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Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function.
Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene.
Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation.
Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.
Figure 1. The Rax C-terminal region contains activation and repression domains. Full-length (FL) or mutated Rax or Rax2 was assayed for the ability to activate expression of a Xenopus rhodopsin gene promoter–luciferase reporter gene (C and D or E, respectively). A: Schematic representation of the Rax constructs assayed. Numbers indicate amino acid residues. Dotted lines indicate internal deletions. Point mutations are marked by an asterisk (*). B: Schematic representation of Rax2 constructs assayed. C, D: Luciferase assay results for Rax constructs diagrammed in A. Reporter gene activity is calculated as firefly luciferase activity normalized to the Renilla luciferase control and is presented relative to activity of a pCS2-only plasmid control. Values represent the mean of three to nine independent experiments (nine replicates per experiment). Error bars represent standard deviation from the mean. All vector-only and Rax activity values are statistically significantly different from full-length Rax activity using a corrected (Bonferroni) critical value of p<0.0017 (*p=4.06 × 10−48, n=81; **p=3.25 × 10−8, n=27; ***p=1.04 × 10−23, n=27; ****p=6.15 × 10−56, n=27; *****p=3.73 × 10−6, n=27; #p=8.75 × 10−18, n=54; ##p=2.42 × 10−10, n=27; ###p=3.28 × 10−20, n=27). E: Rax2 ΔC180 activity is not statistically significantly different from full-length Rax2 activity (Student t test, p=0.02). Abbreviations: HD = homeodomain, OAR = orthopedia, aristaless, rax, OP = octapeptide domain, PPXY = PPXY motif (putative YAP/TAZ binding sequence), RX = Rx domain. *in construct 3PA denotes mutated proline residues (to alanine residues).
Figure 2. The Rax C- and N-terminal regions exhibit functional interaction. Full-length (FL) or mutated Rax was assayed for ability to activate expression of a Xenopus rhodopsin gene promoter–luciferase reporter gene. A: Schematic representation of Rax constructs assayed. Numbers indicate amino acid residues. Dotted lines indicate internal deletions. Point mutations are marked by an asterisk (*). B: Alignment of sequences from X. laevis arx and rax OP motifs. C: Luciferase assay results for Rax N-terminal mutations and deletions diagrammed in A. Reporter gene activity is calculated as firefly luciferase activity normalized to Renilla luciferase control and is presented relative to activity of a pCS2-only plasmid control. Values represent the mean of three to nine independent experiments (nine replicates per experiment). Error bars represent standard deviation from the mean. All vector-only and Rax activity values except ΔN39 are statistically significantly different from full-length Rax activity using a corrected (Bonferroni) critical value of p<0.0017 (*p=4.06 × 10−48, n=81; **p=6.90 × 10−8, n=27; ***p=7.52 × 10−6, n=27; ****p=2.13 × 10−14, n=27; *****p=0.00761, n=27). D: Luciferase assay results for Rax constructs containing C-terminal truncations (indicated on the x-axis) and an intact N-terminus (black filled bars), the L38P mutation (gray filled bars), or ΔOP (hatched bars), as shown in Figure 1A or the lower portion of panel A of this figure. All pairwise activity values are statistically significantly different, except where indicated, using a corrected (Bonferroni) critical value of p<0.0017 (ap=3.34 × 10−39, bp=4.42 × 10−10, cp=3.29 × 10−8, dp=9.50 × 10−38, ep=1.94 × 10−8, fp=6.53 × 10−25, *p=1.87 × 10−17, **p=5.23 × 10−14, ***p=7.12 × 10−19, ****p=8.78 × 10−11; in each case, n=27). Abbreviations: HD = homeodomain, NS = not significant, OAR = orthopedia, aristaless, rax, OP = octapeptide domain, PPXY = PPXY motif (putative YAP/TAZ binding sequence), RX = Rx domain. *in constructs containing L38P denotes mutated leucine residues (to proline).
Figure 3. The Rax C-terminal domain is sufficient to mediate reporter gene expression. Portions of the Rax C-terminal region were fused to a heterologous GAL4 DNA binding domain (GAL4 DBD) and assayed for transactivation of a GAL4-responsive firefly luciferase reporter. Data presentation and abbreviations are as described in Figure 1. The GAL4 DBD–Rax C-terminal fusions are represented schematically at the top of the figure. Values represent the mean of three to ten independent experiments (nine replicates per experiment). Error bars represent standard deviation from the mean. Transactivation activity values for all constructs tested are statistically significantly different from the activity of vector-only or GAL4 DBD alone using a corrected (Bonferroni) critical value p<0.003125 (*p=1.77 × 10−9, n=90; **p=4.36 × 10−40, n=63; ***p=2.29 × 10−51, n=63; ****p=8.33 × 10−15, n=63; ****p=3.76 × 10−12, n=45).
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