Low Affinity Binding Site Clusters Confer Hox Specificity and Regulatory Robustness

Abstract

Summary In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture—clusters of low affinity binding sites—is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

Stable Binding of the Conserved Transcription Factor Grainy Head to its Target Genes ThroughoutDrosophila melanogasterDevelopment

“…Therefore, it remains a challenge to determine which of the identified sites are functional. While it has been demonstrated that low-occupancy regions are less likely to contain regulatory information as compared to highoccupancy regions, there are multiple examples of low affinity binding sites driving specific gene expression (Fisher et al 2012;Ramos and Barolo 2013;Crocker et al 2015). Furthermore, binding events that are temporally dynamic over development are enriched for functional binding events, and are more predictive of activity (Jakobsen et al 2007;Li et al 2008Li et al , 2011Zinzen et al 2009;Wilczynski and Furlong 2010).…”

Synthetic genome readers target clustered binding sites across diverse chromatin states

“…6B). Evolutionarily conserved Hox transcription factors were recently found to rely on clustered low-affinity sites to regulate key developmental genes in Drosophila (46). In this respect, polyamides behave like natural transcription factors when binding genomic loci.…”

“…Similar analyses with transcription factors show limited success, primarily because most transcription factors are dependent on chromatin accessibility (44). Although commonly used bioinformatic methods annotate genomic regions using only the highest affinity consensus sites (45), DNA-binding proteins in cooperative complexes bind weak-and moderate-affinity binding sites that are typically not considered in modeling genomic occupancy profiles (31,46). Consequently, most computational models predict genome-wide binding patterns of natural transcription factors with varying success (47).…”

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

“…83 Low affinity binding sites may be lost as they often do not pass the threshold. As it was 84 shown that low-affinity binding is essential in biology [12,59] [55].…”

“…Incorporating these biologically important binding events [12,59] seems to be relevant 490 for improving the predictions.…”