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

Melnikov, A., Murugan, A., Zhang, X., Tesileanu, T., Wang, L., Rogov, P., Feizi, S., Gnirke, A., Callan Jr, C. G., Kinney, J. B., Kellis, M., Lander, E. S., Mikkelsen, T. S. (2012) Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nature Biotechnology, 30 (3). pp. 271-277. ISSN 10870156 (ISSN)

URL: http://www.ncbi.nlm.nih.gov/pubmed/22371084
DOI: 10.1038/nbt.2137

Abstract

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. © 2012 Nature America, Inc. All rights reserved.

Item Type: Paper
Uncontrolled Keywords: Basal activities Cellular state Genetic analysis High-throughput Human cells Induced activity Regulatory elements Reporter assay Transcription factor binding sites Unique sequence Cloning Dissection Optimization Transcription factors Viruses Cell engineering beta interferon cre recombinase article enhancer region human human cell massively parallel reporter assay nucleotide sequence priority journal regulatory sequence reporter gene sequence analysis
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
organism description > animal > mammal > primates > hominids > human
organism description > animal > mammal
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > transcription factor
CSHL Authors:
Communities: CSHL labs > Kinney lab
Depositing User: Matt Covey
Date Deposited: 30 Jan 2013 15:11
Last Modified: 30 Jan 2013 15:11
PMCID: PMC3297981
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27001

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