The role of Ternary Complex factors in T-cell Positive Selection

Removal of the transcription factor SAP1a member of the Ternary Complex Factor (TCF) group of transcription factors which in conjunction with Serum Response Factor (SRF) has been shown to have a profound effect on positive selection in the thymus. When another TCF Elk1 is knocked out in mice there is no effect on positive selection unless it is on a Sap1a KO background where the phenotype is very severe. We have stimulated isolated double positive T cells (DPs) with anti-CD3 to mimic positive selection and compared basal and stimulated transcription across the four genotypes to discover the downstream targets of Sap1a involved in positive selection. The Affymetrix mouse Chip Mouse430_2 arrays were used to define gene expression profiles in purified DPs with and with out CD3 treatment from 3 mice for each genotype (Wt,SapKO, ElkKO and SAP/Elk double KO)转录因子SAP1a属于三元复合物因子（Ternary Complex Factor, TCF）家族，该因子与血清反应因子（Serum Response Factor, SRF）协同结合后，已被证实对胸腺内T细胞阳性选择具有显著调控效应。若在小鼠体内敲除另一种TCF家族成员Elk1，并不会对T细胞阳性选择产生明显影响；但当该敲除操作处于Sap1a基因敲除的遗传背景下时，则会引发极为严重的异常表型。本研究通过抗CD3抗体刺激分离得到的双阳性T细胞（double positive T cells, DPs）以模拟体内阳性选择过程，针对野生型（Wt）、Sap1a基因敲除型（SapKO）、Elk1基因敲除型（ElkKO）以及SAP/Elk双基因敲除型四种基因型的小鼠，对比分析其纯化DP细胞在未经处理与经CD3刺激后的基础转录与诱导转录组差异，以期筛选出参与阳性选择过程的SAP1a下游靶基因。本实验采用Affymetrix Mouse430_2基因芯片，对每种基因型的3只小鼠的对应样本完成基因表达谱检测。