Functional conservation of Rel binding sites in drosophilid genomes

Abstract

Evolutionary constraints on gene regulatory elements are poorly understood: Little is known about how the strength of transcription factor binding correlates with DNA sequence conservation, and whether transcription factor binding sites can evolve rapidly while retaining their function. Here we use the model of the NFKB/Rel-dependent gene regulation in divergent Drosophila species to examine the hypothesis that the functional properties of authentic transcription factor binding sites are under stronger evolutionary constraints than the genomic background. Using molecular modeling we compare tertiary structures of the Drosophila Rel family proteins Dorsal, Dif, and Relish and demonstrate that their DNA-binding and protein dimerization domains undergo distinct rates of evolution. The accumulated amino acid changes, however, are unlikely to affect DNA sequence recognition and affinity. We employ our recently developed microarray-based experimental platform and principal coordinates statistical analysis to quantitatively and systematically profile DNA binding affinities of three Drosophila Rel proteins to 10,368 variants of the NFKB recognition sequences. We then correlate the evolutionary divergence of gene regulatory regions with differences in DNA binding affinities. Genome-wide analyses reveal a significant increase in the number of conserved Rel binding sites in promoters of developmental and immune genes. Significantly, the affinity of Rel proteins to these sites was higher than to less conserved sites and was maintained by the conservation of the DNA binding site sequence (static conservation) or in some cases despite significantly diverged sequences (dynamic conservation). We discuss how two types of conservation may contribute to the stabilization and optimization of a functional gene regulatory code in evolution.

The Drosophila Baramicin polypeptide gene protects against fungal infection

“…S1A-F). Consistent with this, we found several putative NF-κB binding sites upstream of the BaraA gene (guided by previous reports [17][18][19]). Notably there are two putative binding sites for Relish, the transcription factor of the Imd pathway and three putative binding sites for the Dif/Dorsal transcription factors acting downstream of Toll (Fig.…”

“…It is made The copyright holder for this preprint this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.23.394148 doi: bioRxiv preprint Sequence files were collected from FlyBase [49] and recently-generated sequence data [44,50] and comparisons were made using Geneious R10. Putative NF-κB binding sites were annotated using the Relish motif "GGRDNNHHBS" described in Copley et al [18] and a manually curated amalgam motif of "GGGHHNNDVH" derived from common Dif binding sites described previously [17,19]. Gene expression analyses were performed using primers described in Supplementary data file 1, and further microarray validation for BaraA expression comes from De Gregorio et al [11].…”

NF‐κB: where did it come from and why?

“…Although the discussion below of insect NF‐κB signaling focuses on D. melanogaster and mosquitoes, Relish and Dif homologues have been identified in a variety of other insects including the honeybee Apis mellifera (30), the tsetse fly Glossina morsitans morsitans (110), the silkworm Bombyx mori (111, 112), and the ant Acromyrmex echinatior (113), among several others, and the biological processes that NF‐κB controls in all insects are likely to be similar (111, 114, 115). Furthermore, the sequence and positions of κB sites in the promoters of developmental and immunity genes controlled by NF‐κB/Rel have been shown to be highly conserved among several species of Drosophila (116).…”

Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes

“…Interestingly, transfection of Relish, another Rel family transcription factor, which binds the same or nearly identical DNA sequence motifs as Dorsal (66,67), was able to activate the wt brk, albeit to lower levels as compared with Dorsal. Addition of a TATA box to the brk natural enhancer-promoter with a mutation in the DPE motif could not restore transcription levels in the presence of transfected Relish (Fig.…”