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Heithem M El-Hodiri, Jessica L Buescher; The transcriptional activity of the retinal homeobox (Rax) gene product is dependent on conserved N- and C-terminal domains. Invest. Ophthalmol. Vis. Sci. 2017;58(8):627.
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© ARVO (1962-2015); The Authors (2016-present)
The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite its biological significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. We hypothesized that conserved regions in the N-terminus (including the Octapeptide motif or OP), and the C-terminus (including the Orthopedia-Aristaless-Rax domain or OAR), modulate the transcriptional activity of Rax.
To test our hypothesis, we generated N- and C-terminal deletion and point mutations in Xenopus laevis rax.L using PCR-based methods. We then examined the ability of mutated Rax to transcriptionally activate a rax-responsive luciferase reporter in HEK293T cells (presented as average luciferase units relative to empty vector ± standard deviation).
Wild type Rax weakly activated the reporter (1.5 ± 0.037 X). Deletion of the N-terminal 31 amino acids enhanced activity (2.7 ± 0.067 X, p<2.7 X 10-7) whereas mutation of the N-terminal OP (amino acids 32-39) resulted in unchanged activity (1.5 ± 0.017 X, p<0.063). Moreover, consecutive deletion of the C-terminus generated a corresponding successive increase in transcriptional activity of Rax (2.0 ± 0.05 X, p<0.02, to 4.8 ± 0.14 X, p<1.6 X 10-9). Furthermore, the activity of Rax lacking the C-terminus (up to and including the OAR) was unaffected by simultaneous mutation of the OP (3.1 ± 0.056 X vs 3.1 ± 0.093 X, p<0.85).
Our data indicate that both positive and negative regulatory elements may be present in the Rax N-terminus while the C-terminus contains a strong repressive region which may function independently of the OP. These data support our hypothesis that the highly conserved OP and OAR influence Rax activity, but also indicate the surrounding divergent regions are equally influential. Taken together, these data provide insight into the structural features that regulateRax activity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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