Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise

Abstract

26The binding of transcription factors to short recognition sequences plays a pivotal role in 27 controlling the expression of genes. The sequence and shape characteristics of binding sites 28 influence DNA binding specificity and have also been implicated in modulating the activity 29 of transcription factors downstream of binding. To quantitatively assess the transcriptional 30 activity of dozens of thousands of designed synthetic sites in parallel, we developed a 31 synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically 32 analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) 33 affect transcriptional regulation. Our approach resulted in the identification of a novel 34 highly active functional GR binding sequence and revealed that sequence variation both 35 within and flanking GR's core binding site can modulate GR activity without apparent 36 changes in DNA binding affinity. Notably, we found that the sequence composition of 37 variants with similar activity profiles was highly diverse. In contrast, groups of variants 38 with similar activity profiles showed distinct DNA shape characteristics indicating that DNA 39 shape may be a better predictor of activity than DNA sequence. Finally, using single cell 40 experiments with individual enhancer variants, we obtained clues indicating that the 41 architecture of the response element can independently tune expression mean and cell-to 42 cell variability in gene expression (noise). Together, our studies establish synSTARR as a 43 powerful method to systematically study how DNA sequence and shape modulate 44 transcriptional output and noise. 45 46 47 48 3 Keywords 49 Enhancers, transcriptional regulation, glucocorticoid receptor, transcriptional noise, DNA 50 shape 51 52 53The interplay between transcription factors (TFs) and genomically encoded cis-54 regulatory elements plays a key role in specifying where and when genes are expressed. In 55 addition, the architecture of cis-regulatory elements influences the expression level of 56 individual genes. For example, transcriptional output can be tuned by varying the number 57 of TF binding sites, either for a given TF or for distinct TFs, present at an enhancer [1, 2]. 58 Moreover, differences in its DNA-binding sites can modulate the magnitude of 59 transcriptional activation, as exemplified by the glucocorticoid receptor (GR), a hormone-60 activated TF [3][4][5]. The sequence differences can reside within the 15 base pair (bp) core GR 61 binding sequence (GBS) consisting of two imperfect 6 bp palindromic half-sites separated 62 by a 3 bp spacer. Moreover, sequences directly flanking the core also modulate GR activity 63 [3]. However, these sequence-induced changes in activity cannot be explained by affinity 64 [3]. Instead, the flanking nucleotides induce structural changes in both DNA and the DNA 65 binding domain of GR, arguing for their role in tuning GR activity [3]. 66 Notably, the expression level of a gene is typically measured for p...

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