Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

• Published in: Nature biotechnology Vol. 30; no. 3; pp. 271 - 277
• Main Authors: Melnikov, Alexandre, Murugan, Anand, Zhang, Xiaolan, Tesileanu, Tiberiu, Wang, Li, Rogov, Peter, Feizi, Soheil, Gnirke, Andreas, Callan, Curtis G, Kinney, Justin B, Kellis, Manolis, Lander, Eric S, Mikkelsen, Tarjei S
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.03.2012; Nature Publishing Group
• Subjects: 631/337; 631/61/191; 631/61/32; Agriculture; Base Sequence; Binding Sites; Bioassays; Bioinformatics; Biological and medical sciences; Biological Assay - methods; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cells; Cellular biology; Deoxyribonucleic acid; Diverse techniques; DNA; Enhancer Elements, Genetic; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Genetic engineering; Genetic transcription; Humans; Life Sciences; Models, Genetic; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis; Physiological aspects; Plasmids; Sequence Alignment; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; United States; Human; Enhancer sequence; Transcription; Induction; Regulatory sequence; Functional analysis; Binding site; Mutation; Method; Transcription factor
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/nbt.2137

An improved understanding of enhancers in mammalian genomes could facilitate the design of new regulatory elements. Melnikov et al . synthesize thousands of ~90 nt enhancer variants, assay their activity in human cells and use the data to rationally optimize synthetic enhancers. 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.