Characterization of sequences in human TWIST required for nuclear localization
© Singh and Gramolini; licensee BioMed Central Ltd. 2009
Received: 30 October 2008
Accepted: 17 June 2009
Published: 17 June 2009
Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) 37RKRR40 and 73KRGKK77 in the human TWIST (H-TWIST) protein.
Using site-specific mutagenesis and immunofluorescences, we observed that altered TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays.
Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4.
TWIST1 is a basic helix-loop-helix (bHLH) transcription factor  which forms either homo-or-heterodimers with other bHLH proteins to bind to a core E-box (CANNTG) sequence on the promoter region of target genes through the basic region . Twist is necessary for the development of the mesoderm , cell type determination and differentiation during myogenesis , neurogenesis , cardiogenesis  and also required for formation of the head mesenchyme, somites and limb buds . Twist loses its function as a negative modulator during the differentiation of separate mesodermal layers, myogenesis, osteogenesis or neurogenesis [7, 8]. Twist has also been implicated in neural tube closure and null mice mutants are embryonic lethal at E10.5, whereas TWIST is essential for normal development and promotes autosomal dominant defects characterized by minor skull and limb anomalies in humans . Mutations in TWIST1 result in Saethre-Chotzen Syndrome (MIM #101400, SCS), known as an autosomal dominant craniosynostosis [9, 10]. In addition, mutations in the helix domain of the TWIST1 gene can cause subcellular mislocalization and increased degradation of its protein product . Twist also acts as a key regulator of metastasis, and overexpression of TWIST1 in subsets of sporadic human breast cancer promotes epithelial to mesenchymal transition through down-regulation of E-cadherin which was confirmed in a murine breast tumor model .
Potential functions of TWIST1 are not well defined. Previous studies have shown that Twist functions as a transcriptional repressor and is regulated by its dimerization with other bHLH-containing transcriptional factors. For instance, post-translational modifications such as phosphorylation can alter the dimerization preferences of Twist, promoting either homodimer or heterodimer formation . The Twist heterodimer can also act a transcription repressor, whereas Twist homodimer acts as a transcription activator in Drosophila mesoderm development and in human cranial suture patterning . In order for a protein to function as an activator and/or repressor of transcription of a target gene, efficient nuclear localization is essential . Directed nuclear entry requires the presence of nuclear localization signals (NLSs) that recognize and associate with the nuclear import receptors. Nuclear localization signals (NLSs) mediate active transport of protein into the nucleus selectively and efficiently . Therefore, identification and functional characterization of TWIST NLSs would represent a necessary and important step in understanding the regulation of TWIST and its interaction with other bHLH proteins.
In this study, we identified two NLSs present at the N-terminal region of human TWIST rich in basic amino acids lysine and arginine. Using site directed mutagenesis and immunofluorescence assays, we studied the role of these two NLSs in nuclear localization. Yeast-two-hybrid and immunoprecipitation assays were also performed to identify TWIST-interacting proteins and gain a greater understanding of the mechanism responsible for H-TWIST localization and function.
Comparison of various Twist proteins
Subcellular-localization of H-TWIST-NLS mutants
Subcellular localization of TWISTwt and NLS mutants.
Expressions of the TWIST fusion proteins were also examined by cellular fraction and immunoblotting. A single 28-kDa protein band was recognized for c-myc-TWISTWT by an anti-c-myc antibody (Fig. 2B). pCMV empty vector transfected in cells was used as a negative control along with c-myc-TWISTWT fusion construct in our first set of experiments. No signal was detected in cells transfected with the negative control plasmid (Fig. 2B).
In biochemical experiments, TWIST NLS mutant proteins were in cytoplasmic fractions of transfected cells by immunoblot analyses. In these experiments, there were high levels of expression for the K38R and K73R mutants in the cytoplasmic lysate (Fig. 3B), consistent with our microscopy (Fig. 3A). We detected very low levels of the K76R mutant in the cytoplasm; a finding entirely consistent where the majority of staining is seen restricted to the nucleus. Interestingly, K77R showed expression in the cytoplasm, although our imaging results identified that it was not restricted to the nucleus. These results would indicate that this mutant that loses its nuclear targeting is either not soluble or expressed perhaps in intracellular organelles not solubilized by our protocol.
Rescue of mislocalization of TWIST mutants by co-transfection with heterodimer partner E12
In order to investigate direct interactions between TWIST and E12, the TWISTWT and TWISTNLS1 mutant with eGFP-E12 were transiently transfected into the cells. Here, we observed that the coexpression of TWISTNLS1 mutant K38R with E12 compensates for the mislocalization of this construct and results in the restoration of nuclear expression of the TWIST mutant. The same co-expression results were obtained using K73R and K77R with E12 (Fig. 4). Nuclear localization could be observed despite the non-functional NLSs.
Screening for TWIST interacting partners using yeast two hybrid assay
TWIST interacting proteins were identified by yeast two hybrid experiments.
Gene Bank Acc.No.
mRNA Splicing factor 2
TCF4/SEF2-1A Transcription factor
pre mRNA splicing factor 2
pre mRNA splicing factor 2
Confirmation of the TCF-4 and TWIST interaction by NLS rescue assay
As discussed above, TWIST is a nuclear protein that forms dimers with other HLH or bHLH protein, and mutations in TWIST NLSs result in nuclear mislocalization. Since we hypothesized that the co-transfection of TCF-4 with NLS deficient TWIST will restore protein localization to the nucleus, we performed additional NLS rescue assays, using c-myc TWIST expression constructs and co transfected with a TCF4-GFP construct. The empty GFP was distributed throughout the cell (data not shown), whereas TCF4-GFP expression was predominantly localized to the nucleus of cells (Fig. 5B). In this assay, TWISTNLS1 mutant which was unable to be transported in the nucleus was cotransfected with TCF-4 to check whether the lack of a functional NLS could be compensated by heterodimerization and subsequent co-import of the two proteins. Co-localization of both proteins in the nucleus was detected in the cells co-transfected with both TWISTNLS1 mutant and TCF-4 constructs (Fig. 5B). The same co-expression results were observed using K73R and K77R with TCF-4 (Fig. 5B).
In this study, we identified two putative NLSs at the N-terminal region of TWIST and found that these NLSs mediate the expression of TWIST in the nucleus. An evolutionary alignment of Twist proteins amino acid sequences encoded from different species showed five different highly conserved regions NLS1, NLS2, NSEEE at N-terminal, WR at C-terminal and bHLH DNA binding domain. The HLH region is necessary and sufficient for protein dimerization . Besides having two putative nuclear localization signals, the N-terminal of M-Twist which is an analogue of H-TWIST can also bind to p300/pCAF and decrease its histone acetyl-transferase activity . Moreover, the C-terminus of Twist possesses a WR domain which is a RunX2-binding "Twist Box" and inhibit the RunX2 function during skeletal development .
Nuclear localization signals in TWIST
The regulation of protein transport into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) recognized by protein import receptors . The NLS sequence consists of a cluster of basic residues, either monopartite or bipartite [27, 28]. This sequence is subsequently recognized by the heterodimeric import receptor complex comprising importin α and importin β . Importin α is an adapter protein that consists of a small N-terminal β-binding domain. Importin β does not directly interact with the NLS cargo but acts to direct importin α to the nuclear pore . The nucleocytoplasmic trafficking of large molecules is normally mediated by classical NLSs  and necessary for normal physiology and cellular trafficking. It might be that additional NLS sites exist in other domains of the proteins. Here, we show that N-terminal of H-TWIST contain 2 NLS motifs which are required for nuclear transport of this protein.
We mutated both of the predicted NLS lysine residues to determine their potential role in the importation of TWIST protein. The NLS1 mutant constructs were observed in the cytoplasm indicating that TWISTNLS1 is a functional NLS, which governs the nuclear import of TWIST. Previously, similar results were made for murine Dermo-1, a bHLH protein encoded by the TWIST2 gene, in which engineered replacement of the equivalent NLS (also containing the RKRR sequence) by four alanine residues impaired nuclear import . It is of particular interest that a novel mutation in the NLS1 of TWIST protein reported in SCS patients at C115G, resulting in an Arg39Gly mutation was not restricted to the nucleus, but found expressed in both nucleus and cytoplasm in COS-7 cells . These results are entirely consistent with our findings here, identifying this region as a core NLS motif.
We also examined the functional role of NLS2 by mutagenesis and found that NLS2 likewise NLS1, also played an essential role in the nuclear import of TWIST protein. Although in this study we focused on the lysine residues within the NLS1 and 2 motifs, it is also possible that other residues within these domains may also contribute to nuclear localization. From our findings, it is concluded that sequences at the 38, 73, and 77 amino acid positions within the NLS domains are critical residues. In contrast, the 76 amino acid does not appear to be critical for nuclear import.
Heterodimerization of TCF-4 with TWIST and co-localization in the mammalian cells
H-TWIST belongs to a class of bHLH protein known to form stable heterodimers with members of class a bHLH transcription factors [2, 32]. Interestingly, Twist and MyoD interactions may prevent the specific activation of target genes for osteogenic differentiation, which has been proposed to result in the molecular pathogenesis of the Saethre-Chotzen syndrome . E12 also known as TCF-3, a bHLH protein, has been reported to interact with H-TWIST and this heterodimerization could compensate the nucleus localization of TWIST having mutations in bHLH motif in a co-transfected Cos7 cells . We utilized a similar approach in the case of mutated TWIST-NLSs to confirm the expected nuclear location of the wild-type TWIST protein and the TWIST-NLS mutants with E12 and reported that E12 interaction with TWIST compensates the non-functional NLSs.
In yeast-two-hybrid screening, we identified several novel proteins interacting with TWIST including a gene encoding the class I bHLH protein TCF-4 (transcription factor 4, also known as ITF-2; and SEF-2). The TCF-4 gene is conserved in many species, its expression found in adult heart, placenta, skeletal muscle, lung where TWIST is also highly expressed [33–36]. TCF4 is a bHLH transcription factor and contains two alternatively spliced forms of mouse TCF4 (ITF-2), termed ITF-2A and ITF-2B, that differentially regulate MyoD activation . An alignment of the N-terminal 83 amino acids with the N-termini of full-length E12 and full-length E47 and ITF-2B revealed that this domain is 51% identical among the three E type proteins and HLH domain was also highly conserved in all three proteins . The high expression levels of TCF4 in the brain suggests that it may regulate the activity of some neuron and neuro-endocrinology specific promoters, including the tyrosine hydroxylase enhancer and the somatostatin receptor II promoter .
In this study, we confirmed the heterodimerization of TCF-4 with TWIST in NLS rescue assays and in vitro binding assay. Since the NLS mutant TWIST is unable to localize to the nucleus, this study illustrates that TWIST can form a functional complex together with overexpressed TCF-4, similar to that observed with E12, and that these interactions can restore nuclear localization despite the mutation in NLSs.
It has already been reported that protein transcription factors may possess more than one NLS ; presumably a multitude of NLS may increase the rate of protein factor-transporter protein interactions . Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. These two NLSs may also operate in a cooperative manner for more efficient nuclear translocation. We also conclude that the mislocalization of NLS mutated construct could be restored with other bHLH proteins through heterodimerization with H-TWIST.
TWIST open reading frame (ORF) was amplified by PCR from human genomic DNA using oligonucleotides CGCGGATCCGCGATGATGCAGGACGTGTCC and CCGGAATTCCGGCTAGTGGGACGCGGACAT, containing BamHI and EcoRI sites respectively. After BamHI and EcoRI digestion, it was ligated into a pCMV mammalian expression vector, which contains a c-myc epitope. The E12-ORF was amplified by the PCR using oligonucleotides CCGGAATTCCGGATGGCGCCTGTGGGCACA and CGCGGATCCGCATGTGCCCGGCGGGGTTGT respectively. This was subcloned in Topo TA cloning vector. After EcoRI restriction from Topo vector the E12 ORF subcloned in eGFP-N1 vector in the same GFP reading frame. β-galactosidase open reading frame was digested with BamHI and XhoI from pcDNA4/myc-His/lacZvector and subcloned in pCMV-cmyc mammalian expression vector. For yeast two hybrid assay the TWIST-ORF was amplified from the human genomic DNA using primers CCGGAATTCCGGCTATGATGCAGGACGTGTCC and CGCGGATCCGCGCGTGGGACGCGGACATG 3' containing EcoRI and BamHI sites respectively. After EcoRI and BamHI digestion, it was ligated into a pGKT7 yeast bait vector, which contains a GAL4 DNA binding domain. For the NLS rescue assay, TCF4-ORF was amplified using oligonucleotides CCGGAATTCCGGATGCATCACCAA and CGCGGATCCGCGCATCTGTCCCAT, containing EcoRI and BamHI restriction sites, respectively. After digestion with EcoRI and BamHI, TCF4-ORF was ligated into mammalian expression eGFP-N1 vector. For immunoprecipitation experiments, TWIST-cDNA was subcloned into V5-HIS tag vector (Invitrogen) using clonase, and TCF4 ORF clone was subcloned into the pcTAP mammalian expression vector (Stratagene). All constructs were fully sequenced.
Oligonucleotides used for mutagenesis reactions to alter NLSs sequences in TWIST.
Amino acid substituted in NLSs
Cell culture and transfection assay
Cell chamber slides were seeded with 1.5 × 105 U2-OS (Human osteosarcoma, ATCC) cells, and grown over night in DMEM (Gibco BRL) supplemented with 10% FBS containing 100 μg/ml penicillin and streptomycin. To select a cell line several factors were considered including growth conditions, proliferation ability and ease of transfection. Considering all factors together, several cell lines were used here including OHS, CCL-136, COS7 and U2-OS. Finally, we selected U2-OS cells for further studies. The cells were transiently transfected with 500 ng of plasmid by using Effectene (QIAGEN) according to the manufacturer's instructions.
Culture chamber slides were fixed with 4% paraformaldehyde for 20 min at 25°C, and permeabilized with 0.2% Triton X-100 for 5 min. Subsequently, these slides were incubated for 1 h at 37°C with the mouse monoclonal anti-c-myc primary antibody (Molecular Probes, 1:200 dilution). Slides were further incubated in secondary antibody (Texas Red conjugated goat anti-mouse IgG; Molecular probes, 1:1000 dilution) for 1 h at 37°C. The slides were washed and incubated with phalloidin for 20 min at 25°C and incubated with DAPI at 25°C for 3 min and then mounted by using Vector Shield (Vector Laboratories, CA, USA).
Protein extraction and Western immunoblot analysis
To confirm the expression of TWIST and TWIST NLSs in transfected U2-OS cells for immunoblot analysis, cytoplasmic fractions were isolated from these transfected cells by scraping after 24 h of incubation and then centrifuged for 5 min. The cells were lysed with NP40-containing lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.5% NP-40) to disrupt the cell membrane and then the cell lysate was centrifuged at 500 × g for 5 min at 4°C. The supernatant (cytoplasmic fraction) was removed and the pellet (nuclear fraction) was resuspended in NP-40 containing cell lysis buffer. Proteins were denatured by boiling in sample buffer, separated on 12% SDS-PAGE and then transferred onto the PVDF membrane (Immobilon TM-PSQ, Millipore) and blocked overnight in 5% non-fat powdered milk in TBST (10 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1% (v/v) Tween 20). Mouse monoclonal anti c-myc antibody (1:1000 diluted in TBST) (Molecular probe) used for protein detection. Peroxidase conjugated goat anti-mouse IgG (1:10,000 diluted in TBST) (Sigma, Missouri, USA) was used as a secondary antibody.
The full-length TWIST was used as a bait protein. After transformation of pGBKT7-TWIST into yeast strain AH109, expression of the fusion protein GAL4-BD-TWIST was confirmed by PCR (as described Clonetech Manual). Yeast strainY187 with pretransformed human placental cDNA library was mated with AH109 strain containing pGBKT7-TWIST, and the cells were then plated onto SD/-Ade/-His/-Trp/-Leu plates to screen the expression of ADE2 and His3 genes (Fig 5A). Yeast colonies expressing both of the two genes were subjected colony-lift filter assays to check the LacZ gene expression. Consequently, we isolated 24 positive colonies with β-galactosidase activity following the procedure previously described  and sequenced the plasmids.
Pulldown assays with IgG agarose beads
Mammalian expressed, affinity tagged proteins were purified from transfected cells using IgG agarose beads. Fifty microlitre of a 50% (v/v) agarose beads slurry containing 10–50 μg of purified V5-His-tagged protein were incubated with 100 μg of purified CBP fused protein in a total volume of 200 μl TBS containing 0.1% (v/v) BSA. Following a 2 h incubation with continuous rotation at 4°C, the agarose beads were washed 5 × 10 min at 4°C with the same buffer and 2 × 5 min in TBS without BSA. Agarose beads were mixed with an equal volume of 2× loading buffer and bound proteins were eluted by incubation for 1 h at 4°C and were subsequently separated by SDS-PAGE gels.
We are grateful to Dr. Jürgen Kunz in whose laboratory some of this research was performed (Philipps-University Marburg, Germany). This work was supported by a grant from the International Graduate School "GRK767" funded by the German Research Foundation and the Heart and Stroke Foundation of Ontario (NA-5884 and T-6281). AOG is a New Investigator of the Heart and Stroke Foundation of Canada and Canada Research Chair.
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