Using high-throughput promoter reporter assays to examine regulatory pathways: Hypoxia case study
Shelley Force Aldred1 , Nathan Trinklein1 , Michael Rose1 , Anna Arnaudo1 , Menghang Xia2 , Ruili Huang2 , Jim Hudson3
1SwitchGear Genomics, Menlo Park, CA; 2NIH Chemical Genomics Center, Bethesda, MD; 3Hudson Alpha Institute for Biotechnology, Huntsville, AL
Recent studies have highlighted the benefit of conducting
genome-wide expression studies and transcription factor binding
in parallel. After generating these descriptive data sets, a
number of questions remain. Which genomic elements are
responsible for transcript level changes? What is the effect of a
transcription factor binding event?
Using functional reporter assays to study the behavior of
genomic elements in living cells can help answer many of these
questions. To enable these studies in a high-throughput format,
we have created a genome-wide library of human promoters
and UTRs in a luciferase-based reporter system GoClones.
In this study we applied this resource and approach to the
hypoxia regulatory pathway.
- Assemble a panel of known and potentially novel
- Measure the function of this panel in a variety of different
conditions and cell types
- Measure the effects of naturally occuring sequence variants
- Compare to other hypoxia-related data types
Screening a toxicology panel for HIF1a-dependent hypoxia inducers:VEGF secretion assay and promoter activation patterns identify the same top candidates (Xia et al. manuscript submitted)
We have gained new insight into the regulation of the
human hypoxia pathway by mapping and measuring the
behavior of hypoxia-responsive genomic elements and
overlaying this information on existing datasets.
- Confirming changes in endogenous transcript levels
- Extending the scope of the study to include
Applying the same library resources and strategies
to many other human regulatory pathways
Data on the genome-wide regulatory element library
resource can be found at:
SwitchDB is an open-access database of human
promoters and 3’UTRs.