Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, we showed individual member enhancers act largely independently. We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.

9Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of 10 organisms and are critical for robust developmental patterning. However, their mechanism of 11 action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by 12 buffering upstream noise through a separation of transcription factor (TF) inputs at the individual 13 enhancers. By measuring transcriptional dynamics of several Kruppel shadow enhancer 14 configurations in live Drosophila embryos, we showed individual member enhancers act largely 15 independently. We found that TF fluctuations are an appreciable source of noise that the shadow 16 enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is uniquely 17 able to maintain low levels of expression noise across a wide range of temperatures. A stochastic 18 model demonstrated the separation of TF inputs is sufficient to explain these findings. Our 19 results suggest the widespread use of shadow enhancers is partially due to their noise suppressing 20 ability. 21 22 Stapel, et al., 2017; Raj et al., 2010). For example, organisms use temporal and spatial averaging 42 mechanisms and redundancy in genetic circuits to achieve the precision required for proper 43 development (Stapel, et al.

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Enhancers are stretches of regulatory DNA that bind transcription factors (TFs) and regulate the expression of a target gene. Shadow enhancers are two or more enhancers that regulate the same target gene in space and time and are associated with most animal developmental genes. These multi-enhancer systems can drive more consistent transcription than single enhancer systems. Nevertheless, it remains unclear why shadow enhancer TF binding sites are distributed across multiple enhancers rather than within a single large enhancer. Here, we use a computational approach to study systems with varying numbers of TF binding sites and enhancers. We employ chemical reaction networks with stochastic dynamics to determine the trends in transcriptional noise and fidelity, two key performance objectives of enhancers. This reveals that while additive shadow enhancers do not differ in noise and fidelity from their single enhancer counterparts, sub- and superadditive shadow enhancers have noise and fidelity trade-offs not available to single enhancers. We also use our computational approach to compare the duplication and splitting of a single enhancer as mechanisms for the generation of shadow enhancers and find that the duplication of enhancers can decrease noise and increase fidelity, although at the metabolic cost of increased RNA production. A saturation mechanism for enhancer interactions similarly improves on both of these metrics. Taken together, this work highlights that shadow enhancer systems may exist for several reasons: genetic drift or the tuning of key functions of enhancers, including transcription fidelity, noise and output.