O papel modulador do gene AIRE - capes

D.A.R. Magalhães et al. / Molecular Immunology 42 (2005) 1043–1048 1047mice, encompassing a previous analysis of the genes whichwere only differentially expressed (Cluster and Tree-Viewmethod), and a second analysis clustering only those geneswhich were differentially and significantly expressed (SAMand Cluster and Tree-View methods).By exploring a large set of genes, we intended to identifynovel differentially expressed genes that could be used asmarkers for the individual stages of thymus ontogeny.However, the clustering of raw expression data using theset of 1,261 genes caused a shuffling among RNA samples(cDNA probes) from each day of gestation (Fig. 2).Considering the possibility that the similarity betweensamples from two distant days of gestation, such as 14 and 17days p.c., as seen in the dendrogram of Fig. 2, might not bestatistically significant, we applied a second round of clustering,now using the differentially and significantly expressedgenes. Using this approach, it was possible to distinguish thedays of gestation (Fig. 3), demonstrating the importance, inthis study, of a previous statistical treatment instead of usingonly raw normalized microarray data to run the Cluster andTree-View method.Reclustering showed that the constitution of the gene clusterdiverged for each stage of thymus development, i.e. eachcluster harbored different genes with different expression patterns.The 14th day p.c. RNA sample was considered to be thetest sample for comparisons with the subsequent days of gestation(14 versus 15, 14 versus 16 and 14 versus 17 days p.c.);however, in a given cluster, the repressed genes in a given dayof gestation presented as induced on the following day. Thisis evidence that the genes used for data reclustering may playa role in thymus ontogeny and may represent novel candidategenes participating in the control of T-cell maturation.It was possible to point out seven genes implicated incell signaling. The hematopoietic cell signal transducer gene,Hcst, (accession number NM011827, ID 640698) was inducedin the early stages of thymus development (14–16 daysp.c.) and repressed at 17 days p.c. The Hcst protein is locatedoutside the plasma membrane and is implicated in thecoupling of receptor stimulation to downstream activation ofGTPases. As the maturation of the thymocytes within the thymusdepends on the participation of other cell types, such asstroma (Gill et al., 2003), this could represent evidence for arole of the Hcst gene in cellular communication via molecularsignaling in the early thymus.Genes implicated in the calcium signaling pathwaywere also modulated, such as the proline-rich Gla (Gcarboxyglutamicacid) polypeptide 2 gene, Prrg2, (accessionnumber NM022999, ID 640686), the Down syndromecritical region homolog 1 gene, Dscr1, (accession numberNM019466, ID 640638), and the syntaxin binding protein 3gene, Stxbp3, (accession number NM011504, ID 640484),whose proteins have a role in the inhibition of the calciuminflux pathway via calcineurin. These genes were repressedin the early (15 days p.c.) and late (17 days p.c.) thymus. Thecasein kinase 1,alpha 1 gene, Csnk1a1 (accession numberTable 2Number of ESTs differentially expressed during thymus development of(Balb-c × C57Bl/6) F 1 hybrid mice as reported by the SAM program aThymus development(in days p.c.)Significant ESTsInducedRepressedFDR14–15 20 49 4814–16 10 30 2214–17 10 14 64a Complete file of ESTs available online (http://www.rge.fmrp.usp.br/passos/TRBV81/SAM/table2), FDR = false discovery rate (median).NM146087, ID 640022) whose protein is an inhibitor of calcineurin,was induced at 16 days p.c. These data, suggest thatthe calcium influx pathway could be activated in the thymuswith 15 and 17 days p.c. and down-regulated with 16 daysp.c.All these genes above mentioned participates in the controlof calcium influx pathway mediated by calcineurin, which isimportant in the activation of the nuclear factor of activatedT-cells (NFAT), an transcription factor of T-cells.As calcium signaling is implicated in the induction of T-cell anergy mediated through calcineurin and NFAT, our resultscould be useful to know the genes that induce tolerance(Feske et al., 2001, 2003; Heissmeyer et al., 2004).The Golgi SNAP receptor complex member 2 gene,Gosr2, (accession number NM019650, ID 640152) was inducedin the thymus from 15 to 17 days p.c. This gene hasa role in the intracellular transport of newly synthesized proteinsfrom the endoplasmic reticulum to their destination inthe cell. Since we showed in this study that T-cells within thethymus begin to mature from 14 days p.c., the activation ofthe Gosr2 gene in this phase is of particular importance dueto the necessity of delivering the T-cell receptors chains onthe cell surface.Finally, we have shown that the protein tyrosine phosphatase4a3 gene, Ptp4a3, (accession number NM008975,ID 640437), was induced in the thymus with 17 days p.c.There is evidence for a role of this gene in humans in colorectalcancer metastasis (Saha et al., 2001), i.e. cell migration.As the mature thymocytes should migrate from the thymus toblood stream, this may be evidence for the participation of thePtp4a3 gene in the late stages of T-cell maturation includingmigration from the thymus.The 133 EST sequences reclustered in the dendrogramsof Fig. 3, whose functions were not yet assigned,presented a differential pattern of expression andwere reclustered together with named genes (Table 2)(www.rge.fmrp.usp.br/passos/TRBV81/SAM/table2). Thiscan be evidence for the participation of these ESTs in biologicalprocesses similar of those of the known genes. TheseESTs are being studied by our group with the aim of associatingtheir expression profile with gene function.As the microarrays used in this study were prepared withthymus sequences, we explored their expression levels duringthe development of this organ. Although, the thymus harborsdifferent cell types including macrophages and dendritic