Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Bioinformatics Interdepartmental Graduate Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Cell Syst. 2020 Jul 22;11(1):75-85.e7. doi: 10.1016/j.cels.2020.05.011. Epub 2020 Jun 29.
In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.
在真核生物中，转录因子 (TFs) 通过与 TF 结合位点 (TFBS) 结合，并将转录共调节因子和 RNA 聚合酶 II 定位到顺式调控元件，从而协调基因表达。然而，我们对 TFBS 组成与其定量转录响应之间的关系缺乏基本的了解。在这里，我们通过使用大规模平行报告基因分析 (MPRA) 测量了由 17406 个具有不同组成的模型 TFBS（c-AMP 反应元件 (CRE)）的合成顺式调控元件驱动的表达。我们发现 CRE 数量、亲和力和启动子接近度在很大程度上决定了表达。此外，我们还观察到基于 CRE 之间的间隔和 CRE 与启动子之间的距离的表达调制，其中表达遵循螺旋周期性。最后，我们比较了在染色体外 MPRA 和基因组整合 MPRA 之间的文库表达，其中每个细胞在定义的基因座上检测一个顺式调控元件。这些测定结果彼此之间大体上一致，尽管较弱的非规范 CRE 在基因组环境中表现出更大的活性。
