Transcription factor binding on designed libraries measured by CCRAs

We measured transcription factor binding and expression of designed syththetic promoter libraries using Calling Cards Reporter Arrays (CCRAs). In this study, we showed that CCRAs is able to make quantatitive measurements for many TFs in yeast. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p, and Gcr2p, that act together as a “TF collective”, an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function.本研究采用呼叫卡片报告阵列（Calling Cards Reporter Arrays，CCRAs），对经设计的合成启动子文库的转录因子结合与基因表达水平进行了检测。本研究证实，CCRAs可对酵母中的多种转录因子（transcription factor，TF）实现定量检测。随后，本研究通过检测携带多结合位点的合成启动子上Cbf1p的结合情况，验证了可通过该技术对转录因子协同相互作用开展定量分析。最后，本研究对一组协同发挥功能的转录因子——Tye7p、Gcr1p与Gcr2p构成的“转录因子集合体（TF collective）”的结合与表达特征进行了表征；该集合体是一类重要但研究尚不充分的转录因子协同作用模型。研究发现，Tye7p常可结合不含自身识别位点的启动子，这是由于其可被该集合体中的其他成员招募；而其余成员则必须依赖自身的识别位点才能结合，这提示该类转录因子存在层级招募关系：由其他成员招募Tye7p，而非反向过程。本实验确立了CCRA作为转录因子结合与功能定量研究的有效工具的地位。