Modular Evolution of DNA-Binding Preference of a Tbrain Transcription Factor Provides a Mechanism for Modifying Gene Regulatory Networks

Abstract

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites.

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

“…ChIP was performed as previously described (Cary et al, 2017;Cheatle Jarvela et al, 2014;Mortazavi et al, 2006), with some modifications (see supplementary Materials and Methods). Three independent cultures of S. purpuratus were collected and processed at the mesenchyme blastula stage (24 hpf ) ( Fig.…”

From genome to anatomy: The architecture and evolution of the skeletogenic gene regulatory network of sea urchins and other echinoderms

“…Thus, Alx1 provides a particularly striking example of a specific evolutionary change in transcription factor sequence that has led to a major shift in developmental function (see Lynch & Wagner, ). Another transcription factor in the PMC GRN, Tbr , has also undergone significant protein sequence changes, as evidenced by subtly different DNA binding preferences of the sea star and sea urchin orthologs both in vivo and in vitro (Cary, Cheatle Jarvela, Francolini, & Hinman, ; Cheatle‐Jarvela et al, ).…”

Evolution of transcription factor function as a mechanism for changing metazoan developmental gene regulatory networks

“…Recent work demonstrates that while metazoan developmental transcription factors may not diverge as dramatically as yeast orthologs, they do seem capable of exploiting modular divergence mechanisms used by paralogs [ 74 ]. In this study, it was found that orthologs of a t-box transcription factor, Tbr, from a sea urchin ( Strongylocentrotus purpuratus ) and a sea star ( Patiria miniata ) evolved differences in their secondary binding abilities.…”

“…Because primary and secondary sites have distinct functions, the effects of changing binding to one type of site are less pleiotropic than changing binding to a solitary binding site. Importantly, since alternative binding sites can be gained and lost without affecting a conserved site [ 72 – 74 ], these developmental functions can be uncoupled and evolve independently, thus relieving constraint on developmental processes and allowing for more diverse cell types and structures to arise.…”