Abstract
The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of tens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR’s core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed specific DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.
Highlights
The interplay between transcription factors (TFs) and genomically encoded cis-regulatory elements plays a key role in specifying where and when genes are expressed
When grouping sequences based on their ability to either enhance or blunt GR binding sequence (GBS) activity, we found that each group contained a broad spectrum of highly diverse sequences, but striking similarities in their DNA shape characteristics
To test if we could use the STARR-seq reporter [22] to study how sequence variation of the glucocorticoid receptor (GR) binding site influences GR activity, we first tested if a single GBS is sufficient to facilitate GRdependent transcriptional activation of the reporter
Summary
The interplay between transcription factors (TFs) and genomically encoded cis-regulatory elements plays a key role in specifying where and when genes are expressed. Differences in its DNA-binding sites can modulate the magnitude of transcriptional activation, as exemplified by the glucocorticoid receptor (GR), a hormone-activated TF [3,4,5]. The effects on activity are more modest than those observed for changes within the core, sequences directly flanking the core modulate GR activity [3]. These sequence-induced changes in activity cannot be explained by affinity [3]. The flanking nucleotides induce structural changes in both DNA and the DNA binding domain of GR, arguing for their role in tuning GR activity [3]
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