Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

Abstract

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus.How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation

“…The graded tissue‐level pattern of sim activation was not reflected at the level of individual nuclei. In agreement with a recent study of sim enhancer regulation by NICD , sim nuclear dots were either absent or present and their intensity remained relatively stable (fluctuating around the mean) for the entire duration of cellularization (Fig H), as revealed also by quantitative fluorescent in situ hybridization of nascent transcripts (Fig EV5D–I). These results suggest that a cumulative amount of Notch activation determines the competence of mesectodermal nuclei to express sim , and that the time‐lag between the beginning of cellularization and sim expression is Notch dependent.…”

“…Moreover, once cleaved, NICD molecules move into the nucleus within minutes . While several studies have focused on the mechanisms controlling Notch activation by ligand endocytosis , knowledge about the activation of Notch targets by cleaved NICD and the dynamics involved remains limited . More broadly, the role of signal dynamics in controlling developmental processes and tissue differentiation is only partially understood .…”

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

“…Previous studies on Notch-dependent transcription in the mesectoderm used the MS2-MCP-GFP system to characterize the transcriptional dynamics of two enhancers: the E(spl)m5/m8 mesectoderm enhancer (MSE) that has an SPS site as well as several potential monomeric CSL sites, and the sim MSE enhancer that lacks SPSs but contains monomeric CSL sites (Falo-Sanjuan et al, 2018). Interestingly, the E(spl)m5/m8 MSE and sim MSE enhancers showed very similar transcriptional dynamics and highly correlated transcriptional activity, suggesting that SPS and CSL sites mediate similar transcriptional responses within the mesectoderm.…”

“…For example, the E(spl) genes, many of which contain SPSs, were found to be among the first to respond after a short pulse of Notch activation in Drosophila DmD8 cells (Housden et al, 2013), consistent with SPS-containing enhancers responding quickly to low levels of Notch activation. However, subsequent live imaging studies comparing the activities of enhancers with SPS versus CSL sites revealed that the presence of SPSs did not significantly alter the sensitivity to NICD but instead enhanced transcriptional burst size (Falo-Sanjuan et al, 2018). It should be noted, however, that these studies have largely focused on how the Notch activation complex cooperatively binds to and impacts the regulation of SPS containing enhancers, whereas less is known about whether and how the SPS versus monomeric CSL sites differentially recruit the CSL/co-repressor complexes.…”

Cracking the context-specific PI3K signaling code

“…In addition to the RAS/ERK module, dynamic information transmission has been demonstrated for other signaling pathways including DNA damage-induced TP53 activation (23), nuclear factor B (NF-B) regulation in innate immune signaling (24,25), Ca 2+ -regulated NFAT (26,27), developmental transforming growth factor  (TGF)/NODAL (28)(29)(30)(31), WNT (32), SHH (33,34) and NOTCH (35)(36)(37) signaling, and, as will be detailed below, PI3K-dependent insulin signaling (38,39). Nevertheless, our understanding of the dynamics of the PI3K pathway remains relatively crude.…”