Guerrero-Santoro J et al. (2021), Analysis of the DNA-binding properties of Alx1,...

ECB-ART-49116

J Biol Chem 2021 Jul 01;2971:100901. doi: 10.1016/j.jbc.2021.100901.

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

Guerrero-Santoro J , Khor JM , Açıkbaş AH , Jaynes JB , Ettensohn CA .

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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.

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	Figure 1. Sp-Alx1 ChIP-seq peaks in the Sp-mtmmpb CRM used to identify Alx1-binding sites.A, genome tracks showing the location of the 210-bp Sp-Alx1 ChIP-seq peak (1166) in the 5′ untranslated region of Sp-mtmmpb, overlapping ATAC-seq and DNase-seq differential peaks (i.e., regions of chromatin selectively accessible in skeletogenic PMCs, cells that specifically express Sp-mtmmpb). Four putative Alx1-binding sites are present within ChIP-seq peak 1166. B, previously identified Sp-Alx1 ChIP-seq peak within an intron of Sp-EMI/TM. This region drives PMC-specific reporter expression in transgenic embryos, and expression depends on the first palindromic site (7). The DNA sequence of the first site was used in the EMSA assays. CRM, cis-regulatory module; PMCs, primary mesenchyme cells.
	Figure 2. EMSA analysis of the binding of purified Alx1-HD, Alx1-FL, and Alx1-ΔD2 proteins to the Sp-EMI/TM palindromic site and putative half sites. Binding specificity was confirmed by adding WT or mutant competitor. A, biotin-labeled probes containing the palindromic site, site A, or site B. B, biotin-labeled probes containing the palindromic site, site C, or site D. For the complete sequences of all probes used in this study, see Table S1. A sample of each purified protein was separated on SDS-PAGE gels and visualized by Coomassie staining (Fig. S1). Alx1-FL, full-length Alx1; Alx1-HD, Alx1 HD alone; Alx1ΔD2, a mutant form of Alx1 that lacks the D2 domain; D, dimer; F, free probe; M, monomer.
	Figure 3. Quantitative EMSA analysis of the binding of purified Alx1-FL and Alx1-ΔD2 proteins to the Sp-EMI/TM palindromic site.A, protein titration of Alx1-FL with a constant amount of Cy5-labeled probe containing the Sp-EMI/TM palindromic site. B, protein titration of Alx1-ΔD2 with a constant amount of Cy5-labeled probe containing the Sp-EMI/TM palindromic site. C, quantification of representative gels as shown in panels A and B. Plot of the fraction of probe bound as a function of protein concentration. Data are based on two independent replicates of the binding assay and are represented as the mean ± 1 SD. The filled circles denote Alx1-FL, and the filled squares denote Alx1-ΔD2. D, dimer to monomer ratios based on quantification of the same two independent replicates shown in panel C with data represented as the mean ± 1 SD. Alx1-FL, full-length Alx1; Alx1-HD, Alx1 HD alone; Alx1ΔD2, a mutant form of Alx1 that lacks the D2 domain; D, dimer; F, free probe; M, monomer.
	Figure 4. EMSA analysis comparing the binding of purified Alx1-FL and Alx4-FL proteins to DNA. Probes used in this analysis were all 70 bp in length and included the Sp-EMI/TM palindromic site or two half sites (site A and site B) from the Sp-mtmmpb CRM. A and B, the binding capacities of Alx1, Alx1ΔD2, and Alx4 were analyzed for each biotin-labeled DNA probe. Binding specificity was confirmed by adding WT or mutant competitor. C, protein titration of Alx4-FL with a constant amount of the Cy5-labeled probe containing the Sp-EMI/TM palindromic site. D, plot of the fraction bound as a function of protein concentration, based on quantification of representative gels as shown in panel C. Data are based on two independent replicates of the binding assay and are represented as the mean ± 1 SD. The filled circles denote Alx1-FL, and empty circles denote Alx4-FL. Data for Alx1-FL are also based on two independent replicates, distinct from those shown in Figure 3. E, dimer-to-monomer ratios based on quantification of the same binding assays shown in panel C with data represented as the mean ± 1 SD. For the complete sequences of all probes used in this study, see Table S1. Alx1-FL, full-length Alx1; CRM, cis-regulatory module; D, dimer; F, free probe; M, monomer.
	Figure 5. EMSA analysis demonstrating that binding of Alx1 to closely spaced half sites is noncooperative.A, all probes used were 70 bp in length and included the Sp-EMI/TM palindromic site or two half sites (site A and site B) from the Sp-mtmmpb CRM. Additional nucleotides underlined were inserted between site A and site B to increase the distance between the two sites and alter their relative orientation on the DNA helix. The added sequence was designed by repeating the sequence underlined in the probe containing the two half sites to synthesize site A +++ site B probe. Binding specificity was confirmed by adding WT or mutant competitor. B, protein titration of Alx1-FL with constant amounts of two different Cy5-labeled probes containing sites A and B. C, quantification of the representative gel in panel B. Plot of the fraction bound as a function of protein concentration of two independent replicates with the data represented as the mean ± SD. The filled squares denote the probe site A + site B, and the empty squares denote the probe site A +++ site B. For the complete sequence of all probes used in this study, see Table S1. Alx1-FL, full-length Alx1; CRM, cis-regulatory module; F, free probe.
	Figure 6. Alx1–Alx1 interactions in the absence of DNA.A, GST-tagged Alx1 protein and various deletions mutants were expressed in Escherichia coli and immobilized on glutathione beads. The protein samples were separated on SDS-PAGE gels and visualized by Coomassie staining. B, GST-tagged, immobilized proteins shown in panel A were incubated with purified full-length, His-tagged Alx1. The beads were washed, separated on an SDS-PAGE gel, and analyzed by Western blotting using an anti-His antibody.
	Figure 7. Analysis of the functional role of half sites in vivo using transgenic reporter assays.A, GFP expression in embryos injected with WT Sp-mtmmpb CRM and different Alx1-binding site mutants assayed by fluorescence microscopy. B, representative embryos showing GFP expression. The scale bar represents 50 μm. CRM, cis-regulatory module; PMC, primary mesenchyme cell.

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