Abstract
Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.
Highlights
Haploinsufficiency, or the inability to complete a cellular process with one functional allele of a given gene, manifests in tissue and organ defects with variable penetrance and severity (Wilkie, 1994)
Since prior studies found that including sites for the Grainyhead (Grh) pioneer transcription factor (TF) enhanced Notch reporter activity in Drosophila (Furriols and Bray, 2001) and induced chromatin opening (Jacobs, et al, 2018), we generated fly lines containing CSL and Su(H) paired sites (SPS) reporters with (Figure 1C-E) and without (Figure S1B-C) three copies of a Grh binding element (3xGBE)
To define the SPS-GBE binding site features that contribute to wing notching, we tested additional fly lines and found that: i) 6SG-lacZ caused notched wings when inserted in another locus (Figure S1D-E,H); ii) The relative order of GBE and SPS did not matter (Figure S1E-F,H); iii) The penetrance and severity of wing notching increased as a function of transgene and SPS numbers (Figure 1F-H, and 1J); and iv) Flies with an equal number of Notch monomer (CSL) sites next to 3xGBE did not develop notched wings (Figure 1H-I and 1K)
Summary
Haploinsufficiency, or the inability to complete a cellular process with one functional allele of a given gene, manifests in tissue and organ defects with variable penetrance and severity (Wilkie, 1994). Notch gene dose sensitivity has been observed in a variety of Notch-dependent tissues in both humans and animals. We currently lack a mechanistic understanding of what causes some tissues to be highly sensitive to Notch gene dose and what factors impact the variable penetrance and severity of Notch haploinsufficiency phenotypes (Simpson, et al, 2011; McDaniell, et al, 2006; Krebs, et al, 2004; Witt, et al, 2003)
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