Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness

Michael Z. Ludwig, Manu, Ralf Kittler, Kevin P. White, Martin Kreitman

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long "minimal stripe element" is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped - lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that "robustness" itself must be an evolved characteristic of the wild-type enhancer.

Original languageEnglish (US)
Article numbere1002364
JournalPLoS Genetics
Volume7
Issue number11
DOIs
StatePublished - Nov 2011

Fingerprint

gene expression
fitness
genomics
Gene Expression
gene
binding sites
Binding Sites
viability
Genes
Gene Transfer Techniques
mutants
regulatory sequences
gene activation
genes
Gene Expression Profiling
adulthood
Transgenes
Transcriptional Activation
Drosophila
transgenes

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Ecology, Evolution, Behavior and Systematics
  • Cancer Research
  • Genetics(clinical)

Cite this

Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness. / Ludwig, Michael Z.; Manu, ; Kittler, Ralf; White, Kevin P.; Kreitman, Martin.

In: PLoS Genetics, Vol. 7, No. 11, e1002364, 11.2011.

Research output: Contribution to journalArticle

Ludwig, Michael Z. ; Manu, ; Kittler, Ralf ; White, Kevin P. ; Kreitman, Martin. / Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness. In: PLoS Genetics. 2011 ; Vol. 7, No. 11.
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