Rapid identification and quantitative assessment of plant enhancers in full-genome libraries

Plants respond to environmental stimuli by tightly controlled changes in gene expression, requiring a dynamic regulatory network. Despite this need, a key mode of dynamic regulation, long-range activation or repression, appears to be absent from chromatin interaction datasets (3C, Hi-C) in Arabidopsis thaliana. The apparent lack of distal physical interactions among chromosomes suggests that Arabidopsis may not use typical enhancers, though they are frequent among other higher eukaryotes. We describe the use of an alternative method to identify and functionally characterize enhancers, STARR-seq. This method relies on simultaneously measuring the activity of a library of gene constructs, each containing a fragment of genomic DNA inserted into a transcribed sequence. If a fragment does not possess an enhancer element, the construct is expressed at low level due only to the minimal promoter driving transcription. If a fragment is able to confer distal activation, the gene is expressed at higher level and the functional enhancer sequence, codified in the transcript, increases in abundance.

We have optimized the method for transient expression in plants by testing a known viral enhancer in the experimental context, and demonstrate the scale to which the method can leverage next-generation sequencing to pinpoint functional enhancers. We foresee this method being useful for addressing biological questions of gene regulation, as well as for application to emerging challenges in crop design.

Michael Dorrity

High Throughput Functional Analysis of the Fitness Landscape of a Yeast Promoter

There are multiple ways to increase the expression of a gene, including mutations in cis-regulatory regions and gene amplification. When experimentally-evolved in limited sulfur, yeast reproducibly amplify the locus containing the gene for the high-affinity sulfur transporter, SUL1, increasing Sul1 protein expression. Neither coding nor cis-regulatory mutations have been found in evolved populations. To determine whether non-coding mutations are a viable evolutionary strategy to increase expression of SUL1, we created nearly 100,000 variants of the gene’s ~500 bp promoter and selected the resulting strains for fitness during sulfur limitation.

Most mutations to the SUL1 promoter do not have a significant effect on fitness. Fitness data for single mutants (Figure 1) map functional regions of the promoter (marked in pink), including the TATA box. Using our data, we can identify the transcription factors binding to sensitive regions as Cbf1 and Met32. The wildtype sequences in these regions are not the consensus site for each factor; mutations changing each sequence to the consensus binding site yield 5-10% increases in cellular fitness. We can also identify mutations that create new sites for Cbf1 and Met31 that also lead to 5-10% fitness increases.

Selection in sulfur-limited chemostats is very sensitive and measures Sul1 protein levels in the cell. As such, we can also measure the effect of possible post-transcriptional regulation of SUL1, mainly through the creation of upstream open reading frames (uORFs) in the 5’ untranslated region (Figure 2). New uORFs have little effect if they are not within the 100 bases upstream of the SUL1 start codon. Within 100 bases, most uORFs decrease fitness.  The longest N-terminal fusion initiated at a uORF is 14 amino acids and leads to ~10% decrease in cellular fitness. As these fusions decrease in length, effects on cellular fitness become gradually more neutral.

Matt Rich (with Celia Payen and Maitreya Dunham)

Department of

Genome Sciences
& Medicine,
Univ. of Washington

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