Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic

Abstract

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.

Two promoters integrate multiple enhancer inputs to drive wild-typeknirpsexpression in theD. melanogasterembryo

“…The instantaneous fluorescence of a single mRNA was chosen as the threshold because we reasoned that an increase in fluorescence greater than or equal to that of a single transcript is indicative of an actively producing promoter, just as a decrease in fluorescence greater than that associated with a single transcript indicates that transcripts are primarily dissociating from, not being newly initiated at, this locus. Visual inspection of fluorescence traces agreed well with the burst calling produced by this method (Figure S4) (Waymack et al, 2020).…”

“…The burst frequency, or the inverse of the time between two bursts, depends on both k on and k off . Previous work in the early embryo has shown burst duration (and thus k off ) to be reasonably consistent regardless of enhancer and promoter (Waymack, Fletcher, Enciso, & Wunderlich, 2020; Yokoshi, Cambón, & Fukaya, 2021). Within this regime, burst frequency is mainly dependent on k on .…”

“…To quantify the transcription properties of interest, we used the smoothed traces to determine at which time points the promoter was “on” or “off” (Waymack et al, 2020). A promoter was considered “on” if the slope of its trace, i.e.…”

“…All original MATLAB code used for burst calling, noise measurements, and other image processing are available at the Wunderlich Lab GitHub (Waymack et al, 2020) with a copy archived at https://github.com/elifesciences-publications/KrShadowEnhancerCode. Updates to include calculations of polymerase initiation rate are also available at the Wunderlich Lab GitHub (https://github.com/WunderlichLab).…”

“…This figure is adapted from Waymack, et al with one additional panel (G) added (Waymack et al, 2020). To quantify the burst properties of interest (burst size, burst frequency, burst duration, and polymerase initiation rate), we began by smoothing individual fluorescence traces using the LOWESS method with a span of 10%.…”