In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1
© Silberschmidt et al; licensee BioMed Central Ltd. 2011
Received: 18 November 2010
Accepted: 23 February 2011
Published: 23 February 2011
The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation.
Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1.
The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.
Transcription factors (TFs) bind to DNA and regulate mRNA synthesis in response to different stimuli via multiple protein domains endowing separate functions such as the binding to small ligands, the recognition of specific DNA sequences and the ability to activate or repress transcription. For the latter function it is frequently observed that more than one activation or repression domain can be present in a single TF . In addition, it is well known that transcription factors can be regulated by post-translational modifications, chiefly phosphorylation. However, the function of the diverse activation domains included in a single transcription factor or the role of post-translational modification has been assessed largely in cultured cells. This approach has obvious limitations since many TFs play important roles in diverse cell types and at different stages of development. Thus, whether the diverse functions of a TF could be assigned to separate protein domains or to post-translational modifications is a question that has been rarely addressed and it requires to carry out structure-function relationships studies in whole organisms expressing modified transcription factors lacking only one of its domains and/or mutated in its phosphorylation sites in order to block such post-translational modification. The homeodomain containing transcription factor Nkx2-1 (also called TTF-1, Nkx2-1 or T/EBP) is well suited for this type of studies since it plays important roles in organogenesis and differentiation of several organs such as lung, brain, thyroid and pituitary. In addition, it has been suggested that Nkx2-1 plays diverse roles at different stages in thyroid organogenesis, implying that it may change function, and, hence, target genes, during this process. In keeping with this notion, thyroid specific ablation of the gene encoding Nkx2-1, carried out late in organogenesis, results in altered follicular organization of the thyroid gland, while knock-out of the same gene results in complete absence of the gland.
Nkx2-1 contains two well defined transcription activation domain that appear to be redundant for function in co-transfection assays. Furthermore, Nkx2-1 is post-translationally modified by phosphorylation but both in DNA binding or in co-transfection assays no role could be assigned to this modification. However, that phosphorylation might be important for Nkx2-1 transcriptional activity has been suggested in studies indicating that ERK-mediated phosphorylation of this transcription factor might play a role in Ras-induced loss of its transcriptional activity in an in vitro model of thyroid tumoral transformation. Furthermore, studies in transgenic mice have demonstrated that mice homozygous for a Nkx2-1 allele encoding a phosphorylation-resistent protein are defective for lung cell differentiation, but no data are available on the role of Nkx2-1 phosphorylation in other organs. In this study we tested whether the two activation domains of Nkx2-1 have separate functions in vivo and demonstrate that these domains plays differential roles in thyroid and pitutary. Furthermore, we demonstrate that a Nkx2-1 mutant, encoding a phosphorylation defective protein, brings about critical defects in organization of thyroid follicles, without affecting earlier stages of development that are known to be dependent on the presence of the Nkx2-1 protein.
Taken together these data show that at least some of the multiple functions of Nkx2-1 are endowed into separate domains of the protein and that other functions depend on specific protein modifications. We believe that these data supporting evidence for mechanisms capable of generating novel functions in evolution.
Generation of knock-in mice expressing Nkx2-1 mutants
The expression of ΔNH2 or ΔCOOH mutant proteins in thyroid tissue was evaluated in a mobility shift assay carried out using an oligonucleotide containing a high affinity Nkx2-1 binding site (oligonucleotide C, see "Materials and Methods"). Protein extracts from either +/ΔNH 2 or +/ΔCOOH heterozygous thyroids show two DNA binding activities consistent with the presence of both wild type (wt) and mutant proteins (Figure 1, panel D). The levels of expression of the Nkx2-1 mutants are comparable to those of wild type.
To verify that the rearranged PM allele was not producing any phosphorylated protein, western blot analysis was carried out on lung tissues dissected from E18 PM/PM embryos. Lambda phosphatase treatment of protein extract from wt lung results in an increase in the relative mobility of Nkx2-1 on SDS-PAGE (Figure 1, panel E, lane 1 vs. 2). The PM protein has a mobility identical of the phosphatase treated Nkx2-1 (Figure 1, panel E, lane 3 vs. 2) and shows no further increase in mobility after phosphatase treatment (Figure 1, panel E, lane 3 vs. 4). These data strongly suggest that PM Nkx2-1 is not phosphorylated in vivo, as already demonstrated in cell lines .
Both activation domains and phosphorylation in the Nkx2-1 protein are essential for life
Mice heterozygous for mutant alleles encoding either ΔNH2, ΔCOOH or PM are born and develop normally, without apparent abnormalities in growth or reproduction. On the contrary, no live pups were obtained from mice homozygous for any of the three mutant alleles. These mice die at birth, presumably of respiratory failure. Necropsy of both ΔNH 2 and ΔCOOH homozygous reveals severe lung abnormalities comparable with those described in Nkx2-1 null mice (data not shown); lungs do develop in PM/PM mice but they show severe functional anomalies, as already reported . These results indicate that both transactivation domains of Nkx2-1 as well as its phosphorylation are required for normal development and differentiation of lungs.
Previous studies also demonstrated that at least one copy of a functional Nkx2-1 allele is required for the organogenesis of both pituitary and thyroid. In order to address whether different domains of Nkx2-1 or its phosphorylation have specific roles in the development of these two structures, we decided to analyze in better detail the phenotype of both developing pituitary and thyroid in mutant embryos.
Pituitary organogenesis in wild type and mutant embryos
These data show that for complete pituitary development only activation domain 1 of Nkx2-1 is essential, while both activation domain 2 and phosphorylation are dispensable. No information was collected on the functional differentiation of the pituitary.
Thyroid development in wild type and mutant embryos
At E16.5 thyroid gland has reached its final localization, dorsal to the cricoid cartilage and ventral the trachea (Figure 3, panel F). At this stage, the thyroid is absent in both null, ΔNH 2 and ΔCOOH homozygous embryos (Figure 3, panels G, H and I respectively), while in PM homozygous embryos the thyroid is correctly localized but it is clearly smaller than the gland of age-matched wt embryos(Figure 3, Panels J). Thus, at variance from pituitary, both Nkx2-1 transactivation domains are necessary for thyroid development. The PM mutant does not seem to affect early morphogenesis of the gland but it does influence the size of the gland. Thus we decided to investigate in better detail the thyroid differentiation in these mutants.
Thyroid differentiation in PM/PMembryos
Nkx2-1 phosphorylation is required for folliculogenesis
At E18.5 Tg was well expressed in both wt (Figure 7A) and mutant (Figure 7E) thyroids. However, while in wt thyroids it was most often concentrated in lumina which were clearly defined by the dotty ZO-1 staining (Figure 7B), in mutant mice no lumina were defined by the presence of ZO-1 (Figure 7F) and Tg, although present in significant amount, was not accumulated in the few irregular empty spaces that were observed (Figure 7E). E-cadherin was similarly located on the plasma membrane of wt (Figure 7C) and PM/PM (Figure 7G) thyroids, but only in the former lumina were observed and E-cadherin was confined to the basolateral cell domain. Ksp-cadherin was expressed at much higher levels in wt thyroids (Figure 7D) where it appeared to be confined to the lateral cell domain of most cells.
Overall these data indicate that the organization of follicular structures with lumina is missing in PM/PM mice as indicated by the lack of areas delimited by ZO-1 staining in which thyroglobulin accumulates, like in control thyroids. Lack of follicles correlates with a dramatic reduction in the thyroid/kidney specific Ksp-cadherin. Only in wt thyroids, where follicles form, the E- and Ksp- cadherins segregate to the lateral thyroid epithelial cell domain.
Identification of genes influenced by phosphorylation
The morphology of thyroid gland in PM/PM mice indicates that the first steps of organogenesis (i.e. specification of thyroid anlage and migration) as well as the onset of functional differentiation of thyroid follicular cells is unaffected in these mutant mice, whereas events leading to the follicular organization of the gland are impaired. These data suggest a critical role for phosphorylated Nkx2-1 in this latter process. To identify genes whose expression is responsive to phosphorylation of Nkx2-1, RNAs from E18 PM/PM thyroids and their wild type littermates were compared using the Affymetrix mouse expression set U74Av2.
Downregulated genes in PM/PM versus wt thyroid at E18
Gene onthology category
advanced glycosylation end product-specific receptor
secretoglobin, family 1A, member 1 (uteroglobin)
steroid binding phospholipase A2 inhibitor activity
NMDA receptor-regulated gene 2
calcitonin/calcitonin-related polypeptide, alpha
regulation of transcription
elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2
fatty acid biosynthesis
S100 calcium binding protein A5
calcium ion binding
U7 snRNP-specific Sm-like protein LSM11
proteolysis and peptidolysis
acyl-Coenzyme A dehydrogenase family, member 8
putative proline racemase
napsin A aspartic peptidase
proteolysis and peptidolysis
thyroid hormone generation
chloride intracellular channel 3
folate receptor 1 (adult)
folic acid metabolism
regulation of transcription
transmembrane protein 213
naked cuticle 1 homolog (Drosophila)
calcium ion binding
ras homolog gene family, member f
GTPase mediated signal transduction
PR domain containing 1, with ZNF domain
regulation of transcription
metal ion homeostasis
metal ion homeostasis
arginase type II
SLAIN motif family, member 1
zinc finger pt 28
regulation of transcription
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1
transcription regulator activity/chromatin modification
very low density lipoprotein receptor
RNA binding protein gene with multiple splicing
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2
Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 4
zinc finger protein 26
Upregulated genes in PM/PM versus wt thyroid at E18
Gene onthology category
lymphoid nuclear protein related to AF4-like
transmembrane channel-like gene family 5
a disintegrin and metalloprotease domain 8
forkhead box N2
regulation of transcription
solute carrier family 5 (sodium iodide symporter) member 5
Insulin-like growth factor 2 receptor
transport/growth factor binding
RNA (guanine-7-) methyltransferase
Syntrophin, basic 1
keratin complex 2, basic, gene 4
cytoskeleton organization and biogenesis
poliovirus receptor-related 3
glycosyltransferase 25 domain containing 1
zinc finger matrin type 5
dynein, axonemal, light intermediate polypeptide 1
actin binding LIM protein family, member 3
regulation of transcription/cytoskeleton organization
nuclear receptor subfamily 1, group I, member 3
regulation of transcription
small subunit (SSU) processome component
peptidyl-prolyl isomerase G (cyclophilin G)
sequence similarity 149, member B
tryptophan rich basic protein
guanylate binding protein 7
limb region 1
radial spoke head 9 homolog
cilium axoneme assembly
A kinase (PRKA) anchor protein 6
protein kinase A binding
Classification of the 73 genes whose expression changes in the PM/PM background did not show an enrichment with any specific Gene onthology category. However, the smalll number of genes affected by Nkx2-1 phosphorylation does indicate a specific and restricted pathway controlled by a specific post-translational modification.
We show in this paper that the pleiotropic functions of the transcription factor Nkx2-1 are not a property of the protein as a whole but can be, at least for some of the functions, assigned to specific protein domains or to post-translational modifications. In particular, we focused our attention to the effects of Nkx2-1 in the development of thyroid and pituitary gland using three mutants: a) ΔNH2, a mutant deleted of the transcriptional activation domain 1, located at the amino-terminus; b) ΔCOOH, a mutant deleted of the transcriptional activation domain 2, located at the carboxyl-terminus and c) PM, a mutant where serine residues shown to be target for phosphorylation have been mutated in alanines, thus abolishing phosphorylation of the protein. It should be stressed that all these mutants show similar DNA binding and transcriptional activation potential in co-transfection experiments[7, 8]. In contrast, we show in this study that each mutant shows a distinct phenotype, revealing a level of complexity that could not be predicted by experiments carried out in cultured cells and stressing the relevance of testing the functions, mostly of proteins with pleiotropic functions, in the context of the whole organism. Knock-out experiments have demonstrated that the absence of Nkx2-1 results in impaired thyroid, lung, brain and pituitary development. In the case of lung, thyroid and pituitary, absence of Nkx2-1 shows absence of the structures where Nkx2-1 is expressed, indicating an essential role of this protein in very early steps of organogenesis. While these experiments showed very clearly the important role of Nkx2-1 in the cell types where it is expressed, the very early disappearance of the cognate structures, in particular of lung, thyroid and pituitary, did not allow any further analysis on the function of the protein later in development. The data provided here demonstrate that one of the functional domain of Nkx2-1, indicated as activation domain 2, is dispensable for pituitary organogenesis. In contrast, both activation domains are required for thyroid development. This observation is relevant in two main aspects. The first is that the search for co-factors interacting with Nkx2-1 for pituitary development could be restricted only to one of the two activation domains. The second is concerned with the debate of whether mutations in either transcription factors or in regulatory region are involved in evolutions of organisms. One argument against a putative role of mutations in TFs in evolution is the pleiotropy shown by most, if not all, this type of regulatory molecules. However, we demonstrate here that the pleiotropic action of Nkx2-1 is due to the combination of diverse functions endowed in different domains of the protein. Hence, mutations in one of these domains would not result in changes in all of its activity, thus making easier to envisage their positive role in organismal evolution 
Along these same lines are the data reported here on the role of phosphorylation in NKx2-1 function in thyroid differentiation. We show that mice expressing the PM mutant do develop lungs, pituitary and thyroid gland, at variance from the Nkx2-1 knock-out mouse. The consequences of the PM mutation in lung functions have already been described. We did not address in this study the effect of this mutation in pituitary differentiation, or in specification of diencephalic neurons where Nkx2-1 plays important roles . In the thyroid, PM mutants show a near-normal expression of the genes involved in thyroid hormone biosynthesis. The most relevant phenotype consists in a radically altered follicular architecture, as demonstrated by the deranged localization of E- and Ksp-cadherin[15, 16] and of the tight junction marker Z0-1, even though Nkx2-1 protein is expressed at normal levels and it is properly located in the nuclei. Interestingly, these results are comparable with those obtained with conditional Nkx2-1 KO in thyroid, where also the main defect is the impaired folliculogenesis, suggesting that the dephosphorylated Nkx2-1 is a loss of function mutant. Thus, it can also be concluded from these data that the essential functions that Nkx2-1 plays early in development, as shown by the disappearance of thyroid cell precursors in the Nkx2-1 null mouse, do not require phosphorylation of the protein. In contrast, at later stages, when follicular cells reorganize themselves into follicles, Nkx2-1 is capable of supporting proper organization of thyroid follicular cells only if phosphorylated. Thus, it appears that Nkx2-1 phosphorylation triggers a switch in the function of this transcription factor at later stages in development. Such a notion is plausible since the the cell-cell and cell-extracellular matrix interactions, and hence the adhesiveness, must be different between the early and migrating thyroid cell precursor and the later, not moving and differentiated, thyroid follicular cells.
We show in this study that some of the pleiotropic function of the transcription factor Nkx2-1 can be mapped to distinct portions of the protein or to its phosphorylation. This study shows that many functions can be encoded in diverse portions of the same polypeptide chain and provide an example of how to increase the functional potential of a genome without increasing the gene number.
Animals were kept in an animal house under controlled conditions of temperature, humidity, and lighting and were supplied with standard food and water ad libitum. All animal experimentation respected regulations and guidelines of Italy and the European Union. All the experiments with mice described in this paper have been evaluated and approved (internal ID 0907) from the ethic committee "Comitato Etico per la Sperimentazione Animale" (CESA) of IRSG, Biogem. According to Italian law, the project was sent to the National Authorities on the 11th February 2007.
The colonies of mutant mice were maintained by crossing heterozygous mice with C57BL/6 wild type animals.
Generation of ΔNH 2 /ΔNH 2 and ΔCOOH/ΔCOOHmice
Mouse Nkx2-1 gene was isolated from a strain 129/SV mouse genomic library (Stratagene) using a probe corresponding to the 3′-untranslated region of rat Nkx2-1. To prepare the targeting vector, a fragment extending from bp 4656 to bp 10443 of the reported mouse genomic sequence (GenBank™ accession no. U19755), containing the entire coding sequence for Nkx2-1, was cloned in pBlueScript.
To prepare ΔNH2 targeting vector, a fragment spanning from the translation start site of Nkx2-1 (bp 7957) to the end of homeobox (bp 9480) was removed and replaced by the sequence encoding for the amino acid 159 to 372 of the reported rat Nkx2-1 sequence. The sequence 5'-CCAC-CAATG-3' was added to provide a ribosome entry site and an ATG codon for translation initiation.
To prepare ΔCOOH targeting vector, the fragment spanning from the translation start site of Nkx2-1 (bp 7957) to the end of homeobox (bp 9480) was replaced by the sequence encoding for the amino acid 1 to 221 of the reported rat Nkx2-1 predicted protein sequence.
In both constructs a stop codon and the simian virus 40 poly(A) sequence were inserted downstream of the coding sequence. The targeting vectors include HSV-tk and PGK-neo cassette for selection of transfected ES cells.
The target constructs were introduced by electroporation in RI ES cells and selected as described. Genomic DNA from neomycin resistance clone was digested with BamHI and analyzed by Southern blotting using as a 500-bp probe from nucleotide 10512 to nucleotide 11042 of the 3′-untranslated region of the mouse Nkx2-1 gene (GenBank™ accession no. U19755).
ES cell clones in which the targeting vector had been properly integrated were injected into C57BL/6 blastocysts. Chimeric mice were bred with C57BL/6 mice for germline transmission of the modified allele. The heterozygous ΔNH 2 /+ and ΔCOOH/+ mice were maintained by crossing heterozygous mice with C57BL/6 1 wild type animals.
DNA was extracted from yolk sacs or from a piece of tail of the embryos. The tissue was incubated overnight at 60°C with lysis buffer (50 mm Tris-HCl, 100 mm EDTA, 100 mm NaCl, 1% SDS, 0.5 mg/ml proteinase K), and genomic DNA was extracted by adding 0.3 volumes of 6 m NaCl and then precipitated with isopropyl alcohol. To genotype PM, ΔNH 2 , and ΔCOOH mutants the genomic DNA was digested with BamHI and analyzed by Southern blotting using as a 500-bp probe from nucleotide 10512 to nucleotide 11042 of the 3'-untranslated region of the mouse Nkx2-1 gene. Nkx2-1 +/- mutants were genotyped as described.
Histology, immunohistochemistry and in situhybridization
Animals were killed by cervical dislocation. Staged embryos were obtained by dissection of pregnant females. The day at which the vaginal plug was detected was designed as embryonic day (E)0.5. Thyroids and embryos were fixed overnight at 4°C in 4% paraformaldehyde in PBS at pH 7.2, dehydrated through ethanol series, cleared in xylene, and embedded in paraffin, and 7- μm sections were cut. For histological examinations, sections were dewaxed by standard techniques and stained with Harry's hematoxylin/eosin (BDH) according to manufacturer instructions
For immunohistochemistry studies, slides were. were dewaxed by standard techniques and heat treatment to retrieve the antigen sites was performed.
To quench endogenous peroxidases, the sections were treated with 1.5% hydrogen peroxide in methanol at room temperature. The sections were incubated for 1 h at room temperature with blocking solution (3% BSA/5% goat serum/20 mM MgCl2/0.3% Tween 20 in PBS) and then with primary antibodies overnight at 4°C. Staining procedures and chromogenic reactions were carried out according to the protocols of the Vectastain ABC kit protocol (Vector Laboratories). The primary antibodies used were: anti-rat Nkx2-1, anti-mouse Pax8, anti-human Tg (Dako) and anti-rat NIS.
For in Situ hHybridization the following clones from Deutsche Ressourcenzentrum für Genomforschung (RZPD) were used to prepare a digoxigenin-labeled probe using DIG-labeling RNA kit (Roche Molecular Biochemicals) from T7 promoter according to manufacturer instructions: IMAGp998N18717 (Scgb1a1), IMAGp998L103622 (Sall1); IMAGp998C05710 (Napsa). Hybridization was performed as described.(Dathan2002)
Histological sections were examined with an AXIOPLAN 2 microscope equipped with Axiocam digital camera (Zeiss). Images were processed using Axion Vision software and edited with adobe Photoshop software.
Immunofluorescence and confocal microscopy
Tissue sections were deparaffinized and hydrated through xylenes and graded alcohol series followed by antigen retrieval in sodium citrate buffer [0.01 M (pH 6.0)]. Sections were microwaved for 15 min, washed in PBS and PBS containing 0.2% Triton X-100 for 5 min, and incubated for 1 h with blocking buffer. Tissue sections were then incubated overnight at 4 C with primary antibody diluted in blocking buffer, washed in PBS containing 0.2% Triton X-100 for 5 min and PBS, incubated with the secondary antibody for 1 h at room temperature, washed in PBS containing 0.2% Triton X-100 for 5 min and PBS, and finally mounted in PBS/glycerol (1:1).
Immunofluorescence analysis was performed at a confocal laser scanner microscope (LSM 510; Zeiss, Göttingen, Germany). The lambda of the argon ion laser was set at 488 nm, and that of the HeNe laser was set at 543 nm. Fluorescence emission was revealed by BP 505-530 band pass filter for Alexa Fluor 488 and by BP 560-615 band pass filter for Alexa Fluor 546. Double-staining immunofluorescence images were acquired simultaneously in the green and red channels at a resolution of 1024 × 1024 pixels.
The following antibodies were used: mouse monoclonal antibodies anti-E-cadherin (1:100), Rabbit polyclonal antibodies anti-ZO-1 (1:100) and mouse monoclonal antibodies anti-Ksp-cadherin (1:100) were from Zymed Lab. Inc. (San Francisco, CA). Rabbit polyclonal anti-Titf1 antibodies (1:100) were provided by RDL, mouse monoclonal anti-Tg antibodies (1:100) were from NeoMarkers (Fremont, CA-USA). Alexa Fluor 488 or 543 goat anti-rabbit or anti-mouse were from Molecular Probes (Leiden-NL).
A pool of at least three thyroids at E18 (or two lungs) of the same genotype were homogenized in 100 μl (500 μl) of Buffer P (50 mM Tris HCl pH 7.5, 400 mM NaCl, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 0.01% Brij 35, Sigma protease(P8340) inhibitor cocktail 1 × using a minipotter at 4°C following repeated freeze/unfreeze cycles. After determining proteins (Bio-rad protein assay) 15 μl of homogenate containing 45 μg of total protein were incubated in presence of 200 U of Lambda Protein Phosphatase (New England Biolabs) for 1 hour at 30°C. in presence or absense of phosphatase inhibitors (50 mM sodium fluoride, 10 mM EDTA). 35 μg of total proteins were resolved in a 4 12% Bis-Tris minigel NuPAGE (Invitrogen) at 200 V for 1 hour. Transfer to a PVDF membrane was done with Bio-Rad Mini-TransBlot as described by the manufacturer and western Blot with α-TTF1 (1:10000) were performed as described before.
Cellular extracts were prepared as described before. The binding reaction was carried out in a buffer containing 40 mM Hepes, pH 7.9, 200 mM KCl, 0.5 mM dithiothreitol, and 0.3 mg/ml poly(dI·dC). After 30 min of incubation at room temperature, free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel run in 0.5 × TBE (2 mM EDTA, 90 mM boric acid, 90 mM Tris-HCl, pH 8.0) for 2-3 h at 4°C. The gel was dried and then exposed to an x-ray film at -80°C. Oligonucleotide C, used to measure TTF-1 binding activity, has been described .
The thyroid glands were dissected from E18 embryos, and total RNA was extracted with guanidine isothiocyanate and further purified by the RNeasy minikit (Qiagen). cRNA was generated by using the Affymetrix One-Cycle Target Labeling and Control Reagent kit (Affymetrix Inc., Santa Clara, CA) following the protocol of the manufacturer. The biotinylated cRNA was hybridized to the MG-U74Av2, MG-U74Bv2 and Mg-U74Cv2 Affymetrix DNA chips, containing over 36000 genes and open-reading frames from Mus musculus Genome databases GenBank, dbEST, and RefSeq. Chips were washed and scanned on the Affymetrix Complete GeneChip Instrument System, generating digitized image data files. Reactions were carried out in triplicate. Analysis DAT files (images) were analyzed by MAS 5.0 to generate image data files (CEL files). Probe sets summary intensities were generated using the gcrma algorithm of the BioConductor package. The normalization of the data set was performed by quantile-quantile method of the Bioconductor package. We have filtered out all genes having less than 2 present call in all replicate groups in order to obtain a subset of probe sets resulting expressed in at least one condition. Furthermore we have filtered out all genes showing fold change less than 1.5 in whatever comparison. Ones the final data set is generated, robustness of differential expression was evaluated through statistical validation. We conducted ANOVA on the filtered 1406 probe sets using a cut-off p-value of 0.01.
Resulting gene list obtained with the software GeneSpring contain 81 genes showing differential expression in at least one comparison. These were used in clustering algorithms (k-means and hierarchical clustering).
This work was supported in part by Telethon, Grant GGP05161, "Molecular Genetics of Thyroid Dysgenesis," and by the European Community, Integrated Project CRESCENDO Grant LSHM-CT-2005-01865
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