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

Nat Biotechnol. 2012 Feb 26;30(3):271-7. 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Biological Assay / methods*
  • Enhancer Elements, Genetic*
  • Genes, Reporter*
  • Humans
  • Models, Genetic
  • Molecular Sequence Data
  • Mutagenesis
  • Sequence Alignment
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic

Substances

  • Transcription Factors

Associated data

  • GEO/GSE31982