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TCRy链基因系统是独一无二的，它高度特异地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，Vγ5被重排和表达，而在分娩时的胸腺中，Vγ6表达细胞优先产生。另一方面，在成人胸腺中，Vγ4和Vγ7表达细胞是γδT细胞中的主要类型。Vγ基因的使用顺序与Vγ基因片段在染色体上的定位有关，即首先使用离Jγ1最近的Vγ5基因，然后依次使用距离Jγ1越远的Vγ基因片段，在重排的基因中，观察到未重排的生殖系基因转录进行重排。因此，负责该转录的控制元件可能参与了基因重排的调节。要检验这一假设，…对V-γ基因5‘侧翼区进行了测序和分析。它们显示了ETS转录因子等潜在结合基序的特征分布，并显示了稀疏的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，获得杂交瘤细胞。这些杂交瘤保留了V-γ基因表达的阶段特异性调节以及V-γ基因的甲基化模式，即用HpaII和Mspl限制性内切酶对基因组γ进行酶切分析时，每个表达的V-DNA基因也被去甲基化。最值得注意的是，Vγ4基因上游的甲基化位点包含两个转录因子的基序，该基因是一个成人tγ聚合链基因。克隆了成体胸腺特异性Ets家族转录因子Ets-1、Ets-2、GABP、Fli-1、SAP-1和新发现的PE-L。这些因子在核心ETS基序GGA附近表现出不同的结合特异性，核心ETS基序附近的结合核苷酸序列也有明显的差异。在本工作中，PE-1表现出5‘-A、C、C、G、G、A、A/T、G、N-3’‘的DNA结合特异性，明显地表现出对CC在GGA核心前的偏好。有趣的是，PE-1显示了选择半甲基化DNA的能力，但不能完全结合甲基化的DNA。讨论了这些描述因子作为这些涉及CCGGA的ETS结合位点去甲基化的可能性。。较少

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