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

在免疫系统的基因中，小鼠TcR y链基因系统的独特之处在于其高度特异性表达与小鼠发育阶段相对应的v区片段。在妊娠第15天左右的胎儿胸腺中，Vγ - 5被重新排列和表达，在分娩时的胸腺中，优先产生表达Vγ - 6的细胞。另一方面，在成人胸腺中，表达v γ - 4和v γ - 7的细胞在γδ T细胞中占主导地位。Vγ基因的使用顺序与Vγ基因片段在染色体上的定位有关；即首先使用最接近j - γ1的Vγ 5基因，然后依次使用离j - γ1越来越远的Vγ片段。在重排的基因中，未重排的种系基因的转录被观察到进行重排。因此，负责这种转录的控制元件可能参与基因重排的调节。为了验证这一假设，我们对Vγ基因的5'侧翼区域进行了测序和分析。他们发现了ETS转录因子潜在结合基序的特征性分布，并发现了稀疏的CpG甲基化位点。将各发育阶段的胸腺细胞与胸腺瘤细胞克隆BW5147融合获得杂交瘤。这些杂交瘤保留了Vγ基因表达的阶段特异性调控以及每个Vγ基因的甲基化模式，即当用HpaII和Mspl限制性内切酶消化基因组DNA时，每个表达的Vγ基因也被去甲基化。最值得注意的是，vγ - 4基因上游的一个甲基化位点包含两个ETS转录因子的基序。我们还发现，ETS因子在成人型杂交瘤中表达，而在胎儿型杂交瘤中不表达。克隆了成人胸腺特异性ETS家族转录因子，包括ETS-1、ETS-2、GABP、Fli-1、Sap-1和新型pe -1。这些因子在核心ETS基序GGA旁表现出不同的结合特异性。与核心ETS基序相邻的结合核苷酸序列存在明显差异。在本研究中，PE- 1表现出5′- A CC GGA A/T G T N -3”的DNA结合特异性，明显表现出对GGA核心前CC的偏好。有趣的是，PE- 1显示出选择半甲基化DNA的能力，而不结合完全甲基化的DNA。讨论了这些转录因子作为这些ETS结合位点的去甲基化作用的可能性。少

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