Mutagenesis of GATA motifs controlling the endoderm regulator elt-2 reveals distinct dominant and secondary cis- regulatory elements

Abstract

Cis-regulatory elements (CREs) are crucial links in developmental gene regulatory networks, but in many cases, it can be difficult to discern whether similar CREs are functionally equivalent. We found that despite similar conservation and binding capability to upstream activators, different GATA cis-regulatory motifs within the promoter of the C. elegans endoderm regulator elt-2 play distinctive roles in activating and modulating gene expression throughout development. We fused wild-type and mutant versions of the elt-2 promoter to a gfp reporter and inserted these constructs as single copies into the C. elegans genome. We then counted early embryonic gfp transcripts using single-molecule RNA FISH (smFISH) and quantified gut GFP fluorescence. We determined that a single primary dominant GATA motif located -527 bp upstream of the elt-2 start codon was necessary for both embryonic activation and later maintenance of transcription, while nearby secondary GATA motifs played largely subtle roles in modulating postembryonic levels of elt-2. Mutation of the primary activating site increased low-level spatiotemporally ectopic stochastic transcription, indicating that this site acts repressively in non-endoderm cells. Our results reveal that CREs with similar GATA factor binding affinities in close proximity can play very divergent context-dependent roles in regulating the expression of a developmentally critical gene in vivo.

Evolutionary Dynamics of the SKN-1 → MED → END-1,3 Regulatory Gene Cascade inCaenorhabditisEndoderm Specification

“…Recent work inferred the binding sites for C. elegans END-1 and END-3 as RSHGATAASR and RKWGATAAGR, respectively, which are very similar though not identical (Weirauch et al 2014;Lambert et al 2019). Other work has shown that recombinant DNA-binding domains of C. elegans END-1 and END-3 can bind canonical GATA sites in the promoter of C. elegans elt-2, although END-1 has a higher affinity for such sites (Du et al 2016;Wiesenfahrt et al 2015). From this work, Endoderm GATA Domains (EGDs) immediately upstream of the DBDs show conserved amino acids between END-3s and END-1s but many more that are unique to either EGD ( Figure 10B).…”

“…The E cell generates the entire endoderm (intestine), while its sister cell MS generates many mesodermal cell types, including the part of the pharynx, and many body muscle cells (Sulston et al 1983). Many components of the GRN underlying MS and E development are known with high precision, and in most of cases, regulatory inputs have been confirmed to be direct and cis-regulatory sites have even been identified in upstream regions (Maduro et al 2001;Broitman-Maduro et al 2005;Wiesenfahrt et al 2015;Du et al 2016). This network is therefore a highly suitable system in which to examine questions of GRN evolution and developmental system drift.…”

“…The elt-7 gene encodes a similar GATA factor that shares function and expression with elt-2, but which itself is not essential for normal development (Sommermann et al 2010;Dineen et al 2018). All of END-1, END-3, ELT-2 and ELT-7 have similar DNA-binding properties and interact with canonical GATA binding sites of the type HGATAR (Wiesenfahrt et al 2015;Du et al 2016). Many additional studies have revealed unexpected nuance and complexity to the myriad of factors in this network, confirming that the sum of upstream inputs into elt-2 activation is not merely additive.…”

Two pair‐rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo

“…Additionally, for developmentally regulated genes, 19% of their enhancers were found to drive broader reporter expression than is observed for the corresponding endogenous gene . Similar mechanisms of context dependent activity and nonadditive interactions have been observed for distinct enhancers in Arabidopsis that are acted upon by Polycomb group proteins, and for the Caenorhabditis elegans endoderm regulator elt‐2 …”

“…42 Similar mechanisms of context dependent activity and nonadditive interactions have been observed for distinct enhancers in Arabidopsis that are acted upon by Polycomb group proteins, 43 and for the Caenorhabditis elegans endoderm regulator elt-2. 44 Overall, our experiments with the wg locus provide an additional example of nonadditive interactions between pair-rule responsive enhancers and further suggest that the nonadditive integration of inputs from different enhancers is a widespread aspect of regulating transcription in animal systems.…”