Study on the relationship of T-cell receptor y-chain rearrangement and ETSfamily transcription factors

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Among the genes of the immune system, the murine TcR y-chain gene system isunique in its highly specific expression of V-region segments corresponding to the developmental stages of the mice. In the fetal thymus at around day15 of gestation, Vγ5 is rearranged and expressed, and in the thymus at thetime of delivery, Vγ 6 expressing cells are preferentially generated. On the other hand, in the adultthymus, Vγ4 and Vγ7 expressing cells are the predominant types among γδ T cells. The order of the Vγ gene usage correlates with the localization of the Vγ gene segment on the chromosome ; i.e.the Vγ 5 gene most proximal to the Jγ1 is used first, followed by Vγ segments farther and farther away fiom the Jγ1 in a consecutive manner.Among the genes that rearrange, transcription of the unrearranged germline gene is observed to proceed the rearrangement. The control elements responsible for this transcription may therefore be involved in the regulation of gene rearrangement. To test this hypothesis … More , 5' flanking regions of Vγ genes were sequenced and analyzed. They showed a characteristic distribution of potential binding motifs for ETS transcription factor among others, and revealed sparse CpG methylation sites. Hybridomas were obtained from fusion of thymocytes of each developmental stage with BW5147, a thyoma cell clone. These hybridomas retained the stage specific regulation of Vγ gene expression as well as the methylation pattern of each Vγ gene, i.e. each Vγ gene expressed was also demethylated when analyzed by digestion of genomic DNA with HpaII and Mspl restriction enzymes. Most notably, a methylation site upstream of the Vγ4 gene, an adult tγpey-chain gene, contained two motifs for ETS transcription factors. We also found that ETS factors were expressed in the adult type hybridomas but not in the fetal type hybridomas.Adult thymus specific ETS family transcription factors were cloned including ETS-1, ETS-2, GABP, Fli-1 Sap-1 and novel PE-l. The factors showed different binding specificity next to core ETS motif GGA.There are distinct difference in the binding nucleotide sequences adjacent to the core ETS motif. In the present work, PE- 1 showed DNA binding specificity of 5'- A C C G G A A/T G T N -3'' , clearly showing preference for CC in front of GGA core. Interestingly, PE- 1 showed the capability to select the hemi-methylataed DNA and did not bind completely methylated DNA.The possibility of these hasncription factors acting as the demethylation of these ETS binding sites involving CCGGA is discussed. . Less