Alexandre Melnikov, Anand Murugan, Xiaolan Zhang, Tiberiu Tesileanu, Li Wang et al.
Learning to read and write the transcriptional regulatory code is of central importance to progress in genetic analysis and engineering. Here we describe a massively parallel reporter assay (MPRA) that facilitates the systematic dissection of transcriptional regulatory elements. In MPRA, microarray-synthesized DNA regulatory elements and unique sequence tags are cloned into plasmids to generate a library of reporter constructs. These constructs are transfected into cells and tag expression is assayed by high-throughput sequencing. We apply MPRA to compare >27,000 variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon-β enhancer. We first show that the resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution. We then use the data to train quantitative sequence-activity models (QSAMs) of the two enhancers. We show that QSAMs from two cellular states can be combined to design enhancer variants that optimize potentially conflicting objectives, such as maximizing induced activity while minimizing basal activity.
Alexandre Melnikov, Anand Murugan, Xiaolan Zhang, Tiberiu Tesileanu, Li Wang, Peter Rogov, Soheil Feizi, Andreas Gnirke, Curtis G. Callan Jr., Justin B. Kinney, Manolis Kellis, Eric S. Lander, Tarjei S. Mikkelsen. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. 2012-02-26. https://doi.org/10.1038/nbt.2137
A single-hit scanning library of synthetic cAMP-responsive enhancer (CRE) variants was assayed in HEK293T/17 cells after forskolin induction. Each variant was represented by multiple 10-nt reporter tags, with two biological transfection replicates.
A single-hit scanning library of variants of the human interferon-beta (IFNB) enhancer was assayed in HEK293T/17 cells after Sendai virus infection. Each variant was represented by multiple 10-nt reporter tags in one biological transfection replicate.
A multi-hit sampling library of randomized synthetic CRE variants was assayed in HEK293T/17 cells in paired forskolin-induced and DMSO vehicle states. Each variant was represented by one reporter tag and measured in two biological transfection replicates per state.
A multi-hit sampling library of randomized human IFNB enhancer variants was assayed in HEK293T/17 cells in paired Sendai-virus-induced and mock-infected states. Each variant was represented by one reporter tag and measured in one biological transfection replicate per state.
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