O papel modulador do gene AIRE - capes

R.S. Cardoso et al. / Molecular Immunology 43 (2006) 464–472 471recombination of TRB8.1 [2,3]. The lymphotoxin A gene,accession no. NM010735, chromosome 17, codes for the tumornecrosis factor produced by lymphocytes. This protein iscytotoxic for a wide range of tumor cells in vitro and in vivo.Two genes in cluster 6 are implicated in immune cell activation,cyclin D3 gene (CCND3, accession no. AK046638,chromosome 17) codes for a cdk6 protein kinase essentialfor the control of the cell cycle at the G1/S transition andearly growth response 1 gene (EGR1, accession no. M22326,chromosome 18) codes for a transcriptional regulator (earlygrowth response protein 1) that activates the transcription oftarget genes whose products are required for mitogenesis anddifferentiation.Finally, we found genes in this cluster closely implicatedin the V(D)J recombination mechanism of T-cell receptorgenes and immunoglobulins (Ig). The recombination activatinggene 2 (RAG-2, accession no. AK040375, chromosome2) codes for recombinase 2, a component of the rag enzymaticcomplex (rag complex formed by rag-1 and rag-2 recombinases).This complex recognize and binds to the recombinationsignal sequences (RSSs) at the unrearranged TR or Igloci, consisting of conserved heptamer and nonamer motifsseparated by a spacer region of 12 or 23 bp.V(D)J recombination results in a precise head-to-head ligationof two signal sequences in a joint signal, which maycontain additions or deletions of a few bp. The reaction isinitiated by the formation of DSB, which is resolved by theDNA repair machinery, followed by an exonuclease and/orby terminal deoxynucleotidyl transferase (TdT) before formationof a coding joint.Mice that do not express RAG-1 or RAG-2 genes are unableto form DSB and there is evidence that DNA DSB repairand V(D)J recombination share a partially common mechanism(Schlissel et al., 1993; Chaudhuri and Alt, 2004; Jeggoand Concannon, 2001).DNA-activated protein kinase gene (DNA-PK cs , accessionno. AK088981, chromosome 16), also present incluster 6, codes for a large polypeptide DNA-dependentprotein kinase catalytic subunit and its kinase activity isinvolved in DNA NHEJ required for DSB repair and V(D)Jrecombination. DNA-PK cs forms a complex with twosubunits of the heterodimeric Ku protein (Ku 70 and Ku 80)and must be bound to DNA to express its catalytic properties;cells that lack DNA-PK cs are radiosensitive and defectivein DNA repair, while DNA-PK cs null mice are viable butimmunodeficient, due to inability to complete V(D)J recombination(Lees-Miller and Meek, 2003; Pastwa and Blasiak,2003).The assembly of the DNA-PK cs complex to DNA ends isrequired for NHEJ binding step. Interestingly, the XRCC4repair gene was also observed in cluster 6, and the productof this gene was found to interact with DNA ligaseIV stimulating the ligase activity, in order to complete theNHEJ mechanism and probably the V(D)J recombinationprocess (Lees-Miller and Meek, 2003; Grawunder et al.,1997).Interestingly, the MMTV endogenous retrovirus (accessionno. BU515481, chromosome 19) is also present in thiscluster and displayed overexpression in irradiated FTOCs.We have previously shown an association between the expressionof MMTV and the parallel reduction of the TRBV8.1rearranged segment in vivo during the thymus ontogeny ofthe CBA/J strain (Espanhol et al., 2003).Finally, the DNA cross-link LR1 gene (DNA cross-linkrepair 1B, accession no. BC011094, chromosome 3F2.2),which codes for a protein involved in nucleotide excision repair,was induced in irradiated cultures. The deduced aminoacid sequence of its coded protein was about 30% similarto that of the Artemis protein (Moshous et al., 2001) and itsexpression pattern was similar to that of genes implicated inV(D)J recombination, suggesting a possible role for this genein this process.In conclusion, our results showed for the first time thatthe FTOC model system reproduces the in vivo developmentof the thymus regarding TRBV8.1-BD2.1 V(D)J recombination.Ionizing radiation, a potent physical agent capable of inducingDNA DSB, did not change the onset of recombinationbut increased the amount of the TRBV8.1-BD2.1 rearrangedsegment, suggesting a modulation of genes involved in suchprocess.The hybridization signatures obtained with cDNA microarrayspermitted the identification of genes modulatedduring gamma irradiation of FTOCs. Among them, we foundsome genes related to the V(D)J recombination mechanismand we were able to pinpoint the DNA cross-link LR1 gene(DNA cross-link repair 1B) whose deduced protein sequenceis similar to that of the Artemis protein, and the RUNX3 andSOX4 genes whose coded proteins bind to the enhancers ofT-cell receptor genes.The present data obtained by application of the cDNA microarraymethod indicated several genes participating in themechanism of V(D)J recombination and while some genesare novel candidates, some others are important componentsfor the repair of DSB via NHEJ, thus strongly suggestingan overlap between the two processes. These results opennew perspectives for further studies on the specific roles ofthese genes, using strategies of gene silencing, such as RNAinterference.AcknowledgementsThis research was supported by the Brazilian agenciesFundação de Amparo à Pesquisa do Estado de São Paulo(Fapesp, #99/12135-9) and Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq) and is part ofthe PhD thesis of RSC who received a pre-doctoral fellowfrom Fapesp (#00/09994-9). We would like to thank Mrs.Geneviève Victorero, Mrs. Beatrice Loriod and Mr. FabriceLopez, Unité INSERM ERM 206, Marseille, France, for technicalassistance and discussions.