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Human Molecular Genetics Advance Access originally published online on February 9, 2005
Human Molecular Genetics 2005 14(7):885-892; doi:10.1093/hmg/ddi081
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oupjournals.org

Identification of a novel nuclear localization signal in Tbx1 that is deleted in DiGeorge syndrome patients harboring the 1223delC mutation

Jason Z. Stoller1,2 and Jonathan A. Epstein2,*

1Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA and 2Cardiovascular Division, Department of Medicine, University of Pennsylvania Health System, Philadelphia, PA 19104, USA

* To whom correspondence should be addressed at: 954 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 2158988731; Fax: +1 2155739306; Email: epsteinj{at}mail.med.upenn.edu

Received December 14, 2004; Accepted February 1, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DiGeorge syndrome (DGS) is the most common human chromosomal deletion syndrome and is frequently associated with deletions on chromosome 22q11. Approximately 17% of patients with the phenotypic features of this syndrome have no detectable genomic deletion. Animal studies using mouse models have implicated Tbx1 as a critical gene within the commonly deleted region, and several mutations in TBX1 have been identified recently in non-deleted patients, including missense and frameshift mutations. The mechanisms by which these mutations cause disease have remained unclear. We have identified a previously unrecognized and novel nuclear localization signal (NLS) at the C-terminus of Tbx1 that is deleted by the 1223delC mutation, thus explaining the mechanism of disease in these patients. This NLS is conserved across species, among a subfamily of T-box proteins including Brachyury and Tbx10, and among additional nuclear proteins. By providing functional data to indicate loss-of-function produced by the 1223delC TBX1 mutation, our results provide strong support for the conclusion that TBX1 mutations can cause DGS in humans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DiGeorge syndrome (DGS) comprises phenotypic features such as cardiac outflow tract anomalies, craniofacial dysmorphia, velopharyngeal insufficiency, cleft palate, aplasia or hypoplasia of the thymus and parathyroid glands and neuropsychiatric abnormalities. Cardiac defects, often severe, are present in 75% of patients and significantly contribute to morbidity (1Go–3Go). Most patients with this syndrome have a large (>3 Mb) genomic deletion of chromosome 22q11, including the DiGeorge critical region (DGCR), that is deleted in 90% of DGS patients in whom a deletion is detectable (4Go). Importantly, a significant number of DGS patients (~17%) have no demonstrable chromosomal deletion (4Go) and presumably harbor discrete mutations in a single gene or genes.

Significant investigative activity has focused on the identification of the functionally significant genes located within the DGCR that cause human developmental defects. The genes located within the DGCR on 22q11 are generally conserved in the mouse on chromosome 16 (5Go,6Go). Heterozygous deletion of a 1.5 Mb homologous region of mouse chromosome 16 phenocopies important aspects of DGS, including cardiac, thymus and parathyroid defects (7Go–9Go). Complementation studies and single gene mutational approaches identified Tbx1 as a gene within the murine DGCR that is required for normal pharyngeal and cardiac development. Tbx1 is expressed in early embryonic pharyngeal endoderm, the somite, and in the outflow tract myocardium of the heart, and both mouse and zebrafish models confirm a critical role for Tbx1 in the development of organs derived from these structures (10Go,11Go). Heterozygous mutations in Tbx1 in mice result in outflow tract cardiovascular disease similar to that seen in DGS (7Go,9Go,10Go), though mutation of other genes within the DGCR can also cause abnormalities reminiscent of DGS (12Go–15Go) and could function as genetic modifiers or as components of a multi-gene syndrome.

Tbx1 is a member of the group of transcription factors characterized by a conserved DNA binding domain called a T-box. At least 18 mammalian family members have been identified and several have been associated with human disease. Haploinsufficiency of TBX5 and TBX3 have been identified as the causes of Holt–Oram syndrome and ulnar–mammary syndrome, respectively (16Go,17Go). Mutations in TBX19 and TBX22 are associated with isolated adrenocorticotropic hormone deficiency (18Go) and with cleft palate with ankyloglossia (19Go), respectively. T-box genes, including TBX1, encode nuclear transcription factors that bind to DNA and regulate downstream genes (20Go). T-box family members have been divided into five major phylogenetic subfamilies based on overall sequence similarity and gene structure (21Go). The TBX1 subfamily includes TBX10, TBX15, TBX18, TBX20 and TBX22. The region of highest similarity among related T-box genes is generally restricted to the DNA binding domain, and conserved domains outside of this T-box have not been identified.

Recently, three families were described with TBX1 mutations and classic features of DGS without evidence of a chromosomal deletion at 22q11 (22Go). The specific TBX1 mutations included two missense mutations (F148Y and G310S) and a frameshift mutation (1223delC). However, the functional significance of these mutations was not investigated. If they are truly disease causing sequence alterations, then they should result in altered Tbx1 function. We sought to examine the functional significance of the known human TBX1 mutations and have identified a novel nuclear localization signal (NLS) deleted in an affected family.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional activation properties of Tbx1 mutants
To examine the functional significance of the known human mutations in TBX1, we engineered the corresponding mutations in the murine Tbx1 cDNA. Human and mouse Tbx1 are 87% identical at the amino acid level, and each of the known mutations occurs in conserved regions of the coding sequence. Tbx1 is thought to function by binding to DNA and activating transcription of target genes, though the optimal DNA binding sequence recognized by Tbx1 has not been reported. Therefore, we generated fusion proteins that included the GAL4 DNA binding domain fused to Tbx1, or mutant forms of Tbx1, and we tested the ability of these proteins to activate transcription in transfected HEK-293T cells of a luciferase reporter construct containing GAL4 DNA binding sequences and a minimal promoter. GAL4–Tbx1 was able to induce ~18-fold activation of luciferase expression compared with cells transfected with empty expression vector alone (Fig. 1). Missense mutations corresponding to the human F148Y and G310S alterations had no significant effect on transcriptional activation induced by these GAL4-fusion proteins. However, the ability of the mutated construct that corresponded to the human 1223delC mutation (subsequently referred to as Tbx1delC) to activate transcription of luciferase over baseline levels was completely abolished.



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  		Figure 1. Transcriptional activation of a luciferase reporter by wild-type and mutant Tbx1. GAL4–Tbx1 is able to activate transcription, whereas GAL4–Tbx1delC is not. Missense mutations, GAL4–F137Y and GAL4–G299S, do not disrupt transcriptional activation activity. Results are normalized for transfection efficiency and expressed as mean±SEM of nine independent experiments.

 
Nuclear localization of Tbx1 mutants
The inability of the Tbx1delC mutant to activate transcription from an appropriate reporter construct in transfected cells, even when fused to a known DNA binding domain (GAL4), suggests that the frameshift mutation has resulted in the disruption of a transactivation domain or that the mutant protein is unstable, misfolded or does not translocate to the nucleus. We examined the subcellular localization of the transfected GAL4 fusion proteins and discovered that GAL4–Tbx1delC is located in the cytoplasm, whereas the other GAL4–Tbx1 fusion proteins are located in the nucleus (Fig. 8 inset and data not shown). Hence, we examined the subcellular localization of wild-type and mutant Tbx1 proteins expressed without the GAL4 domain using anti-Tbx1 antibodies to detect transfected protein. Transfected COS7 cells (Fig. 2) and HEK-293T cells (data not shown) were counterstained with DAPI and phalloidin to illustrate nuclear and cytoplasmic domains, respectively. As expected, Tbx1 localized exclusively to the nucleus (Fig. 2A–D). Mutant proteins F137Y and G299S (corresponding to human F148Y and G310S mutations, respectively) also localized to the nucleus (Fig. 2E–L). In contrast, Tbx1delC mutant protein localized predominantly to the cytoplasm (Fig. 2M–P), suggesting that an NLS is normally located at the C-terminus of Tbx1 and has been deleted by this mutation.



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  		Figure 8. Addition of a heterologous NLS to Tbx1delC does not rescue transcriptional activation. GAL4–Tbx1 is able to activate transcription, whereas GAL4–Tbx1delC is not, and NLS–GAL4–Tbx1delC is unable to rescue the ability to activate transcription. Results are normalized for transfection efficiency and expressed as mean±SEM of nine independent experiments. GAL4 fusion proteins were expressed in HEK-293T cells and analyzed by immunocytochemistry with an anti-Tbx1 antibody. Cells were co-stained with DAPI and phalloidin to illustrate nuclei and cytoplasm, respectively. Similar to GAL4–Tbx1, NLS–GAL4–Tbx1delC localizes to the nucleus in contrast to the cytoplasmic expression pattern of GAL4–Tbx1delC (inset).

 


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  		Figure 2. Subcellular localization of wild-type and mutant Tbx1 proteins. Tbx1 proteins expressed in COS7 cells were examined by immunocytochemistry with an anti-Tbx1 antibody. Cells were co-stained with DAPI and phalloidin to illustrate nuclei and cytoplasm, respectively. Wild-type Tbx1 and missense mutants F137Y and G299S localize to the nucleus (A–L). The frameshift mutant Tbx1delC localizes predominantly to the cytoplasm (M–P). Deletion of 31 C-terminal residues (1–448) does not affect nuclear localization, whereas truncation of 86 residues (1–393) removes an NLS and the truncated protein is localized in the cytoplasm (Q–X).

 
The 1223delC mutation in Tbx1 results in a frameshift followed by 51 nonsense codons and a stop codon. We directly tested the effect of C-terminal truncations, in the absence of any additional residues encoded by nonsense codons, by deleting either 31 or 86 C-terminal residues. Deletion of 86 residues corresponds to the number of normally encoded residues missing from Tbx1delC. Deletion of the 31 residues following the Tbx1delC truncation resulted in a truncated protein (1–448) that correctly localized to the nucleus (Fig. 2Q–T). However, deletion of 86 residues resulted in a mutant protein (1–393) that was located predominantly in the cytoplasm (Fig. 2U–X).

These results are consistent with the presence of an NLS in the C-terminus of Tbx1, located between residues 394 and 448. We tested this hypothesis directly by expressing a V5 epitope-tagged peptide that includes these residues (394–479) in COS7 cells and comparing the subcellular localization with that of V5-tagged full length Tbx1. As predicted, the 394–479 fragment was sufficient to direct nuclear localization resulting in a subcellular distribution identical to that of Tbx1 (Fig. 3).



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  		Figure 3. C-terminal fragment of Tbx1 is sufficient to drive nuclear localization. Epitope tagged proteins expressed in COS7 cells were examined by immunocytochemistry with an anti-V5 antibody. Cells were co-stained as in Fig. 2. V5-tagged Tbx1 localizes to the nucleus (A–D). A V5-tagged C-terminal truncated protein (1–393) localizes predominantly to the cytoplasm (E–H). An 86 amino acid C-terminal V5-tagged peptide (394–479) localizes to the nucleus (I–L).

 
A 12 residue motif contained within the C-terminus of Tbx1 is an NLS
To further map the precise location of the NLS within this 55 amino acid C-terminal Tbx1 fragment, we created a series of further deletions. However, because small proteins (<40 kDa) are able to passively enter the nucleus (23Go), we expressed the small Tbx1-derived peptides as fusion proteins with GFP and ß-galactosidase. As a positive control, we utilized the SV40 NLS cloned between GFP and lacZ, and the negative control lacked any exogenous NLS, resulting in cytoplasmic localization (Fig. 4B). Experimental constructs included a series of Tbx1-derived fragments cloned between GFP and lacZ (Fig. 4A). The encoded fusion proteins have a minimum predicted molecular weight of 151 kDa and thus require active transport if they are to enter the nucleus. As predicted by our initial deletion analysis (Fig. 2), fragment 394–448 was sufficient to act as an autonomous NLS (Fig. 4A). Although the fusion proteins containing fragments 394–421 and 422–448 did not localize to the nucleus, the 409–434 peptide was sufficient to drive nuclear localization (Fig. 4A). Further deletion analysis indicates that the 12 amino acid peptide corresponding to fragment 415–426 contains a potent Tbx1 NLS and deletion of three amino acids from the N-terminal end (418–426) destroys NLS activity (Fig. 4).



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  		Figure 4. The Tbx1 NLS maps to a 12 amino acid motif containing a critical arginine residue. (A) Schematic representation of murine Tbx1 and Tbx1delC with the T-box DNA binding domain (blue), the nonsense domain (red) and the frameshift altered C-terminal domain (gold) indicated. A series of peptides from this C-terminal fragment was used to map the Tbx1 NLS. (B) COS7 cells were transiently transfected with expression vectors for the indicated GFP/ß-gal fusion proteins. Cells were fixed, stained with DAPI and visualized by conventional fluorescence microscopy. GFP/ß-gal fusion protein is localized to the nuclear compartment in positive control transfections and to the cytoplasmic compartment in negative control transfections. Fusion protein including fragment 415–426 is restricted to the nucleus but fragment 418–426 is excluded from the nucleus of transfected COS7 cells. Mutation of arginine 417 to alanine in fragment 415–429 results in cytoplasmic localization of GFP/ß-gal fusion protein.

 
Examination of the amino acid sequence of the 12 residue Tbx1 NLS (Fig. 5) that is sufficient to direct nuclear localization reveals no significant homology to previously identified NLS sequences or to other known protein motifs (see, e.g. http://cubic.bioc.columbia.edu/db/NLSdb/ and http://us.expasy.org/prosite/). However, this 12 amino acid region is entirely conserved between human and murine Tbx1 sequences, and a high degree of conservation is retained between human Tbx1 and species as widely divergent as Xenopus laevis, Danio rerio and Branchiostoma floridae (Fig. 5). Interestingly, similar sequences are also found in the closely related T-box proteins, Tbx10 and Brachyury (Fig. 5). In particular, a central proline-rich region containing a core PYPXP motif is common among these sequences, flanked by basic amino acids (shown in red, Fig. 5). The arginine (R), proline (P) and tyrosine (Y) at positions 417, 420 and 421 are conserved among all of these proteins. Therefore, we tested the importance of these residues for NLS function by mutating them each individually to alanine. These three mutations individually destroyed the ability of the Tbx1 NLS to direct nuclear localization (Fig. 4B and data not shown), emphasizing the critical importance of these residues. We then mutated R417 and Y421 to alanine individually and in combination in the setting of wild-type Tbx1. Although the R417A mutation alone did not dramatically affect nuclear localization of Tbx1, the combination of the R417A and Y421A mutations resulted in markedly reduced nuclear localization of full-length protein (Fig. 6). Interestingly, single mutations of the conserved proline residues at positions 422 and 424 had no effect on the ability of this peptide to drive nuclear localization of the GFP/ß-gal fusion protein (data not shown).



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  		Figure 5. Alignment of NLS sequences. Amino acid sequence alignment of the NLS sequences from the indicated T-box proteins is shown. Basic residues are highlighted in red. A shared hydrophilic residue occurs at position 425 (asterisks). Residues with high degree of homology are shaded in gray.

 


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  		Figure 6. Mutation of two critical residues in the Tbx1 NLS abolishes the ability to drive nuclear localization. Tbx1 proteins expressed in COS7 cells were examined by immunocytochemistry with an anti-Tbx1 antibody. Cells were co-stained as in Fig. 2. Full length Tbx1 localizes to the nucleus (A–D), but mutation of R417A and Y421A results in a markedly decreased ability of the protein to enter the nucleus (E–H).

 
Related sequences in Xenopus, Danio and Branchiostoma Tbx1, human and mouse Tbx10 and human and mouse Brachyury are each capable of functioning as NLSs when expressed as fusion proteins with GFP and ß-galactosidase (Fig. 7). These signals all share a hydrophilic residue at position 425 and a core conserved motif consisting of RXXPYPXP. This motif is not restricted to T-box proteins. We searched the GenBank database for identical and related motifs and identified numerous nuclear proteins that harbored related potential NLS sequences. Functional examination of several of these, such as the PSRLAPYPHPATTRG motif in human ATF5, confirmed their ability to function as NLS sequences when expressed as fusion proteins with GFP and ß-galactosidase (data not shown).



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  		Figure 7. Homologous domains in Tbx proteins function as NLSs. COS7 cells were transiently transfected with expression vectors for the GFP/ß-gal fusion proteins corresponding to peptides in Fig. 5. Cells were fixed, stained with DAPI and GFP/DAPI was visualized by conventional fluorescence microscopy. GFP/peptide/ß-gal fusion proteins (green) are localized to the nuclear compartment in all transfections (C–P) in a pattern identical to the human/mouse Tbx1 NLS (A, B). Nuclei are visualized by DAPI staining in blue.

 
The Tbx1delC frameshift results in disruption of a transactivation domain
Although altered translocation of a transcription factor to the nucleus is sufficient to explain a decreased ability to activate transcription, we tested the possibility that the Tbx1delC mutation also disrupted a C-terminal transactivation domain. Addition of the SV40 NLS to the GAL4–Tbx1delC fusion resulted in forced nuclear localization (Fig. 8 inset). We tested the ability of this protein to activate transcription of a luciferase reporter vector in HEK-293T cells. Although GAL4–Tbx1 was able to activate ~17-fold in this experiment, the addition of the heterologous NLS (NLS–GAL4–Tbx1delC) was unable to rescue transcriptional activation ability over baseline levels or the levels of Tbx1delC activation (Fig. 8). This suggests the presence of a transactivation domain between amino acids 394 and 479 of murine Tbx1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Transcription factors and other nuclear proteins are synthesized and undergo posttranslational modifications in the extranuclear compartment. Transport of these nuclear proteins from the cytoplasm to the nucleus is a potential site of functional regulation. Proteins cross the nuclear membrane via discrete multimeric protein channels, the nuclear pore complexes (NPCs). Although small proteins can pass through these pores passively, this mode of translocation is inefficient and unregulated. Larger proteins contain NLSs that mediate active transport across the nuclear membrane. The importin family of carrier proteins recognizes these NLS motifs and facilitate active transport through the NPC. Overall, the nuclear protein NLS motifs are remarkably similar and have been divided into two broad groups, monopartite and bipartite NLSs. The monopartite NLS contains a single cluster of basic amino acids and is typified by the SV40 NLS (24Go). The bipartite NLS, such as the prototypical nucleoplasmin NLS (25Go), is composed of two clusters of basic amino acids separated by a 10–12 amino acid linker. Although the secondary structures of the monopartite and bipartite NLSs are very different, both bind to the importin-{alpha} carrier protein (26Go).

Some nuclear proteins contain motifs apparently unrelated to classic monopartite or bipartite NLS that are both necessary and sufficient to drive nuclear localization (27Go–30Go). The NLS that we describe here is unusual in that it contains a central domain with multiple prolines that are not found in other previously described NLS sequences. Basic residues, especially the arginine at position 417, are functionally important, as they are in many NLS motifs. It remains to be determined whether the Tbx1 NLS binds to importin-{alpha} and whether it is modified (e.g. by phosphorylation) to regulate nuclear localization.

Functional analysis of Tbx1 protein encoded by a mutant allele in a family with DGS has allowed us to identify a novel NLS that is conserved across millions of years of evolution. Truncation of Tbx1 such that the NLS is deleted results in a mutant protein that is predominantly localized in the cytoplasm and is thus unable to mediate nuclear functions. Additionally, this truncation disrupts the transcriptional activation properties of Tbx1 even when the mutant protein is forced to localize to the nucleus by fusion with a heterologous SV40 NLS, indicating that a C-terminal transactivation domain is also disrupted. Frequently, NLS sequences have been identified within or adjacent to DNA binding domains in transcription factors suggesting that these functional domains have been clustered during evolution to provide functional modules for the execution of nuclear activities. Our identification of an NLS located within or near a transcriptional activation domain of Tbx1 is consistent with a similar clustering of functionally related domains.

Although NLS domains have been described in other T-box family members, including Tbx3, Tbx6 and VegT (31Go–33Go), none of these is conserved in Tbx1. One of the two described Tbx5 NLSs (KRKEEECSTTDHPYKK) contained in the C-terminus adjacent to a transcriptional activation domain (34Go) is not conserved in Tbx1. However, a second Tbx5 NLS motif (KAGRRMFPSYKVK) contained within the T-box is partially conserved in Tbx1. Our results indicate that this putative NLS is not sufficient to drive nuclear localization of Tbx1 since the Tbx1delC mutant, including the intact T-box, is predominantly cytoplasmic (Fig. 2U–X). The NLS in T (Brachyury) shown in Fig. 5, which corresponds to the Tbx1 NLS we describe in this paper, partially overlaps with a previously described large domain of T containing several undefined NLSs (35Go). Prior phylogenetic analyses suggest that the TBX1 subfamily is more closely related to the TBX2/3/4/5 and TBX6 subfamilies than to the T subfamily (21Go). The conservation of the Tbx1 NLS among TBX1 and T subfamilies but not among the TBX2/3/4/5 and TBX6 subfamilies suggest that there may be a closer evolutionary relationship between these two subfamilies than has been appreciated.

Animal models have suggested that TBX1 is likely to be responsible for many of the congenital abnormalities seen in DGS. Nevertheless, initial studies of non-deleted DGS patients failed to identify isolated mutations in TBX1 (9Go,36Go–38Go). This left uncertainty regarding a causal relationship between haploinsufficiency of Tbx1 and human DGS. The discovery of affected non-deleted patients harboring TBX1 mutations (22Go) provided promising evidence but it remained unclear if these mutations were polymorphisms closely linked with another gene mutation or if they truly caused functional alterations in Tbx1 that could plausibly account for disease. Our analysis shows that the functional effects of the 1223delC mutation eliminate important nuclear capabilities of Tbx1 and result in functional haploinsufficiency. This provides strong support for a direct causal relationship linking the 1223delC mutation with disease in non-deleted DGS family members.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression constructs
Wild-type Tbx1 expression vector 3.1TOPO-Tbx1 was constructed by PCR amplification of Tbx1 cDNA and cloning with pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen). To generate the mutant expression vectors 3.1TOPO-F137Y, 3.1TOPO-G299S, 3.1TOPO-1178delC, 3.1TOPO-R417A and 3.1TOPO-R417A/Y421A using the Quickchange Site-Directed Mutagenesis Kit (Stratagene), 3.1TOPO-Tbx1 was used as template. The 3.1TOPO-Tbx1 R417A expression vector was generated as described earlier with the following modifications due to the high GC content of the primers: (1) addition of DMSO (6% final concentration); (2) the mutant strand synthesis step was performed separately for each primer, then combined, boiled and slowly annealed. Deletion mutants, 1–393, 1–448 and 394–479, were generated by PCR and TA cloned using the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen). To generate the GAL4 fusion constructs, Tbx1 cDNA was PCR amplified using primers incorporating EcoRI and NsiI ends and cloned into the EcoRI/PstI sites of pM (Clontech). The expression vector pHM830 is a mammalian expression vector encoding a GFP/ß-gal fusion protein separated by a multiple cloning site (MCS) (39Go). pHM840 is a positive control vector with the SV40 NLS cloned into the MCS. Tbx1 cDNA fragments were PCR amplified using primers incorporating SacII and XbaI ends and cloned into the SacII/XbaI sites of pHM830 to generate in-frame fusions. Alternatively, to generate truncation constructs for fragments <75 bp, complementary oligonucleotides encoding the desired peptides flanked by SacII and XbaI restriction sites were generated and cloned directly after annealing and restriction digestion. Coding regions of all vectors were confirmed by direct sequencing. Primer sequences are available by request. All coding sequence corresponds to the following GenBank accession nos: mouse Tbx1 AF349558, human TBX1C NM_080647, Xenopus Tbx1 AF526274, Danio Tbx1 NM_183339, Branchiostoma Tbx1/10 AF262562, human TBX10 NM_005995, mouse Tbx10 NM_001001320, human Brachyury (T) NM_003181, mouse Brachyury (T) NM_009309 and human ATF5 NP_036200.

Immunocytochemistry
COS7 or HEK-293T cells were cultured on glass coverslips to 50% confluency. An aliquot of 3 µg of plasmid DNA per 6 cm dish was transfected with FuGENE 6 transfection reagent (Roche). Following 24 h of incubation, cells were fixed with 4% paraformaldehyde for 5 min, permeabilized with 0.1% Triton X-100 in PBS for 2 min, blocked with 5% goat serum in PBST for 30 min, then probed with either anti-Tbx1 antibody (1 : 500; Zymed) or anti-V5 antibody (1 : 200; Invitrogen) and co-stained with DAPI (1 : 2000; Sigma) and fluorescein-labeled phalloidin (1 : 40; Molecular Probes). Secondary antibodies used were Alexa Fluor 594 goat anti-rabbit (1 : 50; Molecular Probes) and Alexa Fluor 568 goat anti-mouse (1 : 2000; Molecular Probes). Cells transfected with pHM vectors were fixed and permeabilized as described earlier and then stained with DAPI. Images were merged with Adobe Photoshop software.

Luciferase assays
HEK-293T cells were grown in six-well dishes to 30% confluency. The pM expression constructs as described earlier were co-transfected with the pG5luc and phRL-TK reporter vectors (Promega) using FuGENE 6 transfection reagent (Roche). Firefly and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's recommended protocol. All results are normalized for transfection efficiency and represent the average of nine independent experiments.


   ACKNOWLEDGEMENTS
 
We thank Thomas Stamminger and Etienne Weiss for the pHM vectors and the members of the Epstein lab for helpful discussions. This research was supported by the Pediatric Scientist Development Program (NICHD Grant Award K12-HD00850) to J.Z.S. and by NIH 1P01HL075215-01 to J.A.E.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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