Analysis of the DNA-binding properties of Alx1, an evolutionarily conserved regulator of skeletogenesis in echinoderms

Jennifer Guerrero-Santoro,Jian Ming Khor,Ayşe Haruka Açıkbaş,James B Jaynes,Charles A Ettensohn

doi:10.1016/j.jbc.2021.100901

Jennifer Guerrero-Santoro, Jian Ming Khor + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100901

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Abstract

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41–amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.

Highlights

Recent studies have highlighted the central, conserved role of Alx1, a homeodomain (HD)-containing transcription factor (TF), in the formation of the extensive, biomineralized endoskeleton characteristic of all echinoderms [1]
One of the cis-regulatory module (CRM) identified in our ChIP-seq analysis of Alx1binding sites in Strongylocentrotus purpuratus (Sp) embryos [7] contained four putative binding sites—three half sites and one palindromic site
We used an endogenous, palindromic Alx1-binding site located within a CRM associated with Sp-EMI/TM, a gene that encodes a novel, primary mesenchyme cells (PMCs)-specific transmembrane protein (Fig. 1B)

Summary

Results

One of the CRMs identified in our ChIP-seq analysis of Alx1binding sites in Strongylocentrotus purpuratus (Sp) embryos [7] contained four putative binding sites—three half sites and one palindromic site. We used an endogenous, palindromic Alx1-binding site located within a CRM associated with Sp-EMI/TM, a gene that encodes a novel, PMC-specific transmembrane protein (Fig. 1B) This Alx1binding site was previously shown to be required for the activity of a transcriptional reporter in transgenic sea urchin embryos [7]. Alx1-HD bound to the Sp-EMI/TM palindromic site (Fig. 2, A and B, lanes 1–3) and site D (Fig. 2B, lanes 7–9) in both monomeric and dimeric protein–DNA complexes This observation was consistent with what has been reported for other paired-class HDs, which can bind cooperatively to palindromic sites [28, 29, 34]. A slower migrating complex formed in association with each of these sites and was especially prominent in the case of site B

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Discussion

Experimental procedures