Purchase this article with an account.
Amanda G. Kautzman, Irene E Whitney, Benjamin E Reese; Transcriptional Activation Differs Significantly Between the Two Isoforms of Isl1. Invest. Ophthalmol. Vis. Sci. 2014;55(13):703.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Islet1 (Isl1) is a LIM homeodomain transcription factor that plays an essential role in retinal development. It forms a well characterized transcriptional complex with two other proteins, Lhx3 and Ldb1 (Isl1:Lhx3:Ldb1). It has, however, a splicing site that generates two isoforms, Isl1α and Isl1 β, and the Isl1β isoform lacks a critical portion of a protein-binding domain with which Isl1 binds to Lhx3. We recently demonstrated that the two isoforms of Isl1 are expressed in developing retinal tissue, and that some classes of retinal ganglion cell express only the β isoform. The present study sought direct evidence that these two isoforms function distinctly in their ability to regulate gene expression, using a luciferase reporter with Isl1:Lhx3:Ldb1 DNA recognition elements in the promoter region to assay transcriptional activation.
HEK293T cells, which endogenously express Ldb1, were transiently transfected with multiple combinations of plasmids designed to express Lhx3, Isl1α, Isl1β, or GFP. Cells were also co-transfected with a luciferase reporter that has been previously shown to be activated specifically by the Isl1:Lhx3:Ldb1 complex. Cell lysates were assayed for luciferase activity, in three biological replicates, each experiment being performed in triplicate.
The activation of luciferase by Isl1α:Lhx3:Ldb1 was 5 fold greater than Isl1β:Lhx3:Ldb1. A one-way ANOVA and post-hoc comparisons showed the luciferase activation in Lhx3+Islα overexpressing cells to be significantly different from Lhx3+Isl1β and all other conditions, while activation by Lhx3+Isl1β was indistinguishable from Lhx3 alone.
These results demonstrate a functional difference between the two isoforms of Isl1; furthermore, they suggest that the β isoform may not be capable of forming a complex with Lhx3 and Ldb1. This may suggest that Isl1β containing-complexes have unique gene targets from Isl1α. Given our previous finding that Isl1α and Isl1β are differentially expressed in subsets of retinal ganglion cells, these results further suggest that these isoforms likely play distinct roles in neuronal differentiation during development.
This PDF is available to Subscribers Only