. 1B; Supplemental Fig. 1A). A lung biopsy performed at 12 months revealed wide emphysematous areas with destroyed alveolar structure (Fig. 1C) and interstitial fibrosis (Fig. 1D). Clinically, at 14 years of age, he could perform moderate physical activity; he did not require any treatment, although his pulmonary function was severely affected (Fig. 1E). At 17 years of age, he was registered for lung transplantation; however, the disease had stabilized, and surgery was cancelled. It was difficult to measure the patient’s tolerance to efforts owing to the limited motility caused by the involuntary choreic movements. Pulmonary functional tests showed moderate-to-severe dysfunction with an obstructive pattern. The last thoracic computed tomography scan from 2014 showed diffuse affection that was more intense in the superior and middle lobes and in the lingular segment. At the last follow-up examination, the patient was receiving corticosteroids and bronchodilators and had clinical stability with no exacerbation during the previous year. Levothyroxine treatment was continued; the dose was progressively increased to 50 µg/d at 3 months and subsequently gradually increased to 100 μg/d at 14 years. At that time, a thyroid ultrasound scan showed a homogeneous and small thyroid gland. The choreoathetotic movements and psychomotor characteristics were as follows: head control was achieved at 18 months, sitting at 2 years, walking with help at 2 years, 9 months, and autonomous walking at 3.5 years. He pronounced his first words at 2 years and composed sentences at 5 years. Choreoathetosis remained stable with slight improvement at adolescence. At 14 years of age, he showed mild cognitive impairment with adequate school performance and generalized chorea of a nonprogressive course. The cerebral magnetic resonance imaging findings were normal. Since the early neonatal period, the patient presented with a low weight gain curve (Fig. 1F), which became more severe through the years, reaching −2.5 at the chronological age of 14.7 years. This was attributed to chronic undernutrition resulting from feeding difficulties related to the performance of the gastric fundoplication. His height curve was also poor (−2.3 standard deviation; Fig. 1G). The insulinlike growth factor-1 and insulinlike growth factor-binding protein 3 levels were within the low-normal range (Table 1). During follow-up, his bone age was delayed by 2 years, and he started puberty at age 13.1 years with adequate follicle-stimulating hormone, luteinizing hormone, and testosterone values (Table 1). Identification and characterization of an NKX2-1 human mutation Using polymerase chain reaction and direct sequencing of the patient’s DNA, we identified a de novo heterozygous mutation in the NKX2-1 gene (Fig. 2A). The parents were healthy and presented with no NKX2-1 mutations. The mutation was a duplication of a guanine at position 223 of the cDNA (according to the ENST00000498187 transcript), which generates a frameshift, leading to an aberrantly long protein of 407 amino acids, retaining only the first 75 amino acids of the WT protein [c.223dupG/p.(Val75Glyfs*334); Fig. 2B and 2C]. The mutant protein lacks the homeodomain and the C-TAD but conserves most of the N-TAD and the first four of seven phosphorylated serine residues (46). This is one of the most identified amino-terminal frame shift mutations thus far in the NKX2-1 gene. Amino acid prediction of the mutant anticipates a severe impact on protein function based on its reduced conservation of amino acid sequence with respect to the WT protein and the absence of critical domains of this transcription factor. Figure 2. View largeDownload slide Identification of a mutation in the NKX2-1 gene. WT and mutant NKX2-1 protein primary structure, expression, stability, and subcellular localization. (A) NKX2-1 sequence chromatograms from the patient and his unaffected parents. The arrow indicates duplication of a guanine at position 223 in exon 2 of the NKX2-1 complementary DNA (according to the ENST00000498187 transcript). The heterozygous de novo mutation generates a double peak sequence beyond the insertion site, which is absent in the chromatogram of parents, indicating a de novo mutation. (B) Amino acid sequence comparison between NKX2-1 WT protein, the N-terminal mutant frameshift described in the present study [p.(Val75Glyfs*334)], shown in bold, and a C-terminal mutant frameshift [p.(Ala276Argfs*75)], previously described (30). The homeobox (gray box), cysteines for dimerization (plus sign), serines involved in phosphorylation (asterisk), the nuclear localization signal (NLS; underlined), the limit between exon 1 and exon 2 (arrow), the putative TAZ interaction motifs LPPY (yellow) and pSP (green) are shown. The nonsense region of the N-mut (red bold letters) and C-mut (red letters), the newly generated NLS (red underline) and putative pSP motifs (light blue) are also indicated. The most important functional amino acids are absent in the N-mut protein, barring the four phosphorylated serines. (C) Diagrams representing WT, N-mut, and C-mut NKX2-1 proteins. The homeodomain (HD) is shown in black, N-TAD and C-TAD in gray, and the nonsense aberrant region of N-mut and C-mut by diagonal lines. The relative position of NKX2-1 antibody epitope is indicated. (D) Immunoblot of WT, N-mut, or C-mut NKX2-1 proteins. Proteins were transcribed and translated from their respective vectors (including pcDNA3.1 empty vector as control) using TNT reticulocytes. The anti–NKX2-1 antibody detects all three proteins because of the N-terminal location of the epitope (C). Note that the N-mut is larger than that of the other two proteins, according to its predicted sequence. (E) Analysis of WT and mutant NKX2-1 protein stability. Immunoblots of 30 μg total protein extracts from HeLa cells not transfected or transfected with WT, N-mut, or C-mut NKX2-1 expression vectors and lysed at five different time points, as indicated in each lane. All three proteins are expressed, with a maximum peak at 24 hours and are still present at 48 hours after transfection. Tubulin was used as a loading control. (F) Subcellular localization of WT, N-mut, or C-mut NKX2-1 by confocal microscopy. MDCK1 cells were seeded on coverslips, transfected with empty pcDNA3.1 (control), WT, N-mut, or C-mut NKX2-1 expression vectors and stained for immunofluorescence with an anti–NKX2-1 antibody. 4′,6-Diamidino-2-phenylindole was used to stain nuclear DNA. Ab, antibody; COOH, carboxyl. Figure 2. View largeDownload slide Identification of a mutation in the NKX2-1 gene. WT and mutant NKX2-1 protein primary structure, expression, stability, and subcellular localization. (A) NKX2-1 sequence chromatograms from the patient and his unaffected parents. The arrow indicates duplication of a guanine at position 223 in exon 2 of the NKX2-1 complementary DNA (according to the ENST00000498187 transcript). The heterozygous de novo mutation generates a double peak sequence beyond the insertion site, which is absent in the chromatogram of parents, indicating a de novo mutation. (B) Amino acid sequence comparison between NKX2-1 WT protein, the N-terminal mutant frameshift described in the present study [p.(Val75Glyfs*334)], shown in bold, and a C-terminal mutant frameshift [p.(Ala276Argfs*75)], previously described (30). The homeobox (gray box), cysteines for dimerization (plus sign), serines involved in phosphorylation (asterisk), the nuclear localization signal (NLS; underlined), the limit between exon 1 and exon 2 (arrow), the putative TAZ interaction motifs LPPY (yellow) and pSP (green) are shown. The nonsense region of the N-mut (red bold letters) and C-mut (red letters), the newly generated NLS (red underline) and putative pSP motifs (light blue) are also indicated. The most important functional amino acids are absent in the N-mut protein, barring the four phosphorylated serines. (C) Diagrams representing WT, N-mut, and C-mut NKX2-1 proteins. The homeodomain (HD) is shown in black, N-TAD and C-TAD in gray, and the nonsense aberrant region of N-mut and C-mut by diagonal lines. The relative position of NKX2-1 antibody epitope is indicated. (D) Immunoblot of WT, N-mut, or C-mut NKX2-1 proteins. Proteins were transcribed and translated from their respective vectors (including pcDNA3.1 empty vector as control) using TNT reticulocytes. The anti–NKX2-1 antibody detects all three proteins because of the N-terminal location of the epitope (C). Note that the N-mut is larger than that of the other two proteins, according to its predicted sequence. (E) Analysis of WT and mutant NKX2-1 protein stability. Immunoblots of 30 μg total protein extracts from HeLa cells not transfected or transfected with WT, N-mut, or C-mut NKX2-1 expression vectors and lysed at five different time points, as indicated in each lane. All three proteins are expressed, with a maximum peak at 24 hours and are still present at 48 hours after transfection. Tubulin was used as a loading control. (F) Subcellular localization of WT, N-mut, or C-mut NKX2-1 by confocal microscopy. MDCK1 cells were seeded on coverslips, transfected with empty pcDNA3.1 (control), WT, N-mut, or C-mut NKX2-1 expression vectors and stained for immunofluorescence with an anti–NKX2-1 antibody. 4′,6-Diamidino-2-phenylindole was used to stain nuclear DNA. Ab, antibody; COOH, carboxyl. Phenotypic comparison of patients harboring p.(Val75Glyfs*334) and p.(Ala276Argfs*75) We previously identified a frameshift mutation in NKX2-1 [c.825delC/p.(Ala276Argfs*75)] in two sisters of a Spanish family. Unlike the present patient, the two sisters had congenital hypothyroidism and benign chorea but no pulmonary distress (30). The mutant protein from the previous study was shorter than the WT protein but retained much of the normal sequence of the WT and is one of the most described C-terminal frameshift mutations (Fig. 2B and 2C). To search for specific pathogenic mechanisms that might explain the strikingly different lung phenotypes, normal and severe respiratory insufficiency, between the patients with BLTS, we performed a functional comparative study of the two NKX2-1 mutants: p.(Val75Glyfs*334) (herein referred to as N-mut) and p.(Ala276Argfs*75) (herein referred to as C-mut). Protein expression, stability, and subcellular localization of NKX2-1 mutants We first used an in vitro transcription and translation system to synthesize WT, N-mut, and C-mut NKX2-1 proteins. All three proteins generated were detected with a specific N-terminal NKX2-1 antibody, and all three were correctly expressed with a molecular weight compatible with the expected length of the sequence (Fig. 2D). We evaluated their stability in cultured cells and found that the expression of all three was maximal at 24 hours and remained detectable at 48 hours after transfection (Fig. 2E), suggesting that no mutant protein was prematurely degraded with respect to WT. We also performed an in silico analysis using WoLF PSORT, NucPred, and NLStradamus bioinformatic programs, which predicted that the WT and the two mutants would contain consensus nuclear localization sequences (Fig. 2B). Therefore, similar to the WT protein, the mutant proteins retain the capacity to reach the nucleus, which was verified by confocal microscopy of MDCK1 cells transfected with each of the three NKX2-1 constructs (Fig. 2F). Nuclear location was confirmed in a different cell line (NIH3T3; Supplemental Fig. 1B). Transcriptional activity of N-mut and C-mut on three tissue-specific promoters Given the correct nuclear localization of the two mutants, we next studied their transcriptional activity. We first used an electrophoretic mobility shift assay to assess the ability of the mutant proteins to bind DNA using the NKX2-1 binding sites within the TG promoter (oligo C) as a probe (42). The N-mut protein failed to bind to the specific probe (Fig. 3A, lanes 6 to 8). In contrast, strong binding was detected with the WT protein (lanes 3 to 5), and a reduced binding ability was observed for the C-mut protein (lanes 9 to 11), as previously described (30). PCCl3 nuclear extracts were used as a positive control (lanes 12 to 14). Figure 3. View largeDownload slide DNA-binding, transcriptional, and interacting properties of WT and mutant NKX2-1 proteins. (A) WT and mutant NKX2-1 proteins translated from TNT reticulocytes and control PCCl3 nuclear extracts were incubated with the 32P-labeled C oligonucleotide derived from the TG promoter (lanes 3, 6, 9, and 12). Empty pcDNA3.1 vector was used as control for nonspecific bands (lane 2). For competition, a 100-fold excess of unlabeled related C (lanes 4, 7, 10, and 13) or unrelated Sp1 (lanes 5, 8, 11, and 14) oligonucleotides were used. TNT reticulocyte lysates (3 μL) or 7 μg of nuclear extracts were added to each lane. The WT (lanes 3 and 5) and C-mut (lanes 9 and 11) bind the specific probe; however, the N-mut fails to bind (lanes 6 and 8). PCCl3 nuclear extracts were used as a positive control (lanes 12 and 14). The C-mut NKX2-1/DNA complex migrates faster than the WT NKX2-1/DNA complex, in agreement with its smaller size. A nonspecific band related to the TNT lysates was observed in all lanes but was not competed for by the competitor oligos. (B) Transcriptional capacity of N-mut and C-mut over brain-, thyroid-, and lung-specific promoters compared with WT NKX2-1. Light gray bars represent isolated expression of each of the NKX2-1 proteins. Black bars represent transactivation through coexpression of each of them with TAZ. HeLa cells were transiently transfected with 200 ng of WT, N-mut, or C-mut NKX2-1 expression vectors with or without 200 ng of TAZ vector (or empty pcDNA3.1 vector as control). Transcriptional activation was assessed by transfecting 600 ng of each reporter vector: the hTG enhancer/promoter (thyroid), the mLhx6 promoter (brain), and the hSP-B promoter (lung). LUC activity was normalized to Renilla activity to adjust for transfection efficiency. Experiments were performed at least three times in triplicate and expressed as mean ± standard deviation. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). The ns and asterisks over the bars indicate comparison of each condition in the presence or absence of TAZ; ns and number signs over individual conditions indicate comparison with the empty vector. (C) The interaction between NKX2-1 variants and TAZ was analyzed by coimmunoprecipitation assays. HEK293T cells were cotransfected with WT, N-mut, or C-mut NKX2-1 and TAZ-N-FLAG (or pcDNA3.1 as control) expression vectors. Dynabeads® Protein G, cell extracts and antibodies (anti-NKX2-1, anti-TAZ, or anti-FLAG) were allowed to interact overnight at 4°C. Bead–antibody–protein complexes were washed and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. WT, N-mut, or C-mut NKX2-1 was detected by immunoblotting with anti-NKX2-1. TAZ was detected with both an anti-TAZ antibody and an anti-FLAG antibody. The immunoblots represent the (Left) inputs (30 μg of total extracts) and (Right) immunoprecipitated proteins. (Top) Schematic representation of epitope location for antibodies in WT, N-mut, and C-mut NKX2-1 and TAZ proteins. (D) Competition of WT NKX2-1 and mutant variants. HeLa cells were transfected with empty vector or NKX2-1 WT (2 μg), together with increasing amounts of (top) N-mutant protein (0 to 3 μg), Renilla, and TG or (bottom) SP-B promoters driving LUC expression. The results are expressed as relative fold activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). Asterisks indicate comparisons with empty vector; ns and number signs over each condition indicate comparison with the WT vector. (E) PAX8 and mutant NKX2-1 interaction. HeLa cells showed interaction of PAX8 cotransfected with (left) NKX2-1 WT, (center) N-mut, and (right) C-mut. The nucleus stained blue with 4′,6-diamidino-2-phenylindole. Red dots indicate protein–protein interactions detected by polylactide (magnification ×63 images shown). (F) PAX8 rescue of NKX2-1 mutants on the TG promoter. HeLa cells were transfected with equal amounts of the NKX2-1 variants in the presence or absence of PAX8 and with TG promoter-driven LUC and Renilla expression vectors. The results are expressed as the relative fold TG promoter activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk); ns and asterisk over the bars indicates comparisons of each condition in the presence or absence of PAX8 and ns and numbered sign indicate comparison vs the empty vector condition. α, antibody; IB, immunoblotting; IP, immunoprecipitation. Figure 3. View largeDownload slide DNA-binding, transcriptional, and interacting properties of WT and mutant NKX2-1 proteins. (A) WT and mutant NKX2-1 proteins translated from TNT reticulocytes and control PCCl3 nuclear extracts were incubated with the 32P-labeled C oligonucleotide derived from the TG promoter (lanes 3, 6, 9, and 12). Empty pcDNA3.1 vector was used as control for nonspecific bands (lane 2). For competition, a 100-fold excess of unlabeled related C (lanes 4, 7, 10, and 13) or unrelated Sp1 (lanes 5, 8, 11, and 14) oligonucleotides were used. TNT reticulocyte lysates (3 μL) or 7 μg of nuclear extracts were added to each lane. The WT (lanes 3 and 5) and C-mut (lanes 9 and 11) bind the specific probe; however, the N-mut fails to bind (lanes 6 and 8). PCCl3 nuclear extracts were used as a positive control (lanes 12 and 14). The C-mut NKX2-1/DNA complex migrates faster than the WT NKX2-1/DNA complex, in agreement with its smaller size. A nonspecific band related to the TNT lysates was observed in all lanes but was not competed for by the competitor oligos. (B) Transcriptional capacity of N-mut and C-mut over brain-, thyroid-, and lung-specific promoters compared with WT NKX2-1. Light gray bars represent isolated expression of each of the NKX2-1 proteins. Black bars represent transactivation through coexpression of each of them with TAZ. HeLa cells were transiently transfected with 200 ng of WT, N-mut, or C-mut NKX2-1 expression vectors with or without 200 ng of TAZ vector (or empty pcDNA3.1 vector as control). Transcriptional activation was assessed by transfecting 600 ng of each reporter vector: the hTG enhancer/promoter (thyroid), the mLhx6 promoter (brain), and the hSP-B promoter (lung). LUC activity was normalized to Renilla activity to adjust for transfection efficiency. Experiments were performed at least three times in triplicate and expressed as mean ± standard deviation. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). The ns and asterisks over the bars indicate comparison of each condition in the presence or absence of TAZ; ns and number signs over individual conditions indicate comparison with the empty vector. (C) The interaction between NKX2-1 variants and TAZ was analyzed by coimmunoprecipitation assays. HEK293T cells were cotransfected with WT, N-mut, or C-mut NKX2-1 and TAZ-N-FLAG (or pcDNA3.1 as control) expression vectors. Dynabeads® Protein G, cell extracts and antibodies (anti-NKX2-1, anti-TAZ, or anti-FLAG) were allowed to interact overnight at 4°C. Bead–antibody–protein complexes were washed and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. WT, N-mut, or C-mut NKX2-1 was detected by immunoblotting with anti-NKX2-1. TAZ was detected with both an anti-TAZ antibody and an anti-FLAG antibody. The immunoblots represent the (Left) inputs (30 μg of total extracts) and (Right) immunoprecipitated proteins. (Top) Schematic representation of epitope location for antibodies in WT, N-mut, and C-mut NKX2-1 and TAZ proteins. (D) Competition of WT NKX2-1 and mutant variants. HeLa cells were transfected with empty vector or NKX2-1 WT (2 μg), together with increasing amounts of (top) N-mutant protein (0 to 3 μg), Renilla, and TG or (bottom) SP-B promoters driving LUC expression. The results are expressed as relative fold activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk). Asterisks indicate comparisons with empty vector; ns and number signs over each condition indicate comparison with the WT vector. (E) PAX8 and mutant NKX2-1 interaction. HeLa cells showed interaction of PAX8 cotransfected with (left) NKX2-1 WT, (center) N-mut, and (right) C-mut. The nucleus stained blue with 4′,6-diamidino-2-phenylindole. Red dots indicate protein–protein interactions detected by polylactide (magnification ×63 images shown). (F) PAX8 rescue of NKX2-1 mutants on the TG promoter. HeLa cells were transfected with equal amounts of the NKX2-1 variants in the presence or absence of PAX8 and with TG promoter-driven LUC and Renilla expression vectors. The results are expressed as the relative fold TG promoter activation over the empty vector and represent the mean ± standard error of three independent experiments performed in triplicate. Differences were considered not statistically significant at P > 0.05 (ns) and statistically significant at P < 0.05 (asterisk), P < 0.01 (double asterisk), and P < 0.001 (triple asterisk); ns and asterisk over the bars indicates comparisons of each condition in the presence or absence of PAX8 and ns and numbered sign indicate comparison vs the empty vector condition. α, antibody; IB, immunoblotting; IP, immunoprecipitation. We then measured the transcriptional activity of the mutant proteins using representative target promoters specific for the tissues affected in BLTS: Lhx6 (LIM homeobox 6) from brain (basal ganglia), surfactant protein B (SP-B) from lung, and TG from thyroid. Compared with that of the WT protein, the activity of the N- and C-mut proteins over the three promoters was null (Fig. 3B). Although the lack of ability to bind and activate its target promoters suggested that loss of function of the N-mut protein was responsible for the BLTS in the present patient, it does not explain the phenotypic differences between this patient with severe lung disease and the two previously reported patients with normal respiratory function. TAZ/WWTR1 specifically rescues C-mut, but not N-mut, on the lung SP-B promoter TAZ is a transcriptional coactivator that interacts through its WW domain with a large number of transcription factors (37). Among them, it cooperates with NKX2-1 to activate surfactant protein C expression in embryonic and adult mouse lung (25). This observation prompted us to evaluate whether TAZ could be a modifier protein over the transcriptional activity of the NKX2-1 mutant proteins. We found that TAZ coexpression partially rescued the ability of the NKX2-1 C-mut protein to activate the TG and Lhx6 promoters, and, importantly, SP-B promoter activity was completely recovered (Fig. 3B). Coexpression of TAZ with the NKX2-1 N-mut had no effect on the activity of any of the promoters. These findings do much to explain why patients with the C-terminal mutation have a milder brain and thyroid phenotype, with no lung involvement (30), than the present patient with severe pulmonary disease. To rule out the possibility that a mutation in TAZ was present in the patient with the N-terminal mutation, the entire coding region of TAZ/WWTR1 was sequenced, and no pathogenic mutations were identified (data not shown). Overall, these data strongly suggest that TAZ could be a genetic modifier of the severity of the pulmonary phenotype and could explain in part the clinical variability caused by the different NKX2-1 mutations. Physical interaction between TAZ/WWTR1 and NKX2-1 mutants The coactivating function of TAZ is believed to be via binding of its WW domain to the (L/P)PXY amino acid motif present in a large number of transcription factors (37). Accordingly, Park et al. (25) demonstrated that TAZ interacts with the N-TAD of NKX2-1 and suggested that the LPPY amino acid sequence at position 97-100 of NKX2-1 (Fig. 2B) is responsible for this interaction (25). Nevertheless, this motif was found to be not essential for the binding between TAZ and NKX2-1 in another study (47). To investigate to which extent NKX2-1 mutants retain their ability to interact with TAZ, we cotransfected the three NKX2-1 expression vectors, together with FLAG-tagged TAZ into HEK293T cells and measured their interactions by immunoprecipitation. Immunoblotting of immunoprecipitated complexes demonstrated that all three NKX2-1 proteins interacted with TAZ (Fig. 3C), confirming that this binding does not require the LPPY motif of NKX2-1, which is absent in the N-mut protein. One possible candidate binding motif would be pS-P, present in the first 74 amino acids of the NKX2-1 (Fig. 2B), as described by Kanai et al. (37), who showed that TAZ binds to the (L/P)PXY motif but also to pS/T-P residues with slightly lower affinity. Dominant negative activity of the N-mut NKX2-1 protein Our previous work showed that the NKX2-1 C-mut exerts a dominant negative effect over the WT protein and decreases its ability to activate the TG but not the SP-B promoter (30). Because it is possible that truncated versions of NKX2-1 retain their ability to interact with partner proteins and impair the function of the WT protein, we analyzed the activity of TG and SP-B promoters in HeLa cells transfected with the WT protein and increasing amounts of the N-mut protein. We found that the N-mut protein clearly interfered with the ability of the WT protein to activate the TG promoter (Fig. 3D). A similar trend was found for the SP-B promoter, although the difference was not statistically significant. Interaction of PAX8 with NKX2-1 mutants TAZ plays a general role in the activation of NKX2-1 in every tissue in which NKX2-1 has a function. In the thyroid, the cooperation between NKX2-1 and PAX8 to activate thyroid-specific promoters is well known (15). Thus, we analyzed whether the different NKX2-1 mutants retain their ability to bind PAX8. Thus, HeLa cells were transfected with PAX8, together with the WT, N-mut, or C-mut NKX2-1, and a proximity ligation assay was performed to detect protein–protein interactions at a single molecule level. Both mutant proteins were found to bind PAX8 to an extent similar to that of the WT protein (Fig. 3E). To study whether this interaction is functional, we measured the ability of the different mutants to activate the TG promoter in the presence of PAX8. We found that activation of the TG promoter was much weaker by PAX8 than by NKX2-1 (Fig. 3F). PAX8 could partially rescue the ability of the C-mut to activate the TG promoter; however, the increase in the activity of the promoter in the presence of the N-mut and PAX8 was minor. This could explain the more aggressive thyroid phenotype in the patient carrying the N-terminal mutation. Discussion In the present work, we have described a heterozygous mutation in the NKX2-1 transcription factor in a child who developed severe pulmonary complications at the second day of life. Congenital hypothyroidism was diagnosed in the patient in the neonatal screening program, who presented with choreic movements and psychomotor delay from the eight month of life. Therefore, the patient developed full-blown and early expressed BLTS. Patients with this syndrome have been described previously with or without pulmonary dysfunction (48), and the mutations they harbor localize in different domains of NKX2-1. The mutation we have reported is one of the most identified N-terminal frameshift mutations to date in NKX2-1. To understand the molecular basis of the disorder in our patient and to shed further light on the disease, we performed a comparative study using a C-terminal NKX2-1 mutation described previously in two patients with BLTS who did not present with pulmonary involvement (30). Our results showed that both mutant proteins have a similar expression profile and are able to reach the nucleus. Nevertheless, although the C-mut protein retains residual DNA binding activity, the N-mut protein with an N-terminal domain mutation, resulting in loss of the DNA binding domain, is incapable of binding DNA. Both N- and C-mut proteins failed to transactivate specific brain, lung, and thyroid promoters; however, cotransfection of the coactivator factor TAZ/WWTR1 fully rescued the ability of the C-mut, but not the N-mut, protein to transactivate a lung-specific promoter. This tissue-specific action explains, at least in part, the phenotype of the two patients with the C-terminal mutation who did not present with lung complications. This result could also provide an explanation for the phenotype of the patient with the N-terminal mutation, who had severe lung complications. We initially predicted that the N-mut protein, which has lost the LPPY motif, would be unable to interact with TAZ. However, coimmunoprecipitation assays showed that this was not the case. We propose that such an interaction could occur through another candidate-binding motif, the pSer-Pro (pS/T-P), which is conserved in the first 74 amino acids of the NKX2-1 N-mut and has been reported to also bind to TAZ, albeit with lower affinity (37). The most plausible explanation for the molecular mechanism underlying the functional rescue of TAZ on NKX2-1 C-mut and not in N-mut is illustrated in the model depicted in Fig. 4A. As a coactivator, TAZ does not bind DNA directly but rather mediates the transcriptional effects of DNA-binding transcription factors through efficient assembly of the whole transcriptional protein complex. Therefore, TAZ interacts with specific transcription factors through its N-terminal WW domain and with the basal transcriptional machinery through its C-terminal extended coiled–coiled region, acting as a TAD, to stimulate the expression of target genes (35). The results obtained in the present study suggest that two requisites are necessary for NKX2-1 mutants to be amenable for functional rescue by TAZ: (1) to retain sufficient capacity to interact with the WW domain of TAZ; and (2) to retain at least a weak DNA binding ability. As shown in the present study, the NKX2-1 N-mut could only fulfill the first of these requirements, restricting the capacity of TAZ for functional rescue. This contrasts with the C-mutant protein, which can both weakly bind DNA and interact with TAZ. A review of all the mutants of NKX2-1 described to date indicated no correlation is obvious between the mutated region of the protein and the associated phenotype, highlighting the variable penetrance of the different mutations. A summary of the NKX2-1 mutants and the phenotypes associated is shown in Fig. 4B, and the most relevant information for all the mutations described in the reported data can be found in Supplemental Table 1. To compile Fig. 4B, we considered only the mutants from studies that addressed the state of the three organs affected by the syndrome. Together with gene deletions, frameshift mutations more frequently present with the full phenotype, and mutations that generate a stop codon or amino acid substitutions show a weaker association with the full-blown phenotype, which is present only in mutations before or in the homeodomain. Frameshift mutations after the homeodomain that generate a partially aberrant protein can also cause the full phenotype, which might indicate interference between the aberrant tail and the native region. As we have demonstrated in the present study, expression of aberrant NKX2-1 interferes with the ability of the WT to activate target promoters. It is particularly challenging to predict the behavior of the mutants in terms of phenotype probabilities. The structure of the protein and the interaction with principal partner proteins such as TAZ and PAX8 should be studied in detail for every mutant to ascertain the pathogenic mechanism underlying the mutation. Thus, it would be interesting to analyze the functional state of the C-terminal mutants that develop the full phenotype. Our results suggest that NKX2-1 mutations that completely prevent the interaction with TAZ could be inactive on lung-specific target genes, causing severe pulmonary disease. The possibility of such an event among the described NKX2-1 mutations is unknown but could be low because even in the absence of the LPPY motif, the N-mut protein retained sufficient interacting capacity with TAZ, likely through a Ser-Pro motif (37). Figure 4. View largeDownload slide (A) Mechanistic model for the interaction between WT and mutant NKX2-1 with TAZ/WWTR1. Proposed model shown to describe the behavior of WT, N-mut, and C-mut NKX2-1 proteins to explain the differences among the patients studied. (B) Diagrams showing the distribution of the different NKX2-1 mutations described in the reported data and the associated phenotypes. (Upper left) Frameshift mutations, (upper right) STOP codon mutations, (bottom left) amino acid substitutions, and (bottom right) gene deletions. B, brain; BL, brain-lung; BLT, brain-lung-thyroid; BT, brain-thyroid; BTM, basal transcriptional machinery; C, carboxy-terminal; HD, homeodomain; N, amino-terminal; WW, tryptophan-rich protein-binding domain. Figure 4. View largeDownload slide (A) Mechanistic model for the interaction between WT and mutant NKX2-1 with TAZ/WWTR1. Proposed model shown to describe the behavior of WT, N-mut, and C-mut NKX2-1 proteins to explain the differences among the patients studied. (B) Diagrams showing the distribution of the different NKX2-1 mutations described in the reported data and the associated phenotypes. (Upper left) Frameshift mutations, (upper right) STOP codon mutations, (bottom left) amino acid substitutions, and (bottom right) gene deletions. B, brain; BL, brain-lung; BLT, brain-lung-thyroid; BT, brain-thyroid; BTM, basal transcriptional machinery; C, carboxy-terminal; HD, homeodomain; N, amino-terminal; WW, tryptophan-rich protein-binding domain. Regarding the lung phenotype of the patient, it has been recently reported that TAZ is involved in the pathogenesis of pulmonary fibrosis through multifaceted effects on lung fibroblasts (49). Moreover, TAZ is an integral component of the Wnt/β-catenin cascade (50), which has been consistently linked to the pathogenesis of fibrotic disorders (51). With this information, we speculate that TAZ might not only mediate the transcriptional activity of NKX2-1 over surfactant protein promoters but also could be involved in the abnormal alveolarization of the lung (52) and the interstitial fibrosis (53) as the other relevant pathological hallmark of pulmonary disease in the present patient. In support of this concept, transgenic mice lacking TAZ have pathological changes in the lung that resemble human pulmonary emphysema and fibrosis signs (38). Furthermore, interaction with PAX8, a transcription factor that cooperates with NKX2-1 to modulate the expression of thyroid-specific genes, rescues the activity of the C-mutant NKX2-1 but not of the N-mutant NKX2-1, which might explain in part the more severe thyroid phenotype of the present patient with the N-terminal mutation. In conclusion, we found that the c.223dupG/p.(Val75Glyfs*334) mutation in the NKX2-1 gene was responsible for BLTS in a patient with severe lung emphysema. It is one of the most described amino-terminal mutations to date and generates a highly aberrant protein. The described mutation does not alter the nuclear localization of the mutant protein but blocks its DNA-binding capacity and its ability to activate specific promoters of brain, lung, and thyroid. The TAZ coactivator completely recovers the transcriptional activity of a C-terminal mutant [c.825delC/p.(Ala276Argfs*75)] in lung but not in the N-terminal mutant identified in the present study. These data explain the severity of the lung phenotype in the present patient and suggest that TAZ could be a modifier protein of the phenotype in BLTS in patients with NKX2-1 mutations. Abbreviations: BLTS brain-lung-thyroid syndrome C-mut p.(Ala276Argfs*75) LUC luciferase N-mut p.(Val75Glyfs*334) NKX2-1 NK2 homeobox 1 PAX8 paired box 8 PBS phosphate-buffered saline RT room temperature SP-B surfactant protein B TAD transactivation domain TAZ transcriptional coactivator with PDZ-binding motif TAZ-N-FLAG WT mouse TAZ fused to a Flag-tag and cloned into pEF-BOS TG thyroglobulin TSH thyroid-stimulating hormone WT wild-type WWTR1 WW domain-containing transcription regulator protein 1. Acknowledgments We thank all family members for their collaborative participation in this study. We are grateful to Dr. M. Zannini for providing pCMV5-mTAZ and to Dr. S.A. Anderson for providing p5′-Lhx6-IRES-GFP. We are also grateful to Dr. Kenneth McCreath for helpful comments on the manuscript and to Javier Perez from the Imaging Science Service (Biomedical Research Institute) for help with the illustrations. Financial Support: This work was supported by funding from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (grants PI1002160 and PI16/00830 to J.C.M.) and Ministerio de Economía y Competitividad of Spain, Fondo Europeo de Desarrollo Regional (grants SAF2013-44709R and SAF2016-75531R to P.S.). M.A.Z. and P.S. were supported by CIBERONC. C.M.M. was supported by a postdoctoral fellowship from the Research Program of the European Society for Pediatric Endocrinology. Disclosure Summary: The authors have nothing to disclose. References 1. Fernández LP, López-Márquez A, Santisteban P. Thyroid transcription factors in development, differentiation and disease. Nat Rev Endocrinol . 2015; 11( 1): 29– 42. Google Scholar CrossRef Search ADS PubMed  2. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 1, 2018

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