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.

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

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...

Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor, a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after 1 d. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue except for one gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naïve cells.

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs) GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and co-localizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.