Identification of c-Rel and RelA binding regions in stimulated EL4 cells

c-Rel and RelA are members of the NF-kappaB family of transcription factors. They both have important roles in T cell activation. To discover where in the genome c-Rel and RelA bind and hence which genes they may directly target, we used ChIP-chip with EL4 cells stimulated with phorbol ester (PMA) and Ionomycin. We have identified regions in EL4 cells (background strain: C57BL/6N) activated for 2h or 8h by PMA and Ionomycin, that bind the transcription factors c-Rel and RelA. Immunoprecipitated samples from EL4 cells stimulated with PMA and ionomycin for 2 h and 8 h with antibodies against RelA or c-Rel respectively were used for ChIP-on-chip experiments. In addition, samples from total input and mock immunoprecipitation were used as controls. Biological triplicates were used.

c-Rel与RelA均为核因子κB（NF-kappaB）家族转录因子成员，二者在T细胞活化过程中发挥关键作用。为探明二者在基因组中的结合位点，进而明确其直接靶向调控的靶基因，本研究采用佛波酯（PMA）与离子霉素共同刺激EL4细胞，并开展染色质免疫沉淀芯片（ChIP-chip）实验。本研究针对经PMA与离子霉素共同刺激2小时或8小时的EL4细胞（背景品系：C57BL/6N），鉴定出了可结合转录因子c-Rel与RelA的基因组区域。实验中，分别使用抗RelA抗体处理刺激2小时的EL4细胞样本、抗c-Rel抗体处理刺激8小时的EL4细胞样本，所得免疫沉淀产物用于染色质免疫沉淀芯片（ChIP-on-chip）实验。此外，本研究设置了总输入样本与Mock免疫沉淀（Mock immunoprecipitation）样本作为对照，并采用三次生物学重复。